Assessing potential endocrine disrupting effects of municipal effluents on fathead
minnow (Pimephales promelas) populations in Southern Saskatchewan
Graduate Program, University of Saskatchewan, Saskatoon, SK, CA. 2Toxicology Centre, University of Saskatchewan, Saskatoon, SK, CA, 3 School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, CA, 4 Department of Animal and Poultry Science,
University of Saskatchewan, Saskatoon, SK, CA, 5Department of Biological Science, University of Lethbridge, Lethbridge, AB, CA, 6Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, CA
Background
a
a
a
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a
6
a
a
a
a
0.6
a
0.4
0.2
0
era
a
a
a
Site Location & Collection: Wascana Creek, SK, Canada
erb
ar
cyp19b
a
a
A) Males - Brain
a
 40%, 6% and 12% of males had overall tubercle scores >5 at RUS,
RCS, and RDS respectively.
0.4
b
Results – Somatic Indices
0
17bhsd
star
cyp19a
lhr
B) Males - Gonad
ac
era
fshr
a
a
a
1.2
1
0.8
0.6
a
RCS
a
RDS
ab
a
b
b b
erb
ar
vtg
b
b
a
ab
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b
a
a
a
a
a
a
a
ar
cyp19b
1.5
RDS
a
a
a
b b
a
a
star
cyp19a
lhr
fshr
era
erb
ar
vtg
Results – Histopathology
Table 2: Average grade of severity of histological criteria in the gonads
of female adult FHMs from RUS and RDS. RUS: n=10, RDS: n=8.
A
↑ Proportion of spermatogonia
0.8
3.6
Testicular degeneration
0.3
2.5
Incidence of testis-ova
-
-
Interstitial cell
hyperplasia/hypertrophy
-
1.6
Criteria
-
C
1
2
3
RUS
↑ Oocyte atresia
0.2
Perifollicular cell
hyperplasia/hypertrophy
A)
↓ Yolk formation
B)
-
RDS
2.6
1
0.2
Change in gonadal staging Stage 2 Stage 1
A
C
Figure 2: Site map
of Wascana Creek,
Saskatchewan
Sample Processing and Biological Endpoints:
 Plasma: Analyzed for concentrations of 11-Ketotestosterone (11-KT)
and estradiol (E2) using enzyme linked immunoassay (ELISA).
 Tissues: Expression of target genes along the hypothalamus-pituitarygonadal-liver (HPGL) axis quantified by use of qRT-PCR.
 Morphometrics: Somatic (GSI, HSI) and condition indices determined.
 Histopathology: Gonads were analysed for histological alterations
including occurrence of testicular oocytes, mixed sex conditions,
degeneration of tissue, and other abnormalities.
 2° Sexual Characteristics: Scored nuptial tubercle size and location.
C)
B
D
B
D)
D
Female
b
Male
b
3
a
a
1
0
RUS
12
10
B)
RCS
RDS
Female
a
Male
8
GSI
 Brain: Significant decrease in expression of ERβ and aromatase (cyp19b) in both females (3A) and males (4A) at the RDS site.
 Gonad: Significant induction of star and lhr in females (3B) and 17β-dehydrogenase and lhr in males (4B) at the RDS site.
 Liver: Significant down-regulation of AR in females (3C) and ERα and Erβ in males (4C) at the RDS site.
RDS
b
b
2
Figure 4: Abundances of transcripts of genes involved in sex steroid hormone synthesis and signaling in male FHMs collected from RUS, RCS or RDS. (A) Brain, (B)
gonad, (C) liver. Error bars represent SEM. Different letters denote statistically significant differences.(p<0.05; one way ANOVA). RUS: n=10, RCS: n=13, RDS: n=25.
RUS
A)
4
0
17bhsd
6
5
a
b b
 HSI of both females ad male FHMs were significantly greater at RCS
and RDS sites compared to RUS (Figure 3A).
 GSI of both female and male FHMs were significantly lesser at RCS
and RDS sites compared to RUS (Figure 3B.)
a
a
1
0.5
a
0
erb
RUS
2
RDS
4
C) Males - Liver
RCS
RCS
6
2
b
2.5
RUS
8
Fold-Change
1.4
b
10
RUS
1.6
Figure 7: Mature male FHM with clearly visible fat pads collected (A) at RUS
with an overall tubercle score of 57 and (B) nuptial tubercles (C) at RDS with an
overall tubercle score of 0.
HSI
1.8
Criteria
 Receives MWWEs from the City of
Regina’s (pop’n 200,000) outdated
lagoon treatment facility.
 FHMs were collected using seine nets at
three sampling sites:
a
a
a
a
B)
RDS
a
0.8
0.6
RCS
a
a
1
RUS
a
0.2
Table 1: Average grade of severity of histological criteria in the
gonads of male adult FHMs from RUS and RDS. RUS: n=6, RDS: n=7.
Methods
a
a
a
a
C)
A)
C) Females - Liver
Figure 3: Abundances of transcripts of genes involved in sex steroid hormone synthesis and signaling in female FHMs collected from RUS, RCS or RDS. (A) Brain, (B)
gonad, (C) liver. Error bars represent SEM. Different letters denote statistically significant differences.(p<0.05; one way ANOVA). RUS: n=17, RCS: n=17, RDS: n=10.
era
Created by Peter Downing – Educational Media Access and Production © 2011
ab
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1.2
RDS
0
0
Improve our understanding of the potential impacts of EDCs associated
with MWWEs to local aquatic ecosystems in the Canadian prairies
through a case study characterizing the endocrine effects of MWWEs
released into Wascana Creek, SK to a native species the FHM.
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0.2
Research Goals
2
1.4
RCS
b
a
3
b
0.4
Figure 1: City of Regina effluent discharge into Wascana Creek, SK
4
RUS
bc
5
RDS
1
1.6
b
RUS
RCS
0.8
B) Females - Gonad
Fold-Change
Fold-Change
1.2
A) Females - Brain
Fold-Change
1.4
Fold-Change
 Over the past decades, there is an increasing concern regarding the
release of emerging contaminants (ECs) such as pharmaceuticals
and personal care products (PPCPs), endocrine disrupting
compounds (EDCs), and pesticides into the environment.
 One important source of ECs in the environment are municipal
wastewater effluents (MWWEs) as conventional wastewater
treatment plant (WWTP) technologies are often incomplete or
inefficient at removing these contaminants.
 There is particular concern regarding the potential toxicological
impacts of MWWEs to resident organisms, such as fish,that live in
receiving water bodies in the southern prairies as these water
bodies are often small and can consist of up to 100% effluent during
dry seasons (e.g. Wascana Creek, Regina, Saskatchewan, Canada).
RUS: Regina Upstream Site
RCS: Regina City Site
RDS: Regina Downstream Site
Results – 2° Sexual Characteristics
Results
Results–- Gene
Histopathology
Expression
Fold-Change
1 Toxicology
Sara Hanson1,2, Tabata Bagatim2,3, Kean Steeves1,2, Steve Wiseman2, Natacha Hogan2,4, Alice Hontela5, Paul Jones2,3, John Giesy2,6, Markus Hecker2,3
6
Figure 3: Hepato-somatic
indices (HSI, A) and gonadosomatic indices (GSI, B) of
female and male FHMs and at
RUS (n=32), RCS (n=32) and
RDS (n=38). Error bars
represent SEM. Different
letters denote statistically
significant
differences.
(p<0.05; one way ANOVA)
b
4
b
2
a
0
RUS
b
b
RCS
RDS
Conclusions
 Significant physiological, morphological and molecular changes
occurred in FHM populations downstream of Regina’s WWTP.
 No estrogenic effects were observed,
parallel with in vitro (Poster: MP025)
and laboratory in vivo (Poster: MP031)
studies.
 This case study highlights the current
ecological risks of ECs associated with
MWWEs, and the need for
implementing more effective and
affordable measures to remove them
Figure 8: City of Regina MWWE outfall
at WWTPs.
Acknowledgements
Figure 5: Representative photos of histopathological criteria observed in
male adult FHM gonads. Increased spermatogonia: (A) RUS = nonremarkable, (B) RDS = grade 3. Testicular degeneration: (C) RUS = nonremarkable, (D) RDS = grade 2.
Figure 6: Representative photos of histopathological criteria observed in
female adult FHM gonads. Increased oocyte atresia: (A) RUS = nonremarkable, (B) RDS = grade 3. Gonadal staging: (C) RUS = stage 2, (D) RDS
= stage 0.
The authors would like to thank the Hecker lab group for support with field
sampling and analysis. A special thank you to the City of Regina Wastewater
Treatment Plant. Project funding by the Canadian Water Network.