School of Environment and Sustainability
Characterization of the endocrine potencies of
municipal effluents across Canada using in
vitro bioassays
Tabata Bagatim
Supervisor: Dr. Markus Hecker
Tabata Bagatim1, Sara Hanson2, Hongda Yuan2, Kean Steeves2, Steve Wiseman2, Natacha Hogan2,3, Alice Hontela4, Paul Jones1,2, John Giesy2,5, Leslie
Bragg6, Hadi Dhiyebi6, Mark R. Servos 6, Charles Gauthier7 ,François Gagné 8, and Markus Hecker1,2
1School
of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada; 2Toxicology Centre, University of Saskatchewan, Saskatoon, SK,
Canada; 3 Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, Canada; 4Department of Biological Science, University of
Lethbridge, Lethbridge, AB, Canada; 5Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada; 6 Biology Department,
University of Waterloo, Watereloo, ON; 7INRS-ETE et UQTR, Quebec, QC, 8Environment Canada
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Endocrine Disrupting Compounds
•
There is increasing concern about chemicals with the potential to
adversely affect the endocrine system of humans and wildlife.
•
EDCs of primary toxicological concern:
(anti)estrogenic, (anti)androgenic and steroidogenesis disruption properties.
• Receptor mediated processes
• Non-receptor mediated processes
Figure 1 - EDCs mimicking endogenous hormones
CEW 2015
http://www.precisionnutrition.com/all-aboutenvironmental-toxin
Oct 6th, 2015
School of Environment and Sustainability
Sources of EDCs
Adapted from Kirsten Moore et al. 2011
Figure 2 – Sources of EDCs (Adapted from Kirsten Moore et al. 2011)
MWWEs are considered to be the major source of EDCs in Canadian surface waters.
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Emergent Contaminants
Pharmaceuticals
• Birth control
• Cimetidine
Life-Stock Operations
•Hormones
•Pharmaceuticals
Plasticizers
• Bisphenol A
• Phthalates
Flame retardants
•Organobromide
compounds
CEW 2015
Household Cleaning
Products
Oct 6th, 2015
School of Environment and Sustainability
EDCs in MWWEs and challenges
•
Incomplete understanding of the effluents from WWTPs contribution to the
environment in Canadian surface waters.
•
Complex mixture – problematic to identify the specific compounds that
are responsible for biological effects in exposed organisms.
Figure 3 – Saskatoon WWTP
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
EDCs identification approaches
•
Traditional targeted chemical analyses is not able to provide a complete
and objective exposure assessment.
•
Targeted in vitro bioassays can characterize the specific endocrine
activity of complex mixtures, including unknowns chemicals.
•
In vitro bioassays have the potential to serve as predictors of potential
hazards for wildlife.
CEW 2015
Oct 6th, 2015
Toxicology Centre
AIME Overall Project
Assessment of Environmental Impacts of Municipal Effluents (AIME)
In Vivo Studies with
Native Species (FHM)
FHM
Reproductive
Assays
CEW
In Vitro Studies with
Validated Bioassays
Chemical Analytical
Studies
Wild Fish
Study (In
Stream)
October 6, 2015
School of Environment and Sustainability
Objectives
Determine whether MWWEs represent a significant source of EDCs to
aquatic environments in Canada using an in vitro bioassay-directed analysis
approach.

Evaluate and quantify endocrine disrupting activities of MWWEs across Canada.

Characterize the efficiency of WWTPs to remove EDCs.

Evaluate different treatment levels of WWTPs (primary, secondary, etc.).

Determine whether season (temperature) influences EDCs removal efficiency.
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Methods
Saskatoon
260,600 Pop
Regina
232,890 Pop
Guelph
134,894Pop
Kitchener
231,488 Pop
Quebec City
321,221 Pop
Montreal
1,900,000 Pop
Figure 4 – Locations of wastewater treatment plants (WWTPs) in Quebec, Ontario and Saskatchewan, and the population (Pop) they are
serving.
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Methods
MDA
(Anti) androgenicity
Figure 5- Particulate Filtration.
CEW 2015
MVLN
(Anti) estrogenicity
Figure 6 - SPE – Capture of organic
compounds.
H295R Steroidogenesis
Disruption
Figure 7 - SPE – Sample preparation for
bioassay.
Oct 6th, 2015
School of Environment and Sustainability
Cytotoxicity Test
Control
Influent
Effluent
Fold-Change [SC=1]
1.2
*
1
*
*
0.8
*
0.6
*
*
0.4
*
0.2
0
Control
Regina
Saskatoon
Guelph
Kitchener
Montreal
Quebec
City (10x concentrated)
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Dose Response - Saskatoon Spring
Androgenicity
Control
18
Effluent
14
12
% Inhibition
*
16
AEQ [ng DHT]
Influent
Anti-Androgenicity
*
10
8
6
4
2
0
Control
0.1x
0.3x
1x
Concentration
CEW 2015
3x
10x
100
90
80
70
60
50
40
30
20
10
0
Influent
Effluent
*
*
*
*
*
*
*
*
0.1x
0.3x
1x
3x
10x
Concentration
Oct 6th, 2015
School of Environment and Sustainability
WWTPs across Canada - Spring 2014
Androgenicity
AEQ [ng DHT]
100
10
1
Influent
Eflluent
Influent
*
*
*
*
**
*
100
% Inhibition
Control
Anti-Androgenicity
*
80
60
40
20
Eflluent
*
*
*
*
*
**
0
0
*
-20
-40
City (1x concentrated)
CEW 2015
-60
City (1x concentrated)
Oct 6th, 2015
School of Environment and Sustainability
Androgenicity across WWTPs
Spring 2014
Influent
Eflluent
100
AEQ [ng DHT]
*
*
10
1
*
Influent
Eflluent
100
*
*
*
10
*
*
*
*
*
1
City (1x concentrated)
CEW 2015
Control
AEQ [ng DHT]
Control
Summer 2014
City (1x concentrated)
Oct 6th, 2015
School of Environment and Sustainability
Montreal Androgenicity - Summer 2014
AEQ [ng DHT]
Control
Inf Sep 14
Eff Sep 14
Inf Sep 17
Eff Sep 17
100
*
*
10
*
*
*
*
1
0.1
Control
0.1x
0.3x
1x
3x
10X
Fold Concentration [x]
• Montreal Effluent (1x) showed androgenic activity (12-14ng/L AEQ) in spring/summer.
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
WWTPs across Canada - Spring 2014
Anti-Estrogenicity
Estrogenicity
Control
Influent
Eflluent
% Inhibition
EEQ [ng E2]
3
2
1
0
100
90
80
70
60
50
40
30
20
10
0
Influent
Eflluent
*
City (1x concentrated)
City (1x concentrated)
• Montreal Effluent (1x) = 1.78ng/L EEQ in Spring.
• Segner, 2003 - EE2>1.67ng/L
CEW 2015
↓ egg number and fertilization success, delay in time to spawn.
Oct 6th, 2015
School of Environment and Sustainability
Steroidogenesis Disruption across
Canada - Summer 2014
3
Fold-Change [SC=1]
Control
Influent
Effluent
3
2
2
*
*
1
*
1
*
0
Control
Regina
Saskatoon
Guelph
Kitchener
Montreal
Quebec
City (1x concentrated)
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Non-target screening for
contaminants in WWTP Effluents
• Large number of known and unknown
chemicals are present in effluents
• So far, identified approx. 400 different
compounds, ranging from
pharmaceuticals and personal care
products to pesticides
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Chemical Analytical Data (ng/L)
Endpoint
Season
Atrazine
spring
summer
spring
summer
spring
summer
spring
summer
spring
summer
spring
summer
spring
summer
spring
summer
Carbamazepine
Clofibrate
DEET
Diazepam
Ibuprofen
Naproxen
Triclosan
CEW 2015
Regina
<MDL
0.08
7.62
0.91
37.80
<MDL
395.45
25.54
0.22
<MDL
<MDL
<MDL
<MDL
<MDL
28.95
0.37
Saskatoon
0.06
0.16
0.65
8.79
39.82
30.93
10.29
3168.79
<MDL
0.11
<MDL
<MDL
<MDL
<MDL
0.23
8.74
Guelph
0.33
0.21
18.90
16.55
109.35
78.96
74.38
61.14
<MDL
<MDL
<MDL
<MDL
<MDL
<MDL
3.79
2.27
Kitchener
0.61
N/A
1.24
N/A
<MDL
N/A
7.68
N/A
<MDL
N/A
<MDL
N/A
<MDL
N/A
1.76
N/A
Quebec
0.23
<MDL
15.86
2.93
90.77
<MDL
168.15
113.70
0.48
<MDL
<MDL
1637.31
<MDL
320.99
53.36
106.08
Montreal
10.11
1.05
5.33
0.54
<MDL
<MDL
6.98
34.41
0.08
<MDL
687.90
194.13
18.73
<MDL
62.63
13.93
Oct 6th, 2015
School of Environment and Sustainability
Summary
•
Most of the WWTPs had a high removal efficiency of androgenic activity.
•
Selected effluents (e.g. Montreal) had significantly increased androgenic
potencies.
•
Removal efficiencies differed significantly among WWTPs
•
Further analysis need to be completed regarding efficiency of different treatment
levels of WWTPs and determine whether population and temperature affects
EDCs removal efficiency.
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Conclusion
•
In vitro assays supported in vivo findings, suggesting a cost-effective tool for
predicting EDCs in aquatic environments.
•
Targeted chemical analysis did not show a presence of chemicals with
estrogenic properties, aligning with in vitro assays findings.
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Acknowledgement




UofS Toxicology Center
Saskatoon Wastewater Treatment Plant
City of Regina Wastewater Treatment Plant
Department of Biological Science, University of Lethbridge, Lethbridge, AB,
Canada;
 Biology Department, University of Waterloo, Waterloo, ON
 INRS-ETE et UQTR, Quebec, QC, 8 Environment Canada
 Ashley Moate, Craig Baird, Shawn Beitel, Leanne Flahr, J.X. Sun, Bryanna
Eisner
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
Questions?
Fig8: FHM field sampling in Saskatoon Upstream and Downstream
CEW 2015
Oct 6th, 2015
School of Environment and Sustainability
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
CEW 2015
Wilson et al. 2002 - Toxicol Sci 66: 69-81
Wilson et al. 2004 - Toxicol Sci 81: 69-77
Hecker et al. 2006 - Toxicol Appl Pharmacol 217: 114-124
Hecker et al. 2011 – Environ Sci Pol Res 18: 503-515
Mosman et al. 1983 - J Immunol Methods 65: 55-63
Hallgren, 2011 - Ecotoxicology 21: 803–810
Jobling et al., 1996 – Environ. Toxicol. Chem. 15: 194-202
Kime and Nash, 1999 – Aquaculture 177: 345-352
Belfroid et al., 1999 - Sci. Total Environ., 225: 101-108
Kidd et al., 2014 - Phil. Trans. R. Soc. B 369
Segner, 2003 - Ecotox. Environ. Saf. 54: 216–222
Oct 6th, 2015