Assessing potential endocrine disrupting effects of municipal effluents on fathead
minnow (Pimephales promelas) populations in Southern Saskatchewan
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
1 Toxicology
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
Introduction
• Over the past decades, there is an increasing concern regarding the
release of emerging contaminants such as pharmaceuticals and
personal care products (PCPPs), endocrine disrupting compounds
(EDC’s), pesticides and brominated flame retardants (BFRs) into the
environment.1
• Among these chemicals, EDCs have received particular attention as
they have been shown to interact with the endocrine system
resulting in adverse effects on endocrine homeostasis,
reproduction, development and/or behaviour. 2,3
Conclusions
Methods
Site Location & Collection: Wascana Creek, Regina, Saskatchewan
• Receives MWWEs from the City of Regina’s (pop’n 200,000) outdated lagoon treatment facility.
• Collected FHM using minnow traps, seine nets and electro-fishing at three sampling sites:
o
1)RUS: Regina Upstream Site
2)RCS: Regina City Site
3)RDS: Regina Downstream Site
Sample Processing and Biological Endpoints:
• Blood Samples: Blood collected via caudal vein using heparinized needle. Plasma
separated and stored at -80°C until analysis.
• Tissue Samples: Liver, gonads and brain collected and weighed to determine
somatic (GSI, LSI), and condition (KI) indices.
• Histopathology: Gonads fixed in Davidson’s fixative.
• 2° Sexual Characteristics: Scores were given for nuptial tubercle numbers as well
as body color and shape.6
Figure 3: Site map of
Wascana Creek,
Saskatchewan
• Statistics: Significance was determined by 1 Way ANOVA followed by Tukey’s post-hoc test (SPSS 20 software).
Preliminary Results
4.5
A)
12
Female
4
Immature
b
3
Female
a
2
1.5
6
4
1
0
Evaluate reproductive health and endocrine homeostasis of wild
fathead minnow (Pimephales promelas; FHM) populations
downstream of the City of Regina’s wastewater treatment plant.
RUS
RCS
B)
RDS
2.5
Male
b
5
Immature
B)
RUS
RCS
Male
RDS
Immature
2
b
4
HSI (%)
Specific Objectives
6
0
a
GSI (%)
Study Goal
2
0.5
3
1.5
Figure 7: City of Regina
MWWE outfall
Ongoing Work
Immature
8
2.5
• The preliminary data supports the
hypothesis that MWWEs might be
adversely impacting populations of
FHM’s inhabiting receiving waters
around Regina, thus, warranting further
research.
• Similar trends in LSI and GSI were
observed in male FHM’s exposed to
Regina MWWEs in the lab (See TP041).
A)
10
b
3.5
GSI (%)
• One important source of EDCs in the environment are municipal
wastewater effluents (MWWEs)4,5 as conventional wastewater
treatment technologies are often incomplete or inefficient at
removing these contaminants.
• There is concern for 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).
HSI (%)
Figure 1: City of Regina effluent discharge into Wascana Creek
• LSI of both female and male FHMs were significantly greater at RCS and RDS sites compared to RUS (Figures 4A and 4B).
• GSI of both female (Figure 5A) and male (Figure 5B) FHMs were not significantly different. However, a trend towards lower values
was seen at the city site (RCS) and downstream compared to the upstream site.
RUS = Regina Upstream Site RCS = Regina City Site RDS = Regina Downstream Site
• Increased HSI of male and female FHMs collected downstream of the
WWTP compared to upstream could be indicative of an increase in
vitellogenin (VTG) production, a classic biomarker of endocrine
disruption, or an increase in metabolic activity due to contaminant
exposure.
• Lower GSI of male and female FHMs collected downstream of the
WWTP compared to upstream could be explained by alterations in
the sexual maturity of fishes with regards to their ovary or testes
development.
• Non-significant differences of HSI and GSI between RCS and RDS
could be explained by the release of undertreated effluents within
city limits, due to bypassing the WWTP one month prior to sampling.
• Plasma samples: Will be analyzed for VTG and sex steroids (17βestradiol, estrone, testosterone, androstenedione, 11ketotestosterone, progesterone) using enzyme linked immunoassay
(ELISA)
• Tissue Samples: Expression of target genes (VTG, aromatase (AROM),
estrogen receptor (ERα and ERβ), androgen receptor (AR), luteinizing
hormone receptor (LHR) and steroidogenic acute regulatory protein
(StAR) will be quantified by use of qRT-PCR.
• Histopathology: Sections will be analysed for histological alterations
including the occurrences testicular oocytes, mixed sex conditions,
degeneration of tissue, and other abnormalities.
A)
1
B)
2
1. Collect wild fathead minnows during and after the reproductive
season from populations up- and downstream of the City of
Regina wastewater treatment plant (WWTP).
2. Compare maturation status, reproductive health, and endocrine
status of up- and down-stream populations
1
0.5
0
0
RUS
RCS
RDS
Figure 4: Hepato somatic indices (HSI) of sexually immature
and mature female (A) and of sexually immature and mature
males(B) FHMs at RUS (n=32), RCS (n=32) and RDS (n=38). Error
bars represent SEM. Different letters denote statistically
significant differences.
• 40%, 6% and 12% of males had overall tubercle scores >5
upstream of Regina, immediately downstream of the city
and immediately downstream of the City of Regina
wastewater treatment plant (WWTP) respectively.
Figure 2: Electro-fishing
Created by Peter Downing – Educational Media Access and Production © 2011
RUS
RCS
Figure 5: Gonado-somatic indices (GSI) of of sexually immature
and mature female (A) and of sexually immature and mature
male (B) FHMs at RUS (n=32), RCS (n=32) and RDS (n=38). Error
bars represent SEM. Different letters denote statistically
significant differences.
A)
Figure 8: (A) 96 well plate used for ELISA (B) histology section of FHM
RDS
Acknowledgements
•
•
•
Thanks to Ashley Moate and the Hecker lab group for help with field sampling
and analysis.
This project was partially funded through the Canadian Water Network.
A special thanks to the City of Regina Wastewater Treatment Plant.
C)
B)
Figure 6: 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.
References
1. CCME. 2006. 2. Goksøyr A. (2006). Jour. Tox. Environ. Heal., Pt. A:69: 175-184. 3. Campbell C.G., Borglin S.E., Green F.B., Grayson A.,
Wozei E., Stringfellow W.T. 2006. Chemosphere. 65: 1265 – 1280. 4. Desbrow, C., Routledge, E., Brighty, G., et al. 1998. Environ Sci
Technol. 32:1549-1558. 5. Mills,L., and Chichester, C. 2005. Science of the Total Environment. 343: 1-34. 6. US EPA Fish Short-Term
Reproduction Assay. OPPTS 890.1350