Fathead Minnow and Pearl Dace Pilot at Canadian
Experimental Lakes Area
James Lazorchak1, Robert Flick1, Karen Kidd2,3, Vince Palace2, and Robert Evans2
1ORD/NERL, U.S. Environmental Protection Agency, Office of Research and Development,
2Fisheries and Oceans Canada; 3Biology Department, University of New Brunswick
The Story
Methods/Approach
From 1999 through 2004 the Canadian Department of Fisheries
and Oceans Freshwater Research Institute conducted a whole-
lake ecosystem study to evaluate organism through population
effects of exposure to a model endocrine disrupting compound,
the synthetic estrogen ethynyl estradiol. This study at the
Experimental Lakes Area site in Western Ontario was visited by
EPAscientistis for field sampling of exposed indigenous fish
and deployment of unexposed fish in cages, in both curtained
enclosures (limnocorrals) and the lake. Lake water was also
shipped for laboratory exposures of cultured fish at the NERL-
Cincinnati laboratory. EPA diagnostic molecular indicator
measurements showed increased levels of estrogenic activity
across time, in both the water column and in sediments,
consistent with chemical data that were associated with
dramatic changes in fish populations. Invaluable experiences
and data were also gained that provided ORD with the
knowledge of its capabilities in monitoring in very remote
locations, with indigenous and laboratory-cultured fish across
extensive periods of time.
Background Information
¦< Increased concern about impacts of endocrine disrupters on
aquatic organisms
¦< Potent synthetic estrogens used in contraceptives, such as
ethynylestradiol (EE2), are found in waterways downstream
of wastewater treatment plants
¦< Studies have found EE2 in surface waters at concentrations
up to 60 ng/L but more commonly at low ng/L levels
¦< Fisheries and Oceans Canada conducted a whole-lake
experiment at the Experimental Lakes Area (ELA) in
northwestern Ontario
¦< For three summers beginning in 2001, EE2 was added to
surface waters to achieve 5-6 ng/L concentrations in one
lake
¦< Population-, organism-, biochemical- and cellular-level
effects were examined in fathead minnow and pearl dace
exposed to environmentally-relevant concentrations of this
synthetic estrogen
Canadian ELA
58 Designated Research Lakes
and their Watersheds
Detailed Monitoring since 1969
Lake 260 - Estrogen Addition Lake
34 hectares in surface area
Max. depth 14 m
Contains well-defined
populations of lake trout, w
sucker, fathead minnow
and pearl dace
baseline data
reference lake data
Results
2001 Semi-quantitative PCR
Gel Based
2001 Results for Indigenous Male
Fathead Minnows
Vitellogenin Expressi
Conclusions
Field and Lab Methods
Male fathead minnows were collected from dosed Lake 260
and reference lake 114 after 7 weeks, 9 weeks and 12
weeks of dosing. (2001-2003) - minnow traps
Male fathead minnows from reference lake 114 placed into
dosed lake 260 for 13-days. (2001 only)
»-< Water and sediment samples from Lakes 260 and 114
collected and shipped to Cincinnati and tested with lab
•* Livers were collected shipped to Cincinnati for RNA Isolation
and vitellogenin gene expression quantification relative to
1 Study Design
R
recovery?
effects on individuals & populations
ethynylestradiol additions
U.S. EPA Results
x Gene expression in the 2001 fathead minnow deployment
study was detected within 24-hrs after deployment of control
fish into the treated lake and stayed elevated for the entire
13-day study.
x Indigenous male fathead minnows and pearl dace collected
at all time intervals from the dosed lake showed a constant
level of elevation in vitellogenin gene expression in both
males and females and beyond normal breeding season.
x Methods developed to measure vitellogenin gene expression
in fathead minnows were validated for use in another
cyprinid, pearl dace.
x Fathead minnow primers were applied to field collected pear
dace and fathead minnows. This indicates the potential for
assessing exposure to EDCs in other field collected
cyprinids.
x Gene expression was elevated in male adult fathead
minnows exposed to sediment elutriates from sediments
collected in 2004.
Whole Lake Experiment - Main Findings
*< Male and female fathead minnow and pearl dace showed
elevated whole body concentrations of
vitellogenin within 7 weeks of EE2 additions to Lake 260
x Egg development was delayed in fathead minnow and pearl
dace exposed to EE2; testes development
was severely impaired and testes-ova were observed in
males of these species
x Reproductive failure was observed in both of these minnow
species during the second year of additions
Impact and Outcomes
* RT-PCR analyses of total RNA can be used to provide a rapid
and timely estimate of exposure to estrogenic substances to
indigenous organisms and in laboratory animals exposed to
water and sediment samples.
•< Gene expression results in combination with biochemical,
histological and chemical information show that indigenous
fathead minnows and pearl dace continuously exposed to 5
ng/L of EE2 are adversely affected.
U.S. EPA NERL Objectives
¦* To evaluate the performance of U.S. EPA's vitellogenin gene
expression PCR methods (Gel based and QPCR) on
indigenous male fathead minnows exposed to
ethynylestradiol.
•* To develop relationships of gene expression with protein
expression and histopathological effects
¦< To apply the fathead minnow gene expression method to
other cyprinids, i.e. pearl dace
DFO - Study Objectives
-< To assess biochemical through population level effects of a
potent estrogen
* To determine the timing and magnitude of impacts of an
estrogen on aquatic populations
•< To link effects across levels of biological organization
Gel-based measurement of Vg gene expression.
Each column is a separate sample. Size
standards are at left. Vitellogenin PCR
products are separated by charge
(vertical axis) and measured relative to
an internal cell standard (18S rRNA).
2002 Vitellogenin expression
Analyzed by quantitative real-time PCR
2002 Results for Indigenous Male
Fathead Minnows
,Ua
males 2002 Results
J
///f
2003 Results for Indigenous
Male Pearl Dace
1
Future Directions
~< An EDC Multiyear Plan "Annual Performance Measure" report
has been prepared and will be distributed to Office of Water
and Regional Offices to support the development of
environmental policy and water quality criteria and standards
development.
•« A manuscript will also prepared and submitted.
-< Overall, this pilot research activity extended the capabilities of
EPA/ORD EDC monitoring efforts to a successful collaboration
on an international whole-lake ecosystem study at an
extremely remote location.
References
Lattier, D.L., Reddy, T.V., Gordon, D.A., Lazorchak, J.M.,
Smith, M.E., Williams, D.E., Weichman, B., Flick, R.W.,
Miracle, A.L. and Toth, G.P., 2002; 17 d - ethynylestradiol
- induced vitellogenin gene transcription quantified in
livers of adult males, larvae, and gills of fathead minnows
{Pimephaiespromelas). Environ. Toxicol. Chem 21 (11):
2385-2393.
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