Assessing biological effects of municipal wastewater effluent using the fathead minnow reproductive bioassay
Kean Steeves1, Sara Hanson1, Tabata Bagatim2, Steve Wiseman1, Paul Jones1, John Giesy1,5, Markus Hecker1,2, Alice Hontela4, and Natacha Hogan1,3
1Toxicology
Centre, University of Saskatchewan, Saskatoon, SK, Canada; 2 School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada; 3 Department of Animal and
Poultry Science, University of Saskatchewan, Saskatoon, SK, Canada; 4 Department of Biological Science, University of Lethbridge, Lethbridge, AB, Canada; 5Department of Veterinary Biomedical Sciences,
University of Saskatchewan, Saskatoon, SK, Canada
BACKGROUND
RESULTS – Fecundity
Regina Effluent
Municipal wastewater effluent (MWWE) release is a growing
concern in prairie cities as populations increase. Many receiving
environments are small, and effluent release from wastewater
treatment plants (WWTP) can represent a high percentage of water
volume at times of low flow. MWWEs contain a complex mixture of
chemicals, including pesticides, fertilizers and pharmaceuticals and
personal care products (PPCP). These compounds have the
potential to disrupt reproduction and development, and as a result
can have negative impacts to population sustainability of species
occupying receiving environment, including the test species,
fathead minnow (Pimephales promelas).
Figure 2. Cumulative egg production of fathead minnows exposed to Regina and Saskatoon MWWEs. Cumulative egg number shown as
eggs/female/day. Asterisks denotes significant difference from other treatment groups (p<0.05; one-way ANOVA).
• A non-significant trend of decreasing GSI in males was observed
for the Regina effluent. A similar trend was observed in wild
fathead minnows collected downstream of the Regina MWWE
(Poster #TP042).
RESULTS – Somatic Indices
• A non-significant trend of decreasing condition factor was
observed in females exposed to Saskatoon effluent.
Saskatoon Effluent
Regina Effluent
Future Work
*
HSI(%)
METHODS
• A trend towards increasing HSI was observed for females
exposed to Saskatoon MWWE. However, males showed an
opposite trend.
HSI(%)
municipal
different
assessing
minnows
• The study was conducted at the Aquatic Research Facility in The
Alberta Environmental Sustainability and Water Building,
University of Lethbridge.
• Effluents were collected (end-of-pipe) from the Regina and the
Saskatoon WWTPs and immediately transported to Lethbridge.
• Fish were exposed for 21 days to effluent (flow through) in
accordance with USEPA test guidelines
- Control, 10% and 50% effluent
• Endpoints measured:
- fecundity and fertility (daily egg collection)
- somatic indices (liver and gonad), tubercle score, and
condition factor
- Liver, spleen, brain, and gonad tissue were taken for future
histological, molecular, and biochemical analysis.
Figure 1. (A) male fathead minnow, (B) female fathead minnow,
(C) exposure tower.
Created by Peter Downing – Educational Media Access and Production © 2011
• Plasma Hormones: Blood was collected from fathead minnows.
Plasma was separated and will be measured for 17β-estradiol,
testosterone, estrone, progesterone, androstenedione, and 11ketotestosterone.
• Molecular Analyses: Collected tissues will be analyzed for
expression of vitellogenin, aromatase, ERα and ERβ, androgen
receptor (AR), leutanizinghormone receptor (LHR), steroidogenic
acute regulatory protein (stAR).
• Fish were exposed in a flow-through rack system for a two week
pre-exposure period to acclimatize fish and establish baseline
fecundity and fertility data.
• Initial acute toxicity studies were conducted to determine
effluent toxicity:
- 50%, 75%, and 100% treatment
- 100%, and to a lesser extent 75%, Regina effluent was lethal
to minnows
• There was no significant effect of Saskatoon MWWE on fathead
minnow fecundity. Fertilization remained consistent regardless
of treatment, demonstrating a lack of effect (graph not shown).
• Regina MWWE caused a significant decrease in fecundity at both
effluent concentrations. This was demonstrated by the marked
increase in egg production by the controls, while the 10 and 50%
effluent treatments ceased egg laying.
*
HSI(%)
To determine the endocrine disrupting potential of
wastewater effluents from two prairie cities with
treatment technologies (Regina and Saskatoon) by
changes in reproductive physiology of fathead
(Pimephales promelas)
Saskatoon Effluent
*
HSI(%)
OBJECTIVES
Discussion
Figure 3. Hepatosomatic (HSI) and gonadosomatic (GSI) indices of fathead minnows after 21-days of exposure to Regina and Saskatoon MWWEs.
Data are shown as mean ± SEM. Asterisks denotes significant difference from control (p<0.05; one-way ANOVA).
RESULTS – Tubercle Score and Condition Factor
(A) Tubercle Score
(B) Condition Factor - Males
(C) Condition Factor - Females
Regina Effluent
Saskatoon Effluent
Figure 4. Male tubercle score (A), male condition factor (B), and female condition factor (C) of fathead minnows after 21-days of exposure to
Regina and Saskatoon MWWEs. Data are shown as mean ± SEM (p<0.05; one-way ANOVA).
• Gonad Histology: Portions of fathead minnow gonads were fixed
and will be analyzed for various endpoints including: increased
oocyte atresia, increased proportion of spermatogonia, and
perifollicular and interstitial cell hyperplasia/hypertrophy.
REFERENCES
1.
2.
Environmental Protection Agency (2009) OPPTS 890.1350: Fish Short-Term
Reproduction Assay. EPA 740-C-09-007
Tetrault, G., et al. (2012) Aquatic Toxicology. 110-111:149-161
ACKNOWLEDGEMENTS
Thank-you to Ian Kent, Mamun Shamsudin, Andrea Valim for technical support in
housing and exposing fish at the Aquatic Research Facility and to all those who
helped collect effluent and sample fish. To the Aquatic Toxicology and Research
Facility for cooperation in housing of the fathead minnows. Thank you to both the
Regina and Saskatoon Wastewater Treatment Plants for their cooperation in
providing the effluent used in the study. Project funding was provided by the
Canadian Water Network and stipend support to K. Steeves from the Global Institute
of Water Security, University of Saskatchewan.