Bioassay-directed characterization of endocrine disrupting potencies of
municipal effluents in Canada
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
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, 8 Environment Canada
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EDCs have the potential to adversely affect the endocrine system of
humans and wildlife, resulting in impacts on reproduction, growth,
and/or development.
• Solid Phase Extraction (SPE) with Oasis mixed cation exchange (MCX) and Oasis hydrophobic lipophilic balanced (HLB) cartridges, was used to
retain hormones, pharmaceuticals and other EDCs resulting in a 5,000x concentrate.
Conventional wastewater treatment technologies are frequently
incomplete or inefficient at removing EDCs such as
pharmaceuticals, naturally occurring hormones and other toxicants
from wastewater.
• Samples of effluent and influent were/will be collected from six WWTPs in spring, summer, fall, and winter 2014/15 (Map 1).
• Particulates were removed from samples by use of glass microfiber filters that had a 0.6µm pore size rating.
• Statistical differences among samples (influent vs effluent, WWTPs, sampling day) were evaluated using one-way ANOVA (SPSS 20).
Table 1. In vitro test systems for the characterization of endocrine potentials of WWTP effluents and influents.
Effect Type
Cell Line/Test
Cytotoxicity
(Mosman et al. 1983)
MTT (T47D,
MDA, H295R)
Traditional analytical chemistry approaches are limited as they
cannot provide a comprehensive assessment of the multitude of
different compounds in effluents.
(Wilson et al. 2004)
(Anti-)Androgenicity
Bioassays may help overcome these limitations as they
characterize the specific biological activity (e.g. agonistically binding
to the estrogen receptor (ER)) of a complex sample, and thus allow
assessing of all chemicals with specific biological activity in
effluents.
(Wilson et al. 2002)
Endpoints
Method
Cellular metabolic activity
via NAD (P) H-dependant
cellular oxidoreductase
Cells are exposed to increasing sample concentrations for 48h in
triplicate in 96-well plates; MTT assay Dye Solution is added to
each well and incubated for 2-4h to determine cell viability at an
absorbance of 450nm and 620nm.
T47D-Kbluc
Estrogen receptor-mediated Cells are exposed to increasing sample concentrations for 48h in
luciferase reporter gene
quadruplicate in 96-well luminometer plates; luciferase activity is
activity
determined using a microplate luminometer reader.
MDA-KB2
Androgen receptorCells are exposed to increasing sample concentrations for 24h in
mediated luciferase reporter quadruplicate in 96-well luminometer plates; luciferase activity is
gene activity
determined using a microplate luminometer reader.
(Anti-)Estrogenicity
Disruption of
Steroidogenesis
Cells are exposed to increasing sample concentrations for 48h in
Modulation of
triplicate in a 24-well plate; medium is removed at the end of the
steroidogenesis (sex steroid
exposure and production of hormones determined by enzymeconcentrations)
linked immunosorbent assay (ELISA).
H295R
(Hecker et al. 2006 & 2011)
PRELIMINARY RESULTS & DISCUSSION
OBJECTIVES
The main objective of this study is to characterize MWWEs as a
source of emerging contaminants for receiving surface waters using
bioassay-directed analysis.
Specific Objectives are to:
1. Characterize
(anti-)estrogenic,
(anti-)androgenic
and
steroidogenesis disruption potentials of influents and effluents of
selected WWTPs in Saskatchewan (Saskatoon and Regina),
Ontario (Kitchener and Guelph), and Quebec (Quebec City and
Montreal);
2. Determine the efficiency of different WWTPs with different treatment
technologies for removing EDCs;
3. Investigate the influence of factors such as season and temperature
on the efficiency of WWTPs in removing EDCs from raw sewage.
Androgenicity detected by MDA assay
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1
1
1
1
1
1
1
1
Androgenicity detected by MDA assay-Regina and Kitchener
*
6
*
*
4
*
c *
*
2
*
*
1
1
1
1
1
c c
0
Blank
Saskatoon
Regina
Kitchener
Guelph
Montreal
Blank
6
1
1
5
1
1
4
11
Quebec
a
Regina
a
b
a
Kitchener
3
2
1
0
City
Mar-21
Mar-24
Apr-10
Apr-12
Apr-13
Date
Figure 1: Androgenicity (fold-change relative to solvent controls) of extracts (10x
concentrated) of influent and effluent samples collected from selected WWTPs
across Canada as determined by the MDA-KB2 assay. Results are represented
as the mean ± 1SD (n=3). * indicates treatment groups are significantly different
from solvent controls (ISO; p<0.05). c indicates the values are due to cytotoxicity.
Figure 2: Variation of androgenicity (fold-change relative to solvent controls) of
extracts (10x concentrated) of influent and effluent samples collected from both
Regina and Kitchener WWTPs over a six and three days period, respectively.
Results represented as mean ± 1SD (n=3). a indicates treatment groups are
not significantly different between dates and b are significantly different between
dates (p<0.05).
• Samples of influent and effluent collected from the Saskatoon and Guelph WWTPs were cytotoxic at concentrations of 10x and 20x, respectively,
resulting in a significant decrease in androgen receptor (AR)-mediated activity. Determination of exact toxicity thresholds is under way.
• All samples of influent, with exception of Guelph, showed a significant increase in androgenicity relative to the solvent controls, with maximum
activities of 4- to 5-fold greater than activities of samples collected at Regina, Montreal and Quebec (Figure 1).
• Samples of effluent were more variable with regard to induction of androgenicity, suggesting different efficiencies of treatment processes.
Saskatoon
260,600 Pop
Regina
232,890 Pop
Guelph
134,894Pop
Kitchener
231,488 Pop
• Removal efficiency of androgenic activity was greatest for the Quebec WWTP (89%) and lowest for the Montreal WWTP (52%).
Quebec City
321,221 Pop
Montreal
1,900,000 Pop
Map 1 – Locations of wastewater treatment plants (WWTPs) in Quebec, Ontario and Saskatchewan,
and the population (Pop) they are serving.
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6
*
4
*
2
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*
*
0
ISO
0.15625
0.3125
0.625
1.25
Concentration of Influent and Effluent (x-fold)
2.5
17β-estradiol production by H295R Cells
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7
286
165
1a 4
143
1 2
121
00
ACKNOWLEDGEMENTS
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1
1
*
DMSO
DMSO
ISO
ISO
Media
Blank
For 1.0
For 10
Pro 0.3
3.0
Regina Eff Regina Inf Regina Inf
Media
Blank
For
1.0 For
10 Pro
0.3 Pro
Pro
Reg
Eff Reg
Inf Reg
Inf
Sample
5x
5x
1x
Sampl
3
5x
5x
1x
Concentration
e
*
*
*
Saskatoon Saskatoon Saskatoon
SK
Eff SK
Inf SK
Inf
Eff 5x
Inf 5x
Inf 1x
5x
5x
1x
Concentration
Figure 4: Fold-change in 17β-estradiol production by H295R Cells after exposure
to extracts of influent and effluent samples collected from Regina and Saskatoon
WWTPs, and the model inducer and inhibitor forskolin and prochloraz,
respectively. Results are represented as mean ± 1SD (n=2). * indicates significant
difference from solvent controls (DMSO; p<0.05).
• Initial exposure experiments with influent from Regina, and influent
and effluent from Saskatoon significantly increased estradiol
production, suggesting that these samples contain compounds with
the ability to disrupt steroidogenesis.
CONCLUSIONS & OUTLOOK
• WWTPs represent a significant source of EDCs to Canadian surface
waters.
• Removal efficiencies differ significantly among WWTPs as a function
of different levels of treatment (e.g. primary, secondary).
• Selected effluents (e.g. Montreal) had great androgenic potencies
(Figure 1).
• Future analyses will investigate the anti-androgenic, estrogenic, and
anti-estrogenic potentials of samples of influent and effluent.
• Sampling will continue throughout fall and winter 2014/15 to
characterize the influence of different seasons on removal efficiency of
EDCs.
• Detection of specific biological in vitro potentials will inform targeted
chemical analysis pinpoint causative agents in wastewater.
• Exposure data generated under this project will be correlated with
effects on fish investigated in two parallel studies (Posters #TP041
and TP042)
• Samples of both influent and effluent from some WWTPs showed high variation in androgenic potentials among different sampling dates (e.g.
Kitchener) while others were were relatively constant over time (e.g. Regina) (Figure 2).
Canadian Water Network, University of Saskatchewan, City of Saskatoon Wastewater Treatment Plant , City of Regina Wastewater Treatment Plant, University of
Waterloo, INRS-ETE et UQTR, Quebec, Ashley Moate, Craig Baird, Shawn Beitel, Leanne Flahr, Dayna Schultz.
Created by Peter Downing – Educational Media Access and Production © 2011
ISO
• Exposure to samples of influent and effluent from Quebec resulted in
a concentration-dependent increase in androgenicity. Both samples
resulted in a significant increase in AR activity at low extract
concentrations (0.3125x influent and 0.625x effluent).
b
Mar-18
Quebec Effluent
Figure 3: Androgenicity (fold-change relative to solvent controls) of increasing
concentrations (x-fold) of extracts of influent and effluent samples collected from the
Quebec City (QC) WWTP. Results are represented as mean SD (n=4). * treatment
groups are significantly different from solvent control (ISO; p<0.05).
Bioassays:
Although Canada has initiated first steps with the aim of
establishing standardized testing and monitoring criteria for EDC’s
in the environment, our understanding of the contribution of
effluents from wastewater treatment plants (WWTPs) to
environmental endocrine disruption in Canadian surface waters is
incomplete at best.
Quebec Influent
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Fold-Change [SC=1]
Sample Preparation and Extraction:
Fold Change [SC=1]
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Androgenicity detected by MDA assay-Quebec City
There is concern regarding municipal wastewater effluents
(MWWEs) as a major source for endocrine disrupting chemicals
(EDCs) in Canadian surface waters.
Fold Change [SC=1]
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METHODS
Fold Change
BACKGROUND
Fold Change [SC=1]
1School
REFERENCES
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Mosman et al. 1983 - J Immunol Methods 65:55-63
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