Report on Vitellogenin Gene Expression in Fathead Minnows and Pearl Dace from Reference (non-dosed) and Lakes Dosed with EE2 in the Canadian Experimental Lakes Area


EPA/600/R-04/173
September 2004

Report on Vitellogenin Gene Expression in Fathead
Minnows and Pearl Dace from Reference
(non-dosed) and Lakes Dosed with
EE2 in the Canadian Experimental
Lakes Area

Robert W. Flick
James M. Lazorchak
Mark E. Smith

U.S. Environmental Protection Agency
National Exposure Research Laboratory
Ecological Exposure Research Division
Molecular Ecology Research Branch
Cincinnati, OH 45268

Recycled/Recyclable
Printed with vegetable-based ink on
paper that contains a minimum of
50% post-consumer fiber content
processed chlorine-free.

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Notice

The research described in this document has been funded by the United States Environmental Protection
Agency under contract 68D01048 to SoBran, Inc. It has been subjected to Agency peer and
administrative review and approved for publication as an EPA document. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.

The correct citation for this document is:

Flick, Robert W., J. M. Lazorchak, and M. E. Smith. 2004. Report on Vitellgenin Gene Expression in
Fathead Minnows and Pearl Dace from Reference (non-dosed) and Lakes Dosed with EE2 in the
Canadian Experimental Lakes Area. EPA/600/R-04/173. U.S. Environmental Protection Agency,
Cincinnati, OH.

11

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1 ABSTRACT

A whole-lake endocrine disruption experiment was conducted by Fisheries and Oceans Canada
at the Experimental Lakes Area (ELA) in northwestern Ontario for three years beginning in
2001. This experiment examined population, organismal, biochemical and cellular-level effects
in lake trout, white sucker, fathead minnow, and pearl dace exposed to environmentally-relevant
(4-6 ng/L) concentrations of the synthetic estrogen, 17a-ethynylestradiol. The USEPA
collaborated in this study by evaluating vitellogenin (vtg) gene expression in: 1) indigenous
fathead minnows and pearl dace collected from the dosed and reference lakes in 2001 through
2003, before and after dosing; 2) indigenous minnows collected in 2001from the reference lake
and deployed for 1, 3, 7 and 13 days in the dosed and reference lakes; and 3) Cincinnati cultured
minnows exposed to water collected in 2001 through 2004 from ELA lakes and shipped to
Cincinnati. RT-PCR methods were used to measure vtg expression. In addition to water
exposures, embryo-larval fish and adult male fathead minnows were exposed to reference and
dosed lake sediment elutriates. 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. Gene expression in the 2001 fathead minnow deployment study was detected within
24 hours after deployment of control fish into the treated lake and stayed elevated for the entire
13-day study. Highly variable gene expression was found in fathead minnow fry exposed to
dosed lake sediment elutriates, but no significant gene expression was found in fry exposed to
reference lake sediment elutriates. Male adult fathead minnows exposed to elutriates from
sediments collected in 2004 in the previously dosed lake showed significant vitellogenin gene
expression. Results indicate that RT-PCR analyses of total RNA can be used to provide a rapid

and timely estimate of exposure to estrogenic substances.

1

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2 INTRODUCTION

There is increasing concern about the potential impact of endocrine-disrupting
compounds (EDCs) on aquatic organisms. EDCs are compounds that interfere with the normal
functioning of hormones in the body. Among the EDCs that are found in aquatic habitats are
synthetic estrogens, which are used in contraceptives and other pharmaceuticals. These
chemicals enter waterways through sewage treatment plants or septic systems and are also found
in surface waters (Lange et al 2001). One of the most commonly used synthetic estrogens is
17a-ethynylestradiol (EE2). Several studies have found EE2 in waters downstream of sewage
treatment plants. In these rivers, concentrations of EE2 in the low ng/1 levels are common, with
some sites having concentrations as high as 40-60 ng/1 (Desbrow et al 1998; Belfroid et al. 1999;
Larsson et al. 1999; Ternes et al. 1999).

Often the weakest link in determining whether observed adverse effects in humans and/or
wildlife are linked to EDCs is the absence of adequate exposure data. Most exposure
information has focused on the presence of persistent organic pollutants in Europe and North
America. Data on the magnitude and trends of global human or wildlife exposure are limited.
Potential sources of exposure are through contaminated food, contaminated groundwater,
combustion sources, and contaminants in consumer products. Information on exposure during
critical development periods is generally lacking. The exposure data sets that exist are primarily
for various environmental media (air, food, water) rather than the most relevant internal exposure
(blood, tissue). Limited exceptions are human breast milk and adipose tissue samples.
Worldwide, despite large expenditures of money, time, and effort, comparable data sets for
assessing exposures to EDCs for humans or wildlife are not available. Such information is

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essential to evaluate exposure-response relationships adequately in field and epidemiology
studies and to use these relationships to produce credible risk assessments.

Vitellogenin is an egg yolk protein precursor that is produced by female fish prior to
spawning. Its synthesis is initiated in response to rising circulating estrogen levels. Males do not
normally produce this protein, but exposure to estrogenic contaminants initiates its production.
Plasma vtg levels in males exposed to estrogenic substances can increase several thousand fold,
to levels as high as or higher than that found in females (Palace et al. 2002). Although the
presence of plasma vtg protein can be used as an indicator of exposure to estrogenic compounds,
our laboratory has developed a reverse transcription-PCR method for quantifying vtg gene
messenger RNA (Lattier 2002). Because the presence of circulating protein occurs much later
than transcription of the vtg gene and is modulated by numerous control mechanisms,
quantitation of vtg gene transcription is potentially a more sensitive and immediate indicator of
exposure.

In order to investigate the effects of long-term exposure to an endocrine-disrupting
compound on a whole lake ecosystem, Fisheries and Oceans Canada dosed a lake in the
Experimental Lakes Area (ELA) of northwestern Ontario with 17a-ethynylestradiol (EE2) for
three years beginning in 2001. EE2 was added to the lake at environmentally relevant levels
with a target concentration of -4-6 ng/L. The U.S. EPA collaborated in this study by evaluating
vitellogenin gene expression in indigenous fathead minnows (Pimephale promelas) and pearl
dace (Margariscus margarita) collected from a reference lake and a continuously dosed lake and
in laboratory cultured fathead minnows exposed to water and sediments from the dosed lake and
reference lakes. The purpose was to further develop a vitellogenin gene expression assay as an
indicator of estrogen exposure.

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3 METHODS

3.1 EE2 Additions to Lake 260v

Palace et al (2002) described the procedures for EE2 addition and determination of lake
EE2 concentrations. Briefly, EE2 (Schering Pharmaceuticals, Germany) was dissolved in 100%
HPLC grade methanol (Caledon Laboratories), diluted with lake water to 50% solution, and
released into the propeller wash of a boat as it was driven around the lake. The amount of EE2
that was added was calculated based on the depth of the surface water layer (epilimnion) and the
previous week's data for Lake 260 during the open-water season. The target concentration of
EE2 in the epilimnion was 5 ng/L. EE2 was added at a rate of about 5% per addition to the lake
three times weekly. Replicate 1 L water samples were collected weekly at five sites around the
lake. Prior to analysis testosterone was added to each sample as an internal standard. Samples
were filtered and eluted (15-20 ml/min) through pre-cleaned and pre-conditioned C-18 SPE
cartridges (Supelco). EE2 was eluted from the column using 100% methanol. Samples were
dried under pure N2 and reconstituted in enzyme immunoassay (EIA) buffer. EE2 and
testosterone were quantified using radioimmunoassay and EIA plates (Cayman), respectively.
Recoveries of the internal standard ranged from 88% to 109%. EE2 concentrations were
corrected for weekly blank values but not for the recoveries of the internal standard (Palace et al.
2002).

3.2 Study Site and Fish Collections

Figure 1 shows the general study area and the location of the lake to which EE2 was
added (Lake 260), as well as the lakes used as reference systems for the experiment (Lake 114
and Lake 442). Fathead minnows were collected during 2000 through 2004 using live minnow
traps set overnight in epilimnetic waters near shore (<3m) in each of the lakes. Traps were

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emptied into holding pens containing aerated water from the appropriate lake, and fathead
minnows were sorted from the other resident species (Palace et al. 2002). Livers were removed
from males and females, placed in RNAlater and stored at 4° C for at least 24 hours. Samples
were stored at -20°C throughout the dosing season and then shipped to Cincinnati on ice.

3.3 2001 Field Deployment Study (Study 1-21)

Fathead minnows were collected using minnow traps in reference Lake 114 two days prior to
deployment. Males and females were housed together until the day of deployment, at which
time the sexes were separated. Only males were used in the deployment study. Males were
deployed in cages in reference Lake 114 and dosed Lake 260. The cages were suspended several
feet below the water surface and held in place by anchors and buoys. Fish were provided no
food during the period of deployment; however, the cages allowed for the free movement of
water and suspended materials. Minnows were retrieved from cages on days 1, 3, 7 and 13 of
deployment and the livers excised and place in RNAlater. Gel-based RT-PCR was performed on
these samples.

3.4 2001-2004 Studies: Description and Identification of Laboratory Exposures

Several studies were performed with fathead minnows (embryo/larvae, fry and adults) exposed
to water collected from ELA lakes and to sediment elutriates from these lakes. All exposures
were done in an environmental chamber at 25°C. All fish used for laboratory exposures were
reared in-house at the USEPA Cincinnati, Ohio aquatic culture unit maintained by SoBran, Inc.
Moderately hard reconstituted water (MHRW) (hardness of 100 mg/1 as CaCOs) was used as the
control or dilution water for all tests. It was prepared from a standard formula using reagent
grade chemicals and Super-Q® ultrapure water. The water was prepared three days prior to the
start of the test, to allow sufficient time for stabilization.

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3.4.1 Studies 1-11,1-12, and 1-26

3.4.1.1 Lake Water and Sediment Sample Collection

Water samples were collected in polyethylene cubitainers and the sediment samples were
collected in polyethylene bags. The samples were shipped on ice in coolers. All arrived at the
AWBERC facility in good condition. A subsample of each water column sample was collected
and taken to the seventh floor for use in testing. All unused samples were stored at 4° C for use
in any renewal tests. The sediment samples were stored at 4°C until they were used.

No dilutions were prepared for these tests; all samples were tested as 100% receiving
water samples. The water was transferred directly from the cubitainers to the test beakers. Once
the sample or control water was placed into the test beakers, the beakers were covered and
placed into a 25°C environmental chamber, to bring the samples to test temperature. For test
renewal, the water was warmed up to the correct temperature in the cubitainer. For embryo-
larval tests, in addition to the six lake elutriate samples and a 5 ng/1 positive control, a MHRW +
DMSO solvent blank was also included.

The first set of samples was received in good condition, but the laboratory was contacted
by representatives of the ELA to tell us not to use the first set of samples. It was believed they
were collected using a sampling container that might have been contaminated with
hydrocarbons, i.e., oil and gasoline from an outboard motor. The second set of water samples
was received in good condition. Tests with these samples (received 6/22/01) were started on
6/22/01. The two water samples received were from Lake 114, the reference lake, and Lake 260,
the lake being dosed with EE2. Sediment samples were also collected during the first sampling
event. These samples were collected 6/18/01 and received in Cincinnati on 6/20/01.

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3.4.1.2 Elutriate Preparation
Study 1-26: In addition to the water column samples, six sediment samples were received for
analysis using the FHM embryo-larval sediment toxicity testing method. These included one
sample from Lake 114 and five samples from various locations in Lake 260. The samples
consisted of a loose, flock-like material, making it easy for the eggs to sink below the surface.
To deal with this problem, the tests were initiated using mesh screens to support the eggs, but
this did not work, as any handling of the test containers re-suspended the sediment and covered
the eggs. After three days, all eggs exposed on the sediments from both lakes were dead.
Survival after three days was good on the control sand (91.88%), MHRW+DMSO (92.50%) and
the 5 ng/1 EE2 positive control (93.13%). These results indicated mortality was due to the
sediment samples, most likely from physiochemical factors. The decision was made to test these
samples as a sediment elutriate, to provide some data to determine if the EE2 in Lake 260 was
passing into the sediments.

Elutriates were prepared by adding 1L of sediment to 2L of MHRW and mixing on a
shaker table for one hour. The 2:1 water to sediment ratio is a deviation from that described by
US Army Corps of Engineers. This ratio was used so that any contaminants that leached from
the sediment and into the elutriate water would be more concentrated and therefore have a higher
probability of having an effect on the fish. After one hour on the shaker table, the liquid phase
was separated from the solid phase by centrifuging for 20 minutes at 3000 RPM. The liquid
phase (elutriate) was poured off and collected in glass bottles for use in the embryo-larval
toxicity test. These tests were started the day after the elutriate samples were prepared.

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3.4.1.3 Study Animals

The adult fish used in study 1-11 were 13-month-old males that had been separated from females
for four weeks.

The larval fish used in study 1-12 were between 24 and 48 hours old at the start of the
exposures. Fish that are 24 to 48 hours old at the start of a test are close to 96 hours old at the
end of a 48 hour exposure. The embryos used in study 1-26 were 24 to 48 hours old at the start
(so that fertility could be determined). The eggs were treated with 2mg/l methylene blue for 15
minutes to inhibit the growth of fungus. After the treatment, eggs were transferred to fresh water
and held overnight. The eggs were removed from the spawning tiles the day of the test, to
prevent damage from overnight aeration. Infertile eggs were removed prior to test setup.

3.4.1.4 Fathead Minnow Embryo-Larval Sediment Elutriate Exposures
Study 1-26: The test chambers used for the fry exposure procedure (Table 3) were 125 ml
polystyrene cups. The exposure procedure used a 50 ml test solution volume, 40 eggs per
replicate test chamber and four replicate test chambers. All tests lasted five days, with a test
solution renewal after 24 hours. Table 3 contains a summary of the test conditions.

To start the fry test, 40 FHM eggs (24 to 48 hours old) were counted into each of four
replicate test containers. A different pipette was used to add fish to each treatment in order to
prevent cross-contamination of the samples. Once all fish were added, the test chambers were
covered with individual lids to prevent cross-contamination among the test chambers and placed
back into the environmental chamber. Once the test setup was complete, the initial control water
and test dilution samples were analyzed for routine chemistries (pH, dissolved oxygen,
conductivity, and temperature).

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After the first 24 hours of exposure, the water was changed in each sample. Solutions
were brought up to 25°C (the test temperature) before use. After first counting and removing
any dead eggs/fish in each container, water was changed by pouring off at least 40 ml of the old
water and replacing it with fresh test water. An aliquot of water from each replicate test
container was composited to analyze for final test chemical parameters. Once the water was
changed in all containers, they were placed back into the environmental chamber. This same
process was followed for all change days during the test.

3.4.1.5 Fathead Minnow Adult Water Column Exposures

Study 1-11: To start the adult test (Table 2), one adult male fish (from a group isolated from
females) was transferred from a container of adult males to the replicate test chamber using a
coarse mesh nylon net. The net was rinsed in hot water and then deionized water between uses
for each fish, to prevent the contamination of a replicate with DNA from another fish. After the
fish were added, the containers were placed into a 25°C environmental chamber. A pipette
attached to an air source was also placed into each container, to provide for aeration of the test
water. The air was added at the rate of 100 bubbles/minute. The test chambers used for the adult
exposure procedure were 4L glass jars, with 3L of test solution in each replicate. The adult test
used five replicate test chambers, with one fish/replicate. These tests used a 24-hour duration.

3.4.1.6 Fathead Minnow Larvae (Fry) Water Column Exposure

Study 1-12: The test chambers used for the fry exposure procedure (Table 1) were 500 ml glass
beakers. The exposure procedure used a 400 ml test solution volume, 40 fish per replicate test
chamber and 4 replicate test chambers. All tests used a 48-hour duration, with a test solution
renewal after 24 hours. Table 1 contains a summary of the test conditions.

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To start the fry test, 40 FHM larvae (24 to 48 hours old) were counted into each of four
replicate test containers. A different pipette was used to add fish to each treatment, as a means to
prevent cross-contamination of the samples. Once all fish were added, the test chambers were
covered with individual lids, to prevent cross-contamination between the test chambers, and
placed back into the environmental chamber. Once the test setup was complete, the initial
control water and test dilution samples were analyzed for routine chemistries (pH, dissolved
oxygen, conductivity, and temperature).

After the first 24 hours of exposure, the water was changed in each sample. The required
control, solvent blank or toxicant concentration was prepared and all prepared samples or
collected water samples were brought up to the 25°C test temperature. The water was changed
by first counting and removing any dead fish in each, then pouring off at least 200 ml of the old
water and replacing it with fresh test water. An aliquot of water from each replicate test
container was composited, to analyze for final test chemical parameters. Once the water was
changed on all beakers, they were placed back into the environmental chamber.

3.4.1.7 Positive controls

The laboratory control treatment for a test was a MHRW control. In addition, a 5 ng/1 EE2

positive control was conducted concurrently with all tests, to provide a measure of the level of

gene expression using a known toxicant. A DMSO control blank was not included in the water

column test, but a labline water control was, to determine if effects were seen when animals were

placed directly into MHRW from the labline. Labline is the AWBERC in-house culture water,

prepared by carbon-filtering Cincinnati tap water and adding calcium chloride to supplement the

water hardness. The MHRW + DMSO solvent blank was included in the elutriate tests.

Preparation of the solvent blank and/or positive control for all tests required the addition of

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3.57 |lx1 of DMSO or EE2 stock to each liter prepared. The EE2 was prepared such that the
addition of 3.57 |lx1 of stock resulted in a nominal concentration of 5 ng/L EE2.

3.4.2 Studies 1-23 and 1-24

3.4.2.1 Sample collection and exposures

A three-week study was initiated to determine the effects of long-term exposures to EE2 on
vitellogenin gene expression levels in adult FHM males. The study was designed so that adult
FHM males could be exposed to samples shipped from the Canadian Experimental Lake Area
(ELA) over a three-week period, with renewal of the water in the tanks every three to four days.
The water was to be changed a total of five times during the period of the test exposure. In
addition, both the new and old water samples were tested with FHM larvae (24 to 48 hours old).
This provided an estimate of the variation in the gene expression in the larvae each time a new
sample was tested and also an estimate of the level of effect still exhibited by the old water
samples.

The above description is the plan that was to be implemented. Circumstances prevented
that from occurring. The first set of samples was received on 8/2/01. Two coolers, each with
two 20L carboys of sample, were received. The samples received were from Lake 114 (the
reference lake) and Lake 260 (the lake dosed with EE2). The next set of samples was received
on 8/15/01 and used for a water change that day. This was an emergency collection and sample
shipment from 8/14/01. Samples were collected and shipped on 8/7/01, but these were not
received until 8/16/01. It was decided these were too old to be of any use. A replacement for
this set of samples was collected and shipped 8/10/01. These were received on 8/17/01. It was
decided these samples would be used for part of the last water changes with the study. So, due

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to problems with shipping, the samples collected 8/14/01 were received and used for a water
change on 8/15/01. The water left from the 8/14/01samples was then mixed with the water
received on 8/17/01 (collected 8/10/10) and used for the 8/18/01 and 8/20/01 water changes.

3.4.2.2 Study Animals

The adult fish used in study 1-23 were FHM males that were 10 to 12 months old at the
start of the test. These fish had been isolated from female animals for two months prior to the
start of the test.

The larval fish used in study 1-24 were between 24 and 48 hours old at the start of the
exposures. The adult fish used were 13-month-old males that had been separated from females
for 4 weeks.

3.4.2.3 Fathead Minnow Larvae (Fry) Water Column Exposure

Study 1-24: The first test with the FHM fry was conducted using PVC exposure chambers
developed for use in exposing Hyalella azteca in field studies. The purpose for this was to
expose the fry animals in the same tanks as the adults. These chambers are 6 cm long and 4 cm
in diameter. The ends are threaded and accept an open end cap. The ends were closed using
Nitex® mesh screen cut to size. Twenty animals were added to each test chamber, with each
tank receiving eight test chambers. Tests with these chambers were not successful, due to
excessive mortality in all chambers for all samples tested. Due to the mortality problem, a
switch was made to a more conventional fry-exposure method.

The test chambers used for the remaining fry exposure procedures (Table 1) were 500 ml
glass beakers. The exposure procedure used a 300 ml test solution volume, 40 fish per replicate
test chamber and four replicate test chambers. All tests used a 48-hour duration. The tests were

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conducted as static, non-renewal, due to the lack of sufficient lake sample for renewals. Table 2
contains a summary of the test conditions.

No dilutions were prepared for these tests; all samples were tested as 100% receiving
water samples. To limit the amount of sample transported from the sample cold room, located in
room 464, a secondary cubitainer was used to transfer known quantities of sample from the
fourth floor to the seventh floor. All samples were transported inside a cooler, to limit the
possibility of a spill. The lake water was brought up to the test temperature, by placing the
sample cubitainers into the water bath. The control, solvent blank and positive control samples
were prepared in room 783. Since the water in the MHRW carboy was at 24°C, no temperature
adjustment was necessary. These samples were prepared, then either added to the tank to fill or
used for the water renewal.

To start the fry test, 40 FHM larvae (24 to 48 hours old) were counted into each of four
replicate test containers. A different pipette was used to add fish to each treatment, as a means to
prevent cross-contamination of the samples. Once all fish were added, the test chambers were
covered with individual lids to prevent cross-contamination between the test chambers, and
placed back into the environmental chamber. Once the test setup was complete, the initial
control water and test dilution samples were analyzed for routine chemistries (pH, dissolved
oxygen, conductivity, and temperature).

After the first 24 hours of exposure, the water temperatures were checked and any dead
fish removed.

3.4.2.4 Fathead Minnow Adult Water Column Exposures
Study 1-23: The adult fish were tested in glass aquaria, with 20L of test solution in each tank.

The MHRW control and the DMSO solvent blank tests were conducted in 40L glass aquaria.

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The positive control, Lake 114 and Lake 260 tests were conducted in 80L glass aquaria. This
was necessary, so that three tanks (2-40L and 1-80L) could be placed into one of the minnow
cool tanks being used as a water bath. The second minnow cool water bath held two 80L
aquaria. The minnow cools were used as the means to maintain constant temperature. Water at
25°C flowed into these tanks and supplemental heat was provided by electric aquarium tank
heaters. A total of 30 fish was initially exposed in each tank, to provide sufficient numbers of
fish so that five fish could be sacrificed on days 1 and 4. Feeding was done twice each day, but
the feeding was reduced after the water samples failed to arrive. The fish were fed once every
two days, until fresh sample was received, at which point the regular feeding regime was
resumed. Table 1 contains a summary of the test conditions. The tests were started on 8/2/01;
the water was changed on 8/6/01, 8/15/01, 8/18/01 and 8/20/01.

3.4.3 Lake Water and Sediment Sample Collection

Water samples were collected in polyethylene cubitainers and shipped on ice in coolers.
The size of the sample containers prevented the use of a sufficient amount of ice to properly chill
the samples. The delays in sample receipt also prevented the samples' arriving at a temperature
normally considered acceptable. The samples all arrived at temperatures between 18°C and
21°C. Other than the elevated temperatures, the samples arrived at the AWBERC facility in
good condition. A subsample of each was collected and taken to the seventh floor for use in
testing. All unused samples were stored at 4°C for use in any renewal tests.

In addition to the two lake samples, a 5 ng/1 EE2 positive control and a DMSO control
blank were included and treated the same as the lake samples. This provided a means to
compare the results from the lake samples to a series of known samples exposed under the same
conditions.

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3.4.4 Studies 2-19, 2-20, 2-25, 2-27, 2-37, 2-40, 2-56, 2-64 and 2-65

3.4.4.1 Sample collection and exposures

Water and sediment samples were collected three times during 2002; in May, prior to dosing
with ethynylestradiol (EE2), in June, after the 5 ng/1 EE2 had stabilized and finally in October,
the final sample collection period. The reference lake for the first two studies was 114. Due to
Lake 114 freezing, Lake 239 was used as the reference lake in October.

3.4.4.2 Elutriate Preparation

Studies 2-25, 2-40 and 2-56: The elutriate samples used in the FHM embryo-larval test were
prepared using the elutriate methods described by US Army Corps of Engineers. This was done
by mixing 500 ml of sediment with 2000 ml of MHRW, then shaking the sediment:water mixture
for one hour at 100 RPM. After the mixing was completed, each sample was centrifuged for 20
minutes at 5000 RPM and the elutriate water collected. Elutriate was stored at 4°C and aliquots
were removed and warmed to the test temperature (25°C) as needed daily.

3.4.4.3 Study Animals

The adult FHM male and female fish used in studies 2-19, 2-27, 2-56, 2-20, 2-37, and 2-64 were
10-12 months old. These male and female fish had been maintained together throughout the
rearing and holding period.

The FHM embryo used for embryo-larval tests 2-25, 2-40 and 2-65 were 24 to 48-hour-
old embryo obtained from the AWBERC FHM culture unit. The eggs were collected the day
prior to the start of the test and held at 25°C overnight. The infertile eggs were removed and
fertilized eggs were added to the test containers. The larvae used in study 3-37 were between 36

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and 48 hours old at the start of the exposures. The larvae used in study 3-43 were between 24 to
48 hours old at the start of the exposures.

3.4.4.4 Fathead Minnow Embryo-Larval Sediment Elutriate Exposures
Studies 2-25, 2-40 and 2-65: The test conditions for the FHM embryo-larval tests are
summarized in Table 8. The embryo-larval test was conducted using FHM embryo that were 24
to 48 hours old at the start of the test. The test used five replicate test chambers, each chamber
containing 50 ml of test solution. To start the test, 30 FHM embryos were added to each test
container. Each embryo was sorted so that only fertilized, viable embryos were added at the start
of the test. The water was changed daily by removing at least 60% of the existing water and
adding fresh control, solvent blank, positive control or elutriate sample water back to each test
container. Routine chemical parameters were determined on the water that was removed daily.
This was done by compositing samples from each replicate as the water was changed. All fresh
waters used for test renewal were stabilized at the test temperature before use. Duration of the
test is determined by the hatching rate of the embryo. The test was allowed to continue until all
embryos were hatched for at least 24 hours. After this occurred, the test was terminated.

3.4.4.5 Fathead Minnow Adult Water Column Exposures

Studies 2-19, 2-27 and 2-56: The test conditions are summarized in Table 6. Five of the fish
that were used for the initial setup were sacrificed and the livers collected to provide baseline
estimates of the gene expression level activity of the fish at the start of the test. The fish were
fed tropical fish flake food. Exposures were conducted in 4L glass jars, filled with 2L of test
solution. Test duration was 24 hours. The control and each exposure treatment had five
replicate chambers, each containing two adult fish. Once the jars were filled with control water

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or test solution, they were covered and placed into the 25°C (±1°C) environmental chamber.

Once the temperature had stabilized, the fish were added to each replicate and the jar placed back
into the environmental chamber. The remaining initial test solution was analyzed for routine
chemical parameters (pH, dissolved oxygen, conductivity, temperature).

An airtube was placed into each replicate chamber and air bubbled in at the rate of 100
bubbles/minute. The airtube consisted of a 1 ml glass pipette attached to a section of flexible
plastic tubing. All information (number of animals exposed, test temperature, air flow rate,
routine initial chemical parameters, etc.) was recorded on the datasheets.

3.4.4.6 Fathead Minnow Adult Sediment Exposures

Studies 2-20, 2-37 and 2-64: The sediment exposures with the adult FHM followed the
methods described above for the water column exposures, with the following modifications
(Table 7). The sediment toxicity samples were prepared 24 hours prior to the start of the test by
adding 1L of sediment to each replicate test chamber. A 2L volume of MHRW was then added
as the overlying water. The MHRW was added such that the sediment was thoroughly agitated.
It was believed this would provide for the possible release of any contaminants present in the
sediment sample. After the water was added, the test containers were placed into a 25°C (±1°C)
environmental chamber and allowed to stabilize overnight. The next day, the control, solvent
blank and positive control samples were prepared and brought up to temperature. The test was
started as described above. The duration for these exposures was 48 hours, to allow sufficient
exposure time to the sediment samples. The water was not renewed.

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3.4.4.7 Lake Water and Sediment Sample Collection

Samples were collected by Fisheries and Oceans Canada staff onsite at ELA on 5/21/02, 6/18/02
and 10/23/02. The samples were collected in five-gallon plastic cubitainers and shipped in
coolers on ice to AWBERC. Due to the difficulty in shipping overnight from ELA to Cincinnati,
all samples were received two days after sample collection. The tests with the water column
samples were started on the day the samples were received. The tests with the sediment samples
were all started within six weeks of sample collection.

3.4.5 Study 3-37

3.4.5.1 Sample collection and exposures

Three samples from ELA were received, one from Lake 114 at the center buoy and two from
Lake 260 (surface sediments from the nearshore area and from the center buoy). All samples
were received on September 24, 2003, in good condition. Sediment samples were collected in
polyethylene bags and shipped to the laboratory on ice in coolers. All arrived at the AWBERC
facility in good condition within three days of collection. Once received, the samples were
stored at 4°C, until ready for use in the elutriate preparation.

3.4.5.2 Study Animals

The FHM larvae used in study 3-37 were between 36 and 48 hours old at the start of the
exposures.

3.4.5.3 Elutriate Preparation

Study 3-37: Elutriates were prepared by adding 1500 ml of sediment to 1500 ml of MHRW and
mixing on a shaker table for one hour. The 1:1 water-to-sediment ratio is a deviation from that

-------
described by US Army Corps of Engineers. However, this ratio was used so that any
contaminants leached off the sediment and into the elutriate water would be more concentrated
and therefore have a higher probability of having an effect on the fish. After one hour on the
shaker table, the liquid phase was separated from the solid phase by centrifuging for 20 minutes
at 3000 RPM. The liquid phase (elutriate) was poured off and collected in glass bottles for use in
the larval toxicity test. These tests were started the day after the elutriate samples were prepared.
A DMSO control blank was used as the laboratory control treatment for the test. EE2 was
included (1 |lx1 EE2/L MHRW) as a positive control.

3.4.5.4 Fathead Minnow Embryo-Larval Sediment Elutriate Exposures
Study 3-37: The test chambers used for the fry exposure procedure (Table 9) were 500 ml
beakers. The exposure procedure used a 300 ml test solution volume, 40 fry per replicate test
chamber and five replicate test chambers. All tests used a 48-hour duration period, with a test
solution renewal at the 24-hour period. Table 1 contains a summary of the test conditions. At
the end of the test, the fish from each replicate for a concentration were transferred from the test
container, counted and placed into labeled vials containing RNAlater buffer. These were first
stored at 4°C for 24 hours, then stored at -20°C until analyzed.

3.4.6 Study 3-43

3.4.6.1 Sample collection and exposures

The purpose of this test was to determine if the addition of a carrier solvent (dimethyl sulfoxide;

DMSO) had an effect on making EDCs more bioavailable and eliciting more of gene expression

response in the organisms exposed, when compared to a standard sediment elutriate preparation

without DMSO. As part of the investigation, three different amounts of DMSO were used to see

-------
if a higher amount would prove more efficient in releasing the EDCs. Two samples were
received, one from Lake 239 and one from Lake 260. It was not possible to collect samples from
Lake 114, the reference lake routinely used, so samples were collected from Lake 239, another of
the reference lakes. All samples were collected 10/30/03 and stored at 4°C until shipped from
ELA on Monday 11/03/03. The samples were received at AWBERC on 11/05/03 in good
condition. Sediment samples were collected in polyethylene bags and shipped to the laboratory
on ice in coolers. The samples were taken to Room 464, the sample preparation and storage
room. Once received, the samples were stored at 4°C, until ready for use in the elutriate
preparation.

3.4.6.2 Study Animals

The FHM larvae used in study 3-43 were between 24 to 48 hours old at the start of the
exposures.

3.4.6.3 Elutriate Preparation

Study 3-43: Elutriates were prepared by adding 1000 ml of sediment to 2000 ml of
MHRW and mixing on a shaker table for one hour. The 2:1 water to sediment ratio is a
deviation from that described by US Army Corps of Engineers. It was used so that any EDCs
released from the sediments would be more concentrated. A series of 12 elutriate samples was
prepared, using control sand, Lake 239 sediment and Lake 260 sediment. Each sediment was
eluted using 0 |lx1/L DMSO, 1 |lx1/L DMSO, 50 |lx1/L DMSO and 500 |lx1/L DMSO. As stated
above, the various levels of DMSO were used to see if increasing the amount of DMSO provided
for more efficient release of the EE2 into the mixing water. After one hour on the shaker table,
the liquid phase was separated from the solid phase by centrifuging for 20 minutes at 3000 RPM.

-------
The liquid phase (elutriate) was poured off and collected in glass bottles for use in the larval
toxicity test. These tests were started the day after the elutriate samples were prepared. A
DMSO control blank was used as the laboratory control treatment for the test. EE2 was included
(1 |lx1 EE2/L MHRW) as a positive control.

3.4.6.4 Fathead Minnow Embryo-Larval Sediment Elutriate Exposures
Study 3-43: The test chambers used for the fry exposure procedure (Table 10) were 500 ml
beakers. The exposure procedure used a 300 ml test solution volume, 40 fry per replicate test
chamber and five replicate test chambers. All tests used a 48-hour duration period, with a test
solution renewal at the 24-hour period. Table 1 contains a summary of the test conditions. At
the end of the test, the fish from each replicate for a concentration were transferred from the test
container, counted and placed into labeled vials containing Tri reagent. The vials of buffer with
tissue were homogenized; the vials were then stored at -80°C until removed for RNA isolation
and gene expression analysis.

3.4.6.5 Positive controls

17a-ethynylestradiol (EE2) was used as the test positive control chemical. The 10 ng/1
concentration was prepared by weighing out the EE2 and dissolving it in a calculated amount of
DMSO. Once the first stocks were diluted, a 2 |lx1 aliquot was then diluted with 2 ml of DMSO
to prepare the concentrated stock, such that 1 |lx1 of stock was added per liter of MHRW to
prepare the positive control test concentration.

-------
TABLE 1. Summary Test Conditions for Larval Fathead Minnow Gene Expression Exposure
Tests. Studies 1-12 and 1-24.

TEST PARAMETER

CONDITION

Test Type

static-renewal

Test Duration

48 hours

Temperature

25°C (±1°C)

Photoperiod

16 hours light: 8 hours dark

Test Chamber Size

500 ml

Test Solution Volume

400 ml

Renewal of Test Solution

daily

Age of Test Organisms

24-hour to 48-hour-old larvae

No. Organisms/Test Chamber

30

No. Replicate Test Chambers

4

No. Organisms/concentration

150

Feeding Regime

Not fed

Control and/or Dilution Water

Moderately Hard Water/Labline

Endpoint

Based on gene expression data analyzed

Test Acceptability

90% or greater control survival

22

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TABLE 2. Summary Test Conditions for
Exposure Tests. Study # 1-11

TEST PARAMETER

Test Type

Test Duration

Temperature

Photoperiod

Test Chamber Size

Test Solution Volume

Renewal of Test Solution

Age of Test Organisms

No. Organisms/Test Chamber

No. Replicate Test Chambers

No. Organisms/concentration

Feeding Regime

Control and/or Dilution Water

Endpoint

Test Acceptability

head Minnow Male Gene Expression

CONDITION
static
24 hours
25°C (±1°C)

16 hours light: 8 hours dark

4	L
3 L
None

11 to 13-month-old males
1

5
5

Not fed

Moderately Hard Water or MHRW+DMSO
Based on gene expression data analyzed
90% or greater control survival

23

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TABLE 3. Summary Test Conditions for Larval Fathead Minnow Gene Expression
Exposure Tests, Embryo-Larval Elutriate. Study #1-26.

TEST PARAMETER

CONDITION

Test Type

static-renewal

Test Duration

5 days

Temperature

25°C (±1°C)

Photoperiod

16 hours light: 8 hours dark

Test Chamber Size

125 ml

Test Solution Volume

50 ml

Renewal of Test Solution

daily

Age of Test Organisms

24-hour to 48-hour-old embryo

No. Organisms/Test Chamber

40

No. Replicate Test Chambers

4

No. Organisms/concentration

160

Feeding Regime

Not fed

Control and/or Dilution Water

Moderately Hard Water

Endpoint

Based on gene expression data analyzed

Test Acceptability

90% or greater control hatch and survival

24

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TABLE 4. Summary Test Conditions for Long-Term Exposure of Adult Fathead Minnow
Males for Gene Expression Exposure Tests. Study # 1-23.

TEST PARAMETER

CONDITION

Test Type

static

Test Duration

21 D

Temperature

25°C (±1°C)

Photoperiod

16 hours light: 8 hours dark

Test Chamber Size

40L or 80L

Test Solution Volume

20 L

Renewal of Test Solution

Every 3 to 4 days

Age of Test Organisms

11 to 13-month-old males

No. Organisms/Test Chamber

30

No. Replicate Test Chambers

1

No. Organisms/concentration

30

Feeding Regime

Flake food 2X/day

Control and/or Dilution Water

Moderately Hard Water or MHRW+DMSO

Endpoint

Based on gene expression data analyzed

Test Acceptability

90% or greater control survival

25

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TABLE 5. Summary of Test Conditions for Adult Fathead Minnow Gene Expression Exposure
Tests with 2 ELA Water Column Samples.

Study: ELA
TEST PARAMETER
Test Type
Test Duration
Temperature
Photoperiod
Test Chamber Size
Test Solution Volume
Renewal of Test Solution
Age of Test Organisms
No. Organisms/Test Chamber
No. Replicate Test Chambers
No. Organisms/concentration
Feeding Regime
Aeration

Control and/or Dilution Water
Sample/Test Material

Sample Concentration(s)

Endpoint

Test Acceptability

Study #: 2-19, 2-27, 2-56 Date: 05/23/02, 06/20/02, 10/27/02

CONDITION
static-renewal
24 hours
25°C (±1°C)

16 hours light: 8 hours dark

4	L
2 L
None

11, 11, 12-month-old males and females
2, 1 male, 1 female

5

10

Not fed

100 bubbles/min

MHRW, MHRW + DMSO

ELA Lakes 114 (239) and 260, EE2 Positive
control

Lakes-100%, EE2 5 ng/1
Based on gene expression data analyzed
90% or greater control survival

26

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TABLE 6. Summary of Test Conditions for Adult Fathead Minnow Gene Expression Exposure
Tests with 2 EL A Sediment Samples.

Study: EL A
TEST PARAMETER
Test Type
Test Duration
Temperature
Photoperiod
Test Chamber Size
Test Sediment Volume
Test Overlying Water Volume
Renewal of Test Solution
Age of Test Organisms
No. Organisms/Test Chamber
No. Replicate Test Chambers
No. Organisms/concentration
Feeding Regime
Aeration

Control and/or Dilution Water
Sample/Test Material

Sample Concentration(s)

Endpoint

Test Acceptability

Study #: 2-20, 2-37, 2-64 Date: 05/29/02, 02/24/02, 12/16/02

CONDITION
static-renewal
48 hours
25°C (±1°C)

16 hours light: 8 hours dark

4	L

1	L

2	L
None

10, 11-month-old males and females
2, 1 male, 1 female

5

10

Not fed

100 bubbles/min

MHRW, MHRW + DMSO

EL A Lakes 114 (239) and 260, EE2 Positive
control

Lakes-100%, EE2 5 ng/1
Based on gene expression data analyzed
90% or greater control survival

27

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TABLE 7. Summary of Test Conditions for FHM Embryo/Larval Gene Expression Exposure
Tests with Elutriate Samples Prepared from ELA Sediment Samples.

Study: ELA
TEST PARAMETER
Test Type
Test Duration
Temperature
Photoperiod
Test Chamber Size
Test Solution Volume
Renewal of Test Solution
Age of Test Organisms
No. Organisms/Test Chamber
No. Replicate Test Chambers
No. Organisms/concentration
Feeding Regime
Aeration

Control and/or Dilution Water
Sample/Test Material

Sample Concentration(s)

Endpoint

Test Acceptability

Study #: 2-25, 2-40, 2-65 Date: 06/13/02, 08/09/02, 12/18/02

CONDITION
static-renewal

5 to 7 days, depending on egg hatching
25°C (±1°C)

16 hours light: 8 hours dark
150 ml
50 ml
Daily

24 to 48-hour-old embryo
30
5

150

Not fed
None

Moderately Hard Water, MHRW, DMSO

Elutriate from ELA Lakes 114 (239) and
260 and EE2 Positive control

Elutriate-100%, EE2 5 ng/1

Based on gene expression data analyzed

90% or greater control survival

28

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TABLE 8. Summary of Test Conditions for Conducting Larval Fathead Minnow Gene
Expression Exposure Tests with Elutriate Samples. Study # 3-37.

TEST PARAMETER

CONDITION

Test Type

static-renewal

Test Duration

48 hours

Temperature

25°C (±1°C)

Photoperiod

16 hours light: 8 hours dark

Test Chamber Size

500 ml

Test Solution Volume

300 ml

Renewal of Test Solution

daily

Age of Test Organisms

36-hour to 48-hour-old larvae

No. Organisms/Test Chamber

40

No. Replicate Test Chambers

5

No. Organisms/concentration

200

Feeding Regime

Not fed

Control and/or Dilution Water

Moderately Hard Water + DMSO

Endpoint

Based on gene expression data analyzed

Test Acceptability

90% or greater control survival

29

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TABLE 9. Summary Test Conditions for Conducting Larval Fathead Minnow
Gene Expression Exposure Tests with Elutriate Samples. Study # 3-43.

TEST PARAMETER

Test Type

Test Duration

Temperature

Photoperiod

Test Chamber Size

Test Solution Volume

Renewal of Test Solution

Age of Test Organisms

No. Organisms/Test Chamber

No. Replicate Test Chambers

No. Organisms/concentration

Feeding Regime

Control and/or Dilution Water

Endpoint

Test Acceptability

CONDITION
static-renewal
48 hours
25°C (±1°C)

16 hours light: 8 hours dark
500 ml
300 ml
daily

24-hour to 48-hour-old larvae

40

5

200

Not fed

Moderately Hard Water + DMSO
(1, 50 and 500 ug/L)

Based on gene expression data analyzed

90% or greater control survival

30

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3.5	RNA Preparation

Adult fish or fry were sacrificed and adult liver tissues (or whole fry) were suspended in
RNAlater solution (Ambion Inc., Austin, TX). Tissues were homogenized using either a hand-
held homogenizer or a Mixer Mill 200 (Retsch, Germany) with 3mm stainless steel beads
(Qiagen).

Two different techniques were used to extract RNA from individual adult male livers or
fry. In the first protocol, which was used with the samples from 2001, total RNA was isolated
using the guanidinium isothiocyanate method (Chomczynski and Sacchi 1987) followed by
DNAse treatment. For the remaining samples, Tri Reagent was used (Molecular Research
Center, Cincinnati, OH).

The integrity of the RNA was determined by visual inspection of the 18s and 28s
ribosomal bands on a formaldehyde/MOPS gel. The samples were spectrophotometrically
quantified and diluted to a concentration of 1 mg/ml for RT-PCR.

3.6	Gel-based analyses

Reverse transcription polymerase chain reaction (RT-PCR) was performed using
GeneAmp® RNA PCR reagents (PE Applied Biosystems, Foster City, CA). Gene-specific
oligonucleotides were designed from the FHM vtg precursor mRNA sequence (Preziosi 1998)
using Oligo Primer Analysis Software® (Rychlik and Rhoads, 1989). The RT-PCR reactions
contained an empirically determined volume of 18S Competimer®/universal ribosomal RNA
(rRNA) primer mix (Ambion Inc., Austin, TX) in a muliplex reaction. Reactions also contained
Advantage-2 DNA polymerase (Clontech Laboratories, Palo Alto, CA, USA.). Secondary PCR
reaction mix was added to the 20 |lx1 reverse transcription reaction for a final PCR volume of 50
(0.1. The secondary master mix contained 21.5 |lx1 of H20, 5 |lx1 of 10X Advantage 2 PCR buffer, 1

31

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|Lxl of Advantage 2 DNA polymerase, 150 nM of each Vg oligonucleotide primer, and 0.5 |lx1 of
5:5 (fry) or 7:3 (liver) ratio of competimer/18S oliginucleotide per reaction.

Thermal cycle number for log linear amplification of vitellogenin gene cDNA for fry and
adult liver was determined to be 32 and 17 cycles, respectively. The PCR profile used for both
adult liver and fry was: 95° for 1 min; 32 or 17 cycles of 95° for 30 s, 64° for 30 s, and 72° for
30 s; and 1 cycle of 94° for 30 s, 64° for 30 s, and 72° for 10 min to complete any unfinished
transcripts.

The amplification products were separated electrophoretically on 1.8% agarose gels.
Following electrophoresis, gels were stained in IX TAE containing SYBR® Green I (Molecular
Probes, Eugene, OR) and scanned using a Flurolmager® 595 system (Molecular Dynamics,
Sunnyvale, CA). Relative intensities for both the Vg and 18S ribosomal bands were analyzed
with ImageQuant® (Molecular Dynamics) software.

Relative gene expression was expressed as the ratio of pixel density of the vitellogenin
band to the sum of the pixel densities of the vitellogenin and 18S bands [Vg/(Vg+18S); Lattier
2001], Across replicates, averages and standard deviations were calculated using routines
available in Lotus 123 (Cambridge, MA, USA) and Microsoft Excel (Redmond, WA, USA).
3.7 Quantitative Real-time PCR

Total RNA (2 |Lxg) was reverse-transcribed in a 20 |lx1 reaction mixture containing PCR II
buffer (Perkin Elmer), 5 rnM MgC^. 2.5 rnM total dNTP, 2.5 |jM random hexamers, 20 U RNase
inhibitor and 2.5 U MuLV reverse transcriptase. Prior to addition of the reverse transcriptase,
the reactions were heated to 70°C for 5 min followed by a 15-min ramp to 23°C. This insured
denaturation of RNA secondary structure and annealing of the RNA to the random hexamers.
Reverse transcriptase was added and the reactions incubated at 48°C for 30 min in a GeneAmp

32

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9600 thermal cycler, followed by 5 min at 95°C to inactivate the enzyme. No template reactions
and no reverse transcriptase reactions were run with each set of reactions as controls.

Prior to real-time RT-PCR, 80 |lx1 of sterile, DNase-free, RNase-free water was added to
each 20 |lx1 cDNA sample. Real-time RT-PCR was performed using DyNAmo PCR mix
(Finnzymes). Reactions (25 |lx1 total) consisted of SYBR Green PCR mix, 3 |lx1 diluted cDNA
and 20 pmol primer. Real-time RT-PCR was performed on an MJ Opticon. Reactions were run
both with vitellogenin primers and 18S primers. Vitellogenin primers and cycling conditions
were developed in this laboratory and will be published in a research article within the next year.
The 18S primers used were QuantumRNA 18S universal primers (Ambion) were used as internal
standards.

4 RESULTS

4.1 Lake 260 EE2 Concentrations

The target concentration of EE2 in Lake 260 was 5 ng/L. In the first year of dosing, the
overall mean epilimnetic concentration of EE2 from 30 May to 4 October 2001 was 6.0±2.8
(SD) ng/L with a high of 8.9 ng/L and a low of 4.0 ng/L (Figure 2). In 2002, mean EE2
concentrations (blank corrected) were 5.0 ± 1.8 ng/L from 30 May to 1 October. Over the
addition period in 2003, mean EE2 concentrations (blank corrected) were 4.8 ±1.0 ng/L between
26 May to 8 October and similar to the two previous years.

4.2 Studies Conducted in 2001

In order to determine the kinetics of vitellogenin expression during the initial period of

exposure in 2001, male fathead minnows were collected from Lake 260 after seven weeks, nine

-------
weeks and three months of dosing. Male fathead minnows were collected at the same time from
reference Lake 114. Gel-based RT-PCR was performed on samples and vitellogenin expression
quantified relative to 18s ribosomal RNA expression.

Vitellogenin was induced in males collected from Lake 260 at all time points (Figure 3).
Males collected from Lake 114 had little to no vitellogenin mRNA. Vitellogenin expression in
males was comparable to that of females collected from Lake 114 on July 25. The level of
expression of vitellogenin in male fathead minnows collected from Lake 260 was statistically
different from that of males from Lake 114.

The deployment study was originally supposed to last for three weeks, with five fish
withdrawn from each lake after 1,7, 14 and 21 days of deployment. Due to unexpectedly high
levels of mortality, the experiment had to be terminated early. There were not enough fish to
continue the study in Lake 114 through day 14, so all seven remaining fish were pulled on day 7.
The experiment was also terminated early in Lake 260 since only four fish were remaining on
day 13. Male fathead minnows exhibited an increase in vitellogenin mRNA levels after only one
day of deployment in Lake 260 (Figure 4). Vitellogenin mRNA levels remained high throughout
the study to day 13. Response to EE2 by males was variable, with some fish showing high levels
of expression and others showing very little expression. The standard deviations for these
samples are quite high. Males in Lake 114 showed no significant expression on days 3 and 7
(Figure 4). However, on day 1 there was a single fish that had elevated levels of vitellogenin
mRNA. The other four fish showed no Vg expression.

Elutriate from Lake 114 did not induce vitellogenin in fry, whereas elutriates from
sediment from the dosed Lake 260 did (Figure 6). Vitellogenin induction was highly variable in

-------
these samples. Unfortunately, it appears that fry did not respond to water with 5 ng/L EE2. This
makes it difficult to interpret these results.

4.3 Studies Conducted in 2002

4.3.1	Fathead Minnows

Fathead minnows were collected at various times throughout the year in 2002 from dosed
and reference lakes. Vitellogenin gene expression was determined using QPCR.

Males collected from reference lakes 114, 302 and 442 showed no vitellogenin gene
expression throughout the year (Figure 7). Males collected from the dosed Lake 260 on May 27
did not express vitellogenin mRNA, but those collected in June, July and November did.
Induction of expression was variable among these fish.

Females collected from reference Lake 442 showed elevated vitellogenin expression in
May, while vitellogenin expression in females from Lake 114 was high during June (Figure 8).
This may represent differences in breeding season between these two lakes. In contrast, females
from the dosed lake 260 continued to have high levels of expression into October, well beyond
the breeding season for this species.

4.3.2	Pearl Dace

Pearl dace were collected from dosed lake 260 and reference lakes 114 and 442 at various
times during May, June and September in 2002. Vitellogenin gene expression was determined
using QPCR.

No vitellogenin gene expression was detected in males collected from Lake 114 June or
September (Figure 9). Fish exposed to EE2 in Lake 260 showed variable response in gene

35

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expression. Reference Lake 442 had some fish with elevated gene expression: one fish of the
two collected on May 14 and one fish of the seven collected on September 24.

Females in Lake 260 did not exhibit an increase in vitellogenin expression relative to
females from other lakes (Figure 10). One female from Lake 442 showed an extremely high
level of vitellogenin. However, an examination of the data revealed that this was due to an
abnormally low 18S value, which suggests that a failed PCR reaction may be skewing the results
for this sample.

The vitellogenin primers used for quantitative real time RT-PCR were originally
designed for use with fathead minnows, the sequence of the primers having been based on the
fathead minnow vitellogenin gene. These primers appeared to work well with pearl dace
samples. We sequenced the pearl dace vitellogenin cDNA to determine its similarity to fathead
minnow vitellogenin. The two sequences were 89% similar based on a BLAST search of the
sequence. Since the sequencing was done in only one direction, more careful sequencing would
likely increase the estimate of similarity.

4.4 Studies Conducted in 2003

4.4.1 Fathead Minnows

Male fathead minnows collected from Lake 260 in May of 2003, prior to the third year of
dosing, showed elevated levels of vitellogenin expression (Figure 11). Males from lakes 114 and
442 showed no expression. Female fathead minnows collected from lakes 442 and 260 in May
of 2003 both showed elevated levels of vitellogenin expression (Figure 12). No female fathead
minnows were collected from Lake 114 in May of 2003.

-------
4.4.2 Pearl Dace

Male pearl dace collected from Lake 260 in May 2003 (prior to dosing) exhibited
significant vitellogenin gene expression, while fish from reference lakes 114 and 442 had no
vitellogenin mRNA (Figure 13). Female pearl dace showed differences in levels of vitellogenin
expression between the dosed and reference lakes (Figure 14). Whether this may be due to
differences in breeding season among the lakes or a consequence of the EE2 present cannot be
determined. Female fathead minnows showed similar patterns of vitellogenin gene expression
between reference Lake 442 and dosed Lake 260.

4.5 Studies Conducted in 2004

No dosing of Lake 260 occurred in 2004. Male fathead minnows exposed to water from
Lake 260 did not show an increase in vitellogenin gene expression, with levels similar to those
from fish exposed to water from Lake 114 and other control males. Female fathead minnows
exposed to water from ELA lakes and the control females showed high variability in vitellogenin
expression.

Fry exposed to elutriates prepared from sediments collected from Lake 260 in 2004
showed no increase in vitellogenin gene expression; levels were similar to those from fry
exposed to elutriates prepared from lake 114 sediment. In this set of exposures, fry responded to
10 ng/L EE2, although variably.

In 2004 male fathead minnows exposed to sediment elutriate from Lake 260 did show
increased vitellogenin expression relative to Lake 114 elutriate. Although results from
individual fish varied, one male had a vitellogenin mRNA level that was much higher than that
of any males exposed to 5 ng/L EE2.

-------
4.6 Summaries for individual studies from 2001 through 2004

4.6.1 Laboratory Exposures

Study 1-11: The results from the FHM adult male gene induction tests with the MHRW
control, labline water, 5 ng/1 EE2 and both Canadian water column samples showed no toxicity
to the fish in any sample. All fish exposed survived and were sacrificed for necropsy and liver
collection at the end of the test. Table 12 contains a summary of the animal survival data, as
well as all routine chemical parameters.

Study 1-12: The results for the FHM fry gene induction tests with the MHRW control,
labline water, 5 ng/1 EE2 and both Canadian water column samples showed toxicity to the fish
exposed in Lake 114 (survival = 83.1%, CV = 11.3%) when compared to the fish in the MHRW
control (survival = 96.9%, CV = 2.5%), Mann-Whitney Rank Sum Test, P=0.029. The survival
of the fish in Lake 260 was not statistically different from that in the control. Table 11 contains a
summary of the survival and routine chemical data for these samples.

Study 1-23: The results from the adult FHM male long-term exposure tests (Tables 15-
19) are unusual, due to the problems encountered in receiving the shipments of fresh lake water
for renewal. On 8/13/01, all fish in the DMSO control tank were found dead. No cause was
apparent, so any reason given would be speculation. The water in the tank was 7 days old, but
the water in the other tanks was also the same age, so this does not explain the mortality. The
next problem was toward the end when the Lake 260 fish began to die. This mortality forced the
test to be shut down a day early, to make certain at least 5 fish were still alive for the final
necropsy. For the remaining three tanks (control, 5 ng/1 EE2 and Lake 114), only 5 ng/1 EE2 had
any mortality, 2 fish died. No fish died in the control or Lake 114 (the site control). On 8/21/01,

38

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the last fish were necropsied, one day ahead of schedule. At this time, all live fish remaining in
each tank were sacrificed, necropsied and the liver tissue collected. The tissue samples from all
necropsies were stored in labeled tubes of RNAlater, for future RNA isolation and gene
expression analysis.

Study 1-24: The only problem encountered in the FHM fry tests (Tables 20-24) were
with the fry exposed in the fish tanks in the mesh-covered chambers. As stated previously,
survival in this system was poor, with no sample meeting the minimum control survival criteria
of 90% or greater. Control survival was 85%, control + DMSO was 84.4%, 5 ng/1 EE2 was
65%), Lake 114 was 82.5% and Lake 260 was 85%. The fish from all treatments were collected
and placed into labeled tubes of RNAlater buffer for gene expression level analysis. The next
test started 8-6-01, using the remainder of the samples received to start the tests. Survival was
acceptable in all treatments. The next set of fry tests started when the next fresh sample
(collected 8/14/01) was received on 8/15/01. At this point, the decision was made to test both
the new water used for renewal and the old water from the tanks for gene expression level
effects. This would provide a means to judge what effects (if any) the old water (now over 2
weeks old and in the tanks for 10 days) would have on the fry. The survival in all samples
tested, old and new 114, old and new 260, control, control+DMSO and 5 ng/1 EE2, was
acceptable. The next renewals, and fry tests, were performed 8/18/01, with samples collected
8/10/01 and received 8/17/01. The water left from the 8/15/01 renewals was mixed in as well.
For these tests, both new and old 114 and 260 waters were tested, as well as control, control +
DMSO and 5 ng/1 EE2. Again, survival in all samples was acceptable. The final tests began
8/21/01. These tests included the last batch of new (?) water for both lakes, old lake water from

-------
8/20/01 and 8/21/01, control, control + DMSO and 5 ng/1 EE2. Survival was again acceptable
across all samples.

At the end of all fry tests, the fish were removed from the test vessels and transferred into
labeled tubes containing RNAlater buffer. They were then stored for future RNA isolation and
gene expression level analysis. Due to the lack of sufficient sample, all tests were conducted as
static, non-renewal. A set of routine chemical parameters (pH, dissolved oxygen, conductivity
and temperature) were determined for the new water used to start the test (initial) and this same
water after the 48-hour exposure period (final).

Once the tests were completed, the vials containing the fish or liver samples were stored
at 4°C for 24 hours, then at -20°C until processed for RNA isolation and gene expression
analysis.

Study 1-26: The results from the FHM embryo-larval tests with MHRW control,
MHRW + DMSO solvent blank, 5 ng/1 EE2 positive control and the 6 elutriate samples were
successful, with acceptable survival in the control (97.5%), positive control (92.5%), and solvent
blank (94.4%), Table 13. Survival in the sediment elutriate samples ranged from 10.6% (Lake
260 #1) to 94.4% (Lake 260 #2), Table 13. The survival in the Lake 260 #1 sample was
determined to be statistically different from the control for survival, t-test, alpha = 0.05. The p
value for the sample was 0.04, just below the significance level of 0.05. This was the only
sample to show significant toxicity. Table 13 contains a summary of the survival data for these
samples. Table 14 contains a summary of the routine chemical analysis data associated with
these samples.

-------
Once the tests were completed, the vials containing the fish or liver samples were stored
at 4°C for 24 hours, then at -20°C until processed for RNA isolation and gene expression
analysis.

Studies 2-19, 2-20 and 2-25: The tests with the May water column and sediment
samples were successful, with acceptable survival in the control samples for all tests. Survival
was 100% for all animals exposed in Study 2-19, the first FHM adult gene expression test in the
lake water samples, Table 25. After 24 hours of exposure, the fish were sacrificed and the liver
tissue collected and placed into RNAlater. The tissue samples were stored at 4°C for 24 hours,
then moved to -20°C storage until prepared for gene expression analysis. Survival was 100% for
all animals exposed in Study 2-20, the first FHM adult gene expression sediment conducted with
the lake sediment samples, Table 26. After 48 hours of exposure the fish were sacrificed and the
liver tissue collected and placed into RNAlater. The tissue samples were stored at 4°C for 24
hours, then moved to -20°C storage until prepared for gene expression analysis. Control survival
was 94.4% (Table 27) for the fry exposed to the elutriate samples prepared from the lake
sediment samples (Study 2-25). Survival in the other 4 samples ranged from 88.1%> to 95.0%.
None were found to be statistically different from the control for survival. At the end of the test,
all live fry were collected and preserved in RNAlater buffer. The tubes were stored at 4°C for 24
hours, then moved to -20°C storage until prepared for gene expression analysis.

Studies 2-27, 2-37 and 2-40: The tests with the June water column and sediment
samples were successful, with acceptable survival in the control samples for all tests. Survival
was 100%o for all animals exposed in Study 2-27, the second FHM adult gene expression test in
the lake water samples, Table 28. After 24 hours of exposure, the fish were sacrificed and the
liver tissue collected and placed into RNAlater. The tissue samples were stored at 4°C for 24

-------
hours, then moved to -20°C storage until prepared for gene expression analysis. Survival was
100% for all animals exposed in Study 2-37, the first FHM adult gene expression sediment
conducted with the lake sediment samples, Table 29. After 48 hours of exposure the fish were
sacrificed and the liver tissue collected and placed into RNAlater. The tissue samples were
stored at 4°C for 24 hours, then moved to -20°C storage until prepared for gene expression
analysis. Control survival was 98.8% (Table 30) for the fry exposed to the elutriate samples
prepared from the lake sediment samples, Study 2-40. Survival in the other 4 samples ranged
from 93.6% to 98.2%. None were found to be statistically different from the control for survival.
At the end of the test, all live fry were collected and preserved in RNAlater buffer. The tubes
were stored at 4°C for 24 hours, then moved to -20°C storage until prepared for gene expression
analysis.

Studies 2-56, 2-64 and 2-65: The tests with the October water column and sediment
samples were successful, with acceptable survival in the control samples for all tests. Survival
was 100%) in all but one of the samples for Study 2-56, the FHM adult gene expression test in the
third set of lake water samples, Table 31. Survival was 90% in the Lake 239 water sample, due
to the death of the female fish in replicate 1. After 24 hours of exposure, the fish were sacrificed
and the liver tissue collected and placed into RNAlater. The tissue samples were stored at 4°C
for 24 hours, then moved to -20°C storage until prepared for gene expression analysis. Control
survival was 90% in Study 2-64, the FHM adult gene expression sediment conducted with this
set of lake sediment samples, Table 32. This was due to the loss of the female fish in replicate 5.
Survival was 100% in the remaining samples. After 48 hours of exposure the fish were
sacrificed and the liver tissue collected and placed into RNAlater. The tissue samples were
stored at 4°C for 24 hours, then moved to -20°C storage until prepared for gene expression

-------
analysis. Control survival was 97.0% (Table 33) for the fry exposed to the elutriate samples
prepared from the lake sediment samples, Study 2-65. Survival in the other 4 samples ranged
from 94.0% to 98.0%>. None were found to be statistically different from the control for survival.
At the end of the test, all live fry were collected and preserved in RNAlater buffer. The tubes
were stored at 4°C for 24 hours, then moved to -20°C storage until prepared for gene expression
analysis.

Study 3-37: Results from the sediment elutriate test showed excellent survival of the fry
in all but Lake 260 CB (Center Buoy) treatment after 2 days of exposure. The survival in Lake
114 sediment elutriate was 99.5% and in Lake 260 SS (Shore Sediment) was 96.5%. Lake 260
CB failed however, with a survival of only 8.5%, which was found to be statistically different
from that in the control, t-test, alpha = 0.05. All replicates of Lake 260 CB were composited into
one tube (#21) due to the poor survival. The DMSO control survival was 99.5%, with survival in
the EE2 positive control of 99.5% as well. Table 34 contains a summary of the survival data
and statistical analysis results.

Study 3-43: Results from the sediment elutriate test showed acceptable survival of the
fry. The survival in all exposures exceeded the 90% acceptability criteria. Tables 35 and 36
Contain routine chemistry data. Table 37 contains a summary of the survival data and statistical
analysis results.

At the end of the test, the live larvae were collected and placed into labeled tubes with Tri
reagent. The vials of buffer with tissue were homogenized, then the vials were then stored at -
80°C until removed for RNA isolation and gene expression analysis.

43

-------
Vitellogenin Expression
ELA 3-Week Study (All Data)
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The only problem encountered in all of the FHM fry tests (Appendix Tables 6-10) were
with the fry exposed in the fish tanks in the mesh covered chambers. As stated previously,
survival in this system was poor, with no sample meeting the minimum control survival criteria
of 90% or greater. Control survival was 85%, control + DMSO was 84.4%, 5 ng/1 EE2 was
65%, Lake 114 was 82.5% and Lake 260 was 85%. The fish from all treatments were collected
and placed into labeled tubes of RNAlater buffer for gene expression analysis. The next test
started 8/6/01, using the remainder of the samples received to start the tests. Survival was
acceptable in all treatments. The next set of fry tests started when the next fresh sample
(collected 8/14/01) was received on 8/15/01. At this point, the decision was made to test both
the new water used for renewal and the old water from the tanks for gene expression level
effects. This would provide a means to judge what effects (if any) the old water (now over 2
weeks old and in the tanks for 10 days) would have on the fry. The survival in all samples

44

-------
tested, old and new 114, old and new 260, control, control+DMSO and 5 ng/1 EE2, was
acceptable. The next renewals, and fry tests, were performed 8/18/01, with samples collected
8/10/01 and received 8/17/01. The water left from the 8/15/01 renewals was mixed in as well.
For these tests, both new and old 114 and 260 waters were tested, as well as control, control +
DMSO and 5 ng/1 EE2. Again, survival in all samples was acceptable. The final tests began
8/21/01. These tests included the last batch of new water for both lakes, old lake water from
8/20/01 and 8/21/01, control, control + DMSO and 5 ng/1 EE2. Survival was again acceptable
across all samples.

Once the tests were completed, the vials containing the fish or liver samples were stored
at 4°C for 24 hours, then at -20°C until processed for RNA isolation and gene expression
analysis. Males exposed to labline water, water with DMSO, and Lake 114 water showed no
vitellogenin expression (Figure 5). Males exposed to Lake 260 water showed extensive increase
in vitellogenin levels, even higher in some cases than males exposed to 5 ng/L EE2. Males had
variable response to both the 5ng/L water and the Lake 260 water. Two of the fish exposed to 5
ng/L EE2 showed no increase in expression of vitellogenin. One male exposed to Lake 260
water showed no expression, while two had expression levels comparable to that of fish exposed

-------
to 5 ng/L EE2. The remaining fish had very high levels of expression, roughly five times that of
the high level responders to the 5 ng/L EE2.

5 DISCUSSION

Vitellogenin gene mRNA was upregulated in indigenous male fathead minnows after
only 24-hour exposure to EE2 and was still high after four months of exposure. Similarly,
indigenous male pearl dace had high vitellogenin levels after four months of exposure to EE2.
Female fathead minnows continued to have high vitellogenin levels into the fall, well beyond the
normal breeding season, although pearl dace females in this study did not follow this trend. In
females, vitellogenin protein is sequestered in eggs. In males and non-reproductive females (i.e.,
those without eggs) vitellogenin has no sink, and the protein continues to circulate in the blood.
High levels of protein can cause problems for these fish. After four months of continuous
exposure to EE2, fathead minnows exhibited widespread fibrosis and inhibited development of
testicular tissues (Palace 2002). Also, EE2-exposed fish had kidney anomalies including edema
within and between the kidney tubules, and hyaline deposits in the tubule cells. Liver tissues
showed a loss of glycogen stores and increased liver cell size. These latter effects on the kidney
and liver were likely due to the production and accumulation of VTG in these tissues.

-------
One of the difficulties encountered in working with wild fish populations was sex
determination. Determining the sex of fathead minnows from the ELA lakes was more difficult
than determining sex in laboratory-reared animals. The wild fish were generally smaller than
those used in lab exposures, which is likely a consequence of the very large populations that
were in evidence. Male fathead minnows did not have well-developed secondary sex
characteristics (swollen fat pad on the nape, nuptial tubercles around the mouth and nose) that
facilitate sex determination in laboratory-reared fish. Internal anatomy (identification of gonadal
tissues as ovary or testis) was of little use in diagnosing sex since the females were not in
breeding condition, so eggs were not present.

Determining the sex of pearl dace was even more problematic. In this species, the only
external secondary sex characteristic is a slightly thickened first spine on the pelvic fins, which is
seen as a slightly darker brown anterior edge of the pelvic fin. Doubts about this method were
voiced. Again, internal anatomy proved of little value in most fish, since the females were not in
breeding condition, and the lack of eggs made the identification of gonads nearly impossible.
For individuals with more experience, confidence in sex determination seemed higher than for
us. Because of the difficulties in distinguishing between sexes, there exists the very real
possibility that some of the fish that we identified as males were in fact females. The inclusion
of female fish from reference lakes, with their relatively high vitellogenin levels, in the male
samples, would minimize the difference in vitellogenin expression between males from dosed
and undosed lakes. This would explain why some males from the reference lakes exhibited high
vitellogenin levels, whereas laboratory-reared fathead males rarely show even a small level of
expression when not exposed to EE2. Within a group of EE2-exposed fish it is expected that not
all will respond equally. There are often high expressing fish and fish that do not respond at all.

-------
Although trends can be seen between exposed and nonexposed fish, these differences often are
not statistically significant.

6 CONCLUSIONS

Gene expression in indigenous male fathead minnows and pearl dace collected at all time
intervals from the dosed lake showed a constant level of elevation. Gene expression in the 2001
fathead minnow deployment study was detected within 24 hours after deployment of control fish
into the treated lake and stayed elevated for the entire 13-day study. Highly variable gene
expression was found in fathead minnow fry exposed to dosed lake sediments, but no significant
gene expression was found in fry exposed to reference lake sediments. Male adult fathead
minnows exposed to sediment elutriates from sediments collected in 2004 in the previously
dosed lake showed significant vitellogenin gene expression. Results indicate that RT-PCR
analyses of total RNA can be used to provide a rapid and timely estimate of exposure to
estrogenic substances.

48

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7 REFERENCES

Belfroid, A. C., A. Van der Horst, A. D. Vethaak, A. J. Schafer, G. B. J. Rijs, J. Wegener, and
W. P. Cofino. 1999. Analysis and occurrence of estrogenic hormones and their glucuronides in
surface water and waste water in the Netherlands. Sci Total Environ. 225:101-108.

Chomczynski, P. and N. Sacchi. 1987. Single step method of RNA isolation acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159.

Desbrow, C., E. Routledge, G. C. Brighty, J. Sumpter, and M. Waldock. 1998. Identification of
estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological
screening. Environ. Sci Technol. 32:1549-1558.

Lange, R., T. H. Hutchinson, C. P. Croudace, F. Siegmund, H. Schweinfurth, P. Hampe, G. H.
Panter, and J. P. Sumpter. 2001. Effects of the synthetic oestrogen 17a-ethynylestradiol on the
life-cycle of the fathead minnow (Pimephalespromelas). Environ. Contam. Toxicol. 20:1216-
1227.

Larsson, D. G. J., M. Adolfsson-Erici, J. Parkkonen, M. Pettersson, A. H. Berg, P-E.Olsson, and
L. Forlin. 1999. Ethinylestradiol-an undesired fish contraceptive. Aquat. Toxicol. 45:91-97.

Lattier, D. L., D. Gordon, D. J. Burks, and G. P. Toth. 2001. Vitellogenin gene transcription: A
relative quantitative exposure indicator of environmental estrogens. Environ. Toxicol. Chem. 20
(9): 1979-1985.

49

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Lattier, D. L., T. V. Reddy, D. A. Gordon, J. L. Lazorchak, M. E. Smith, D. E. Williams,
B. Wiechman, R. W. Flick, A. L. Miracle, and G. P. Toth. 2002. 17a-ethynylestradiol-induced
Vitellogenin Gene Transcription Quantified in Livers of Adult Males, Larvae, and Gills of
Fathead Minnows (Pimephalespromelas). Environ. Tox. Chem. 21(11)2385-2393.

Palace, V., R. E. Evans, K. Wautier, L. Vandenbyllardt, W. Vandersteen, and K. Kidd. 2002.
Biochemical and physiological effects in wild fathead minnows from a lake experimentally
treated with the synthetic estrogen, ethynylestradiol. Water Qual Res J Can 37 (3) 637-650.

Preziosi, P. 1998. Endocrine disrupters as environmental signallers: an introduction. Pure Appl
Chem 70:1617-1631.

Rychlik, W. and R. E. Rhoads. 1989. A computer program for choosing optimal
oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic
Acids Res 17:8543-8551.

Ternes, T.A., M. Stumpf, J. Mueller, K. Haberer, R. D. Wilken, and M. Servos. 1999.

Behaviour and occurrence of estrogens in municipal sewage treatment plants-1. Investigations in
Germany, Canada and Brazil. Sci. Tot. Environ. 225:81-90.

50

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Field
Station

Experimental Lakes Area

Designated Research Lakes shaded Blue

51

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Mean EE2 Concentrations in Lake 260

Figure 2. Concentration of EE2 (ng/L) in Lake 260 during the three years of dosing.

52

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Figure 3. Study 1-21: vitellogenin gene expression in adult male fathead minnows collected from Lake 260 in
2001. PCR performed on liver for 17 cycles. Error bars represent ±1 standard deviation.


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Figure 4. Study 1-21: vitellogenin gene expression in adult male fathead minnows deployed in Lake 114 and
Lake 260 in 2001. PCR performed on liver for 17cycles. Each sample consisted of five fish, except *n=7;
**n=4. Error bars represent ±1 standard deviation.

53

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DMSO

Lab Line* Lake 114 5 ng/L EE2** Lake 260

Figure 5. Study 1-11: vitellogenin gene expression in laboratory reared males exposed to water collected from
ELA lakes in 2001. PCR performed on liver for 17 cycles. Each sample consisted of four fish, except *n=3;

*n=5. Error bars represent ±1 standard deviation.

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Figure 7. Study 2-62: vitellogenin gene expression in male fathead minnows collected in 2002. PCR
performed on liver for 17cycles. Error bars represent ±1 standard deviation.

Lake 114 Lake 114 Lake 114
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Figure 8. Study 2-62: vitellogenin gene expression in female fathead minnows collected in 2002. PCR
performed on liver for 17cycles. Error bars represent ±1 standard deviation.

55

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Figure 9. Study 2-63: vitellogenin gene expression in male pearl dace collected in 2002. PCR performed on
liver for 17cycles. Error bars represent ±1 standard deviation.

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Figure 10. Study 2-63: vitellogenin gene expression in female pearl dace collected in 2002. PCR performed
on liver for 17cycles. Error bars represent ±1 standard deviation.

56

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Figure 11. Study 3-45: vitellogenin gene expression in male fathead minnows collected in May 2003. PCR
performed on liver for 17cycles. Error bars represent ±1 standard deviation.

Figure 12. Study 3-45: vitellogenin gene expression in female fathead minnows collected in May 2003. PCR
performed on liver for 17cycles. Error bars represent ±1 standard deviation.

57

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4

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Figure 13. Study 3-45: vitellogenin gene expression in male pearl dace collected in May 2003. PCR
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deviation.

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Figure 14. Study 3-45: vitellogenin gene expression in female pearl dace collected in May 2003. PCR
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Figure 15. Study 4-19: quantitative real time PCR analysis of vitellogenin gene expression in female fathead
minnows collected in May 2004. Sample size is five for each group. Error bars represent ±1 standard
deviation.

Figure 16. Study 4-19: quantitative real time PCR analysis of vitellogenin gene expression in male fathead
minnows collected in 2004. Sample size is five for each group. Error bars represent ±1 standard deviation.

59

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Figure 18. Study 4-20: quantitative real time PCR analysis of vitellogenin gene expression in male fathead
minnows exposed to elutriate prepared from sediment collected in 2004. Sample size is six except *n=5. Error
bars represent ±1 standard deviation.

60

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Appendix 1

The description of the EE2 dosing of the ELA lake from the Department of Fisheries and Oceans
Canada project plan, courtesy of Karen Kidd:

Despite the overt physiological evidence that fish are being adversely impacted by
endocrine disrupting substance (EDS), it remains unclear whether these compounds are affecting
aquatic organisms at the population level. It has been recognized nationally and internationally
that there is a need to determine whether the molecular and cellular effects, such as egg protein
(vitellogenin) production in males, observed in fish exposed to estrogen mimics are indicative of
changes in the viability of the population. Though significant progress has been made in
characterizing the effects of estrogenic contaminants in individuals, population level approaches
to identify and quantify effects are lacking (US EPA 1997, Jobling et al. 1998). The following
study has been designed to address this need, and to provide information that will complement
lab and field studies being conducted by DFO, other government agencies, and academic
institutions.

We are conducting a multi-year whole-ecosystem study at the Experimental Lakes Area
(ELA) in northwestern Ontario that will expose well-defined aquatic populations to the synthetic
estrogen 17-alpha-ethynylestradiol (EE2). This study has been designed to determine whether
aquatic populations are adversely impacted by an EDS, and to validate and calibrate the
relationship between organism- and population-level responses to hormone mimics. EE2 was
chosen for this experiment because it is known to be a potent estrogen mimic in fish and other
vertebrates (Routledge et al. 1998), and it is found at low ng/L levels in river waters downstream
of sewage treatment plants (Belfroid et al. 1999).

In the first two years of this study, baseline information was collected on the populations
of fish, amphibians, benthic and pelagic invertebrates, microorganisms and algae in the study
(Lake 260) and reference lakes (Lakes 375, 114, 305, 224 and 442). For three consecutive
summers (2001 through 2003), EE2 was added continuously to Lake 260 over the ice-free season
to maintain the water at a concentration known to induce biochemical responses in fish (5 to 6 ng
EE2 L-l). This lake was chosen for the EE2 amendments because it contains lake trout, white
sucker, pearl dace and fathead minnows (a commonly used species in EDS assays), allowing for
contrasts between large and small fish species. It also has long-term records on fish populations
(since 1984), and historical water quality and zooplankton data. Impacts of EDS exposure on
small and large fish populations will be assessed using age and size distributions, sex ratios, age
to maturity, condition factors, abundance, growth rates, annual survival and recruitment, and
fertilization studies. Organism-level responses in fish are also being examined and include
impacts on circulating and tissue hormone levels, gonadal development (histological
assessments) and in vitro steroid synthesis, and vitellogenin production. These responses will be
used to evaluate the utility of the different exposure markers commonly used in field screening
programs, and will contribute to our understanding of the underlying mechanisms that mediate a
fish's response to estrogenic compounds. Changes in the populations of algae, microorganisms,
zooplankton, benthic invertebrates and tadpoles will also be evaluated after EE2 amendments,
using both baseline and reference lake data collected this past field season and over the next

61

-------
several years. In summary, this study will provide much-needed information on the magnitude
of the effects of a potent EDS on aquatic populations.

62

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Appendix 2

Table 10. Results from a FHM fry gene induction test performed on 6-22-01 with moderately
hard reconstituted water (MHRW), labline water, 5 ng/1 EE2, Lake 114 and Lake 260. Test
duration of 48 hours. Lake 114 and 260 samples collected 6-20-01. Study # 1-12.



pH

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8.16

8.3

7.9

300

331

24.2

24.1

Labline

1-4

160

155

8.07

7.92

8.0

7.9

452

395

24.1

24.2

5 ng/1
EE2

1-4

160

156

8.01

8.12

8.3

8.0

298

422

24.0

24.0

Lake 114

1-4

160

133

6.67

7.01

8.4

8.0

14

36

24.0

24.3

Lake 260

1-4

160

143

6.75

7.07

00
00

8.1

21

46

24.2

24.0

Table 11. Results from a FHM adult male gene induction test performed on 6-22-01 with
moderately hard reconstituted water (MHRW), labline water, 5 ng/1 EE2, Lake 114 and Lake
260. These tests used 1 fish in each of the 5 test replicates. Test duration of 24 hours. Lake 114
and Lake 260 samples collected 6-20-01. Study #1-11.



pH

DO

Conductivity

Temp

Cone.

rep

exp

live

I

F

I

F

I

F

I

F

Lab Cont

1-5

5

5

8.00

7.73

8.3

6.9

300

308

24.2

24.2

Labline

1-5

5

5

8.07

7.77

8.0

6.9

452

464

24.1

24.1

5 ng/1
EE2

1-5

5

5

8.01

7.72

8.3

6.8

298

306

24.0

24.1

Lake 114

1-5

5

5

6.67

6.98

8.4

6.8

14

26

24.0

24.1

Lake 260

1-5

5

5

6.75

8.86

00
00

6.6

21

32

24.2

24.1

63

-------
Table 12. Results from sediment elutriate tests conducted with Canadian ELA samples using P.
promelas embryo/larvae. Elutriate samples were prepared after the embryo-larval tests on the
whole sediment resulted in 100% mortality for the lake sediments. Tests were 5 days duration,
conducted at 25°C. Water renewed daily. Tests included MHRW control, MHRW + DMSO
solvent blank, 5 ng/1 EE2 positive control and six lake samples. Study #1-26.

Conc./Site I.D.

rep

exp

live

RNAlater
Tube#

% Sur

CV

t-test*

Lab Cont

1-4

160

156

1-4

97.5

2.09

N/A

MHRW + DMSO

1-4

160

151

5-8

94.4

2.54

0.094

5 ng/1 EE2

1-4

160

148

9-12

92.5

2.21

0.073

Lake 114

1-4

160

127

13-16

79.4

25.51

0.125

Lake 260 #1

1-4

160

113

17-20

70.6

29.24

0.041

Lake 260 #2

1-4

160

151

21-24

94.4

4.52

0.235

Lake 260 #3

1-4

160

134

25-28

83.8

19.88

0.152

Lake 260 #4

1-4

160

139

29-32

86.9

11.60

0.084

Lake 260 #5

1-4

160

121

33-36

75.6

29.37

0.097

* Samples analyzed using t-test function in Lotus, alpha = 0.05. Site I.D. and t-test result in bold
denotes sample found to be statistically different from the control.

64

-------
Table 13. Routine initial and final chemical analysis of elutriate samples prepared from
Canadian ELA Lake samples. Listed are the initial chemical determination for the start of the
test and the ranges of final chemistry results for each analyte. Study # 1-26.



pH

DO

Conductivity

Temp

Cone.

I

F

I

F

I

F

I

F

Lab
Cont

8.32

8.14-8.46

8.2

7.4-8.1

319

317-345

25.1

24.8- 25.6

MHRW +
DMSO

8.28

8.11-8.26

8.3

7.1-7.8

314

318-333

25.2

24.9- 25.5

5 lift/1 EE2

8.31

8.10-8.23

8.2

7.3-7.6

312

319-336

25.2

25.0- 25.5

Lake
114

7.37

7.53-7.94

7.8

7.2-7.8

235

249- 262

24.7

24.9- 25.4

Lake 260
#1

6.97

7.47- 7.74

8.5

7.1-7.7

214

224- 242

24.4

24.8- 25.3

Lake 260
#2

6.96

7.33-7.68

8.4

7.1-7.8

234

235- 268

25.0

24.9- 25.2

Lake 260

#3

6.58

7.30-7.53

8.5

7.0-7.8

230

230-259

24.9

25.0- 25.3

Lake 260
#4

6.64

7.33-7.48

8.5

7.2-7.8

202

212-251

24.9

24.8- 25.2

Lake 260
#5

6.65

7.29-7.50

8.5

7.4-7.8

215

228- 238

24.8

24.9- 25.1

Table 14. Results from the Day 1 necropsy of fish for the Canadian ELA 3-week study (8/2/01).
Fish were randomly selected for use in the necrospy procedure and water samples were collected
to analyze for final water chemistries. Study #1-23.



pH

DO

Conductivity

Temp

Cone.

rep

# N

Tube

#

I

F

I

F

I

F

I

F

Lab Cont

1

5

6-10

8.01

7.91

8.1

7.4

301

323

24.2

24.6

Cont +
DMSO

1

5

16-20

8.04

7.68

8.0

6.8

303

313

24.4

24.8

5 ng/1
EE2

1

5

11-15

8.09

7.74

8.1

7.0

304

317

24.3

25.0

Lake 114

1

5

21-25

6.65

7.55

8.9

6.9

50

65

24.2

24.5

Lake 260

1

5

26-30

7.13

7.18

8.4

6.3

62

52

24.2

24.6

# N = number necropsied.

65

-------
Table 15. Results from the Day 4 necropsy of fish for the Canadian ELA 3-week study (8/6/01).
Fish were randomly selected for use in the necrospy procedure and water samples were collected
to analyze for final water chemistries. Water was changed at this time. Study #1-23.



pH

DO

Conductivity

Temp

Cone.

rep

# N

Tube

#

I

F

I

F

I

F

I

F

Lab Cont

1

5

31-35



7.66



7.7



372



24.8

Cont +
DMSO

1

5

36-40



7.57



7.7



372



24.7

5 ng/1
EE2

1

5

41-45



7.69



7.8



364



24.9

Lake 114

1

5

46-50



7.14



7.8



104



24.6

Lake 260

1

5

51-55



7.17



7.8



113



24.7

Table 16. Results from the Day 7 necropsy of fish for the Canadian ELA 3-week study (8/9/01).
Fish were randomly selected for use in the necrospy procedure and water samples were collected
to analyze for final water chemistries. Water was not changed at this time. Study #1-23.



pH

DO

Conductivity

Temp

Cone.

rep

# N

Tube

#

I

F

I

F

I

F

I

F

Lab Cont

1

5

56-60



7.99



7.7



345



24.6

Cont +
DMSO

1

5

61-65



7.96



7.7



371



24.7

5 ng/1
EE2

1

5

66-70



8.09



7.8



372



24.6

Lake 114

1

5

71-75



7.58



7.8



78



24.3

Lake 260

1

5

76-80



7.61



7.8



82



24.4

66

-------
Table 17. Results from the Day 13 necropsy of fish for the Canadian ELA 3-week study
(8/15/01). Fish were randomly selected for use in the necrospy procedure and water samples
were collected to analyze for final water chemistries. Water was changed at this time. No data
for DMSO control, all fish dead by this point. Study #1-23.



pH

DO

Conductivity

Temp

Cone.

rep

# N

Tube#

I

F

I

F

I

F

I

F

Lab Cont

1

5

81-85



8.28



7.9



381



24.0

Cont +
DMSO

1

5

N/A



N/A



N/A



N/A



N/A

5 ng/1
EE2

1

5

86-90



8.15



7.8



402



24.2

Lake 114

1

5

91-95



7.93



7.8



102



24.3

Lake 260

1

5

96-100



7.70



7.6



125



24.3

Table 18. Results from the Day 20 necropsy of fish for the Canadian ELA 3-week study
(8/21/01). Fish were randomly selected for use in the necrospy procedure and water samples
were collected to analyze for final water chemistries. This completed the study. All remaining
fish were necropsied at this time. Study #1-23.



pH

DO

Conductivity

Temp

Cone.

rep

# N

Tube#

I

F

I

F

I

F

I

F

Lab Cont

1

10

101-110



7.88



7.1



354



24.5

Cont +
DMSO

1

0

N/A



N/A



N/A



N/A



N/A

5 ng/1
EE2

1

8

111-118



7.68



7.2



363



24.4

Lake 114

1

10

119-128



7.78



6.8



100



24.8

Lake 260

1

5

129-133



7.63



7.0



121



24.6

67

-------
Table 19. Results from the first fry test with fresh samples from Lakes 114 and 260, 8/2/01. Fry
were exposed in the fish tanks in screen covered PVC exposure chambers. Tests were 48 hours
duration. No renewal of test solution. Fry used were 24 to 48 hours old. As is apparent,
survival was below acceptable levels, so future tests were conducted in 500 ml glass beakers.
Study #1-24.



pH

DO

Conductivity

Temp

Cone.

rep

sur

Tube#

I

F

I

F

I

F

I

F

Lab Cont

1-4

136/160

1-4

8.10

7.75

8.0

7.6

315

338

24.1

24.8

Cont +
DMSO

1-4

135/160

5-8

8.08

7.72

8.0

7.5

310

335

24.2

24.9

5 ng/1
EE2

1-4

104/160

9-12

8.00

7.64

8.0

7.2

311

329

24.1

25.1

Lake 114

1-4

132/160

13-16

7.78

7.14

8.4

6.7

61

71

24.1

24.6

Lake 260

1-4

136/160

17-20

7.24

7.16

8.5

6.7

50

81

24.2

24.8

Table 20. Results from the second fry test with samples from Lakes 114 and 260, 8/6/01. Fry
were exposed in glass beakers, 300 ml of test solution. Tests were 48 hours duration. No
renewal of test solution. Fish used were fry 24 to 48 hours old. Study #1-24.



pH

DO

Conductivity

Temp

Cone.

rep

sur

Tube#

I

F

I

F

I

F

I

F

Lab Cont

1-4

159/160

21-24

8.32

8.33

8.4

7.8

280

306

24.8

25.1

Cont +
DMSO

1-4

160/160

25-28

8.13

8.25

8.4

7.5

287

306

24.8

25.2

5 ng/1
EE2

1-4

160/160

29-32

8.13

8.17

8.2

7.5

287

312

24.7

24.9

Lake 114

1-4

158/160

33-36

6.92

7.83

8.2

7.4

17

60

24.9

24.6

Lake 260

1-4

159/160

37-40

7.05

7.70

8.4

7.5

38

83

24.9

24.4

68

-------
Table 21. Results from the third fry test with samples from Lakes 114 and 260, 8/15/01. For
this test, both the water used to renew the tanks and the old water from the tanks was analyzed
using the fry exposure test method. Fry were exposed in glass beakers, 300 ml of test solution.
Tests were 48 hours duration. No renewal of test solution. Fish used were fry 24 to 48 hours
old. Study #1-24.



pH

DO

Conductivity

Temp

Cone.

rep

sur

Tube#

I

F

I

F

I

F

I

F

Lab Cont

1-4

160/160

41-44

8.22

8.80

8.5

7.5

290

314

24.3

25.2

Cont +
DMSO

1-4

159/160

45-48

8.22

8.71

8.2

7.5

293

310

24.4

25.2

5 ng/1
EE2

1-4

160/160

53-56

8.22

8.25

8.5

7.1

290

310

24.1

24.6

Lake 114
new

1-4

160/160

57-60

7.58

7.26

8.2

7.2

21

32

24.3

24.6

Lake 260
new

1-4

160/160

61-64

7.50

7.48

8.4

7.4

14

46

24.1

24.7

Lake 114
old

1-4

160/160

49-52

7.93

8.28

7.8

7.9

102

121

24.3

25.0

Lake 260
old

1-4

148/160

65-68

7.70

7.24

7.9

6.2

125

134

24.4

24.5

Table 22. Results from the fourth fry test with samples from Lakes 114 and 260, 8/18/01. For
this test, both the water used to renew the tanks and the old water from the tanks were analyzed
using the fry exposure test method. Fry were exposed in glass beakers, 300 ml of test solution.
Tests were 48 hours duration. No renewal of test solution. Fish used were fry 24 to 48 hours
old. Study #1-24.



pH

DO

Conductivity

Temp

Cone.

rep

sur

Tube#

I

F

I

F

I

F

I

F

Lab Cont

1-4

160/160

69-72

8.01

8.26

8.0

7.6

300

3307

24.1

25.1

Cont +
DMSO

1-4

160/160

73-76

8.05

8.16

8.1

7.5

304

300

24.0

25.0

5 ng/1
EE2

1-4

159/160

85-88

8.10

7.94

8.0

7.2

301

301

24.2

25.0

Lake 114
new

1-4

159/160

81-84

7.43

7.71

00
00

7.3

20

48

24.1

25.0

Lake 260
new

1-4

159/160

89-92

7.40

7.00

8.7

7.0

15

80

24.3

24.9

Lake 114
old

1-4

160/160

77-80

7.78

7.72

6.8

7.0

100

85

24.2

25.0

Lake 260
old

1-4

160/160

93-96

7.63

7.31

7.0

7.0

121

45

24.4

25.0

69

-------
Table 23. Results from the fourth fry test with samples from Lakes 114 and 260, 8/21/01. For
this test, both the water used to renew the tanks and the old water from the tanks were analyzed
using the fry exposure test method. Fry were exposed in glass beakers, 300 ml of test solution.
Tests were 48 hours duration. No renewal of test solution. Fish used were fry 24 to 48 hours
old. Study #1-24.



pH

DO

Conductivity

Temp

Cone.

rep

sur

Tube#

I

F

I

F

I

F

I

F

Lab Cont

1-4

159/160

97-100

8.43

8.14

8.1

7.6

293

310

24.2

24.7

Cont +
DMSO

1-4

160/160

101-104

8.42

8.20

8.1

7.7

298

326

24.8

25.2

5 rift/l EE2

1-4

156/160

105-108

8.35

8.17

8.2

7.5

300

317

24.6

25.2

Lake 114
new

1-4

148/160

113-116

7.06

7.50

8.3

7.7

19

46

24.4

24.7

Lake 260
new

1-4

159/160

109-112

7.54

7.56

8.4

7.7

26

47

24.3

24.6

Lake 114
8/20 old

1-4

159/160

117-120

7.24

7.67

7.9

7.7

30

83

24.4

25.2

Lake 260
8/20 old

1-4

159/160

121-124

7.13

7.41

7.8

7.1

44

88

24.3

25.1

Lake 114
8/21 old

1-4

160/160

125-128

7.26

7.47

8.0

7.4

21

65

24.8

24.6

Lake 260
8/21 old

1-4

153/160

129-132

7.16

7.21

8.0

7.0

33

68

24.6

24.7

70

-------
Table 24. Results for the Adult FHM Gene Expression test with 2 ELA Lake samples.
Conducted 5/23/02. Treatments included MHRW control, MHRW + DMSO, 5 ng/1 EE2
positive control sample and samples from Lake 114 and 260. Duration was 24 hours.
Study #2-19.



pH

DO

Conductivity

Temp

Cone

Rep

sex

exp

live

Tube#

I

F

I

F

I

F

I

F

MHRW

1-5

m

5

5

1-5

8.03

7.57

8.3

5.5

300

307

25.0

24.1

MHRW

1-5

f

5

5

21-25

















Cont + DMSO

1-5

m

5

5

6-10

8.08

7.96

8.3

7.7

297

314

25.0

24.0

Cont + DMSO

1-5

f

5

5

26-30

















5 ng/1 EE2

1-5

m

5

5

11-15

8.10

7.75

8.5

7.2

290

304

25.0

24.2

5 ng/1 EE2

1-5

f

5

5

31-35

















Lake 114

1-5

m

5

5

16-20

7.53

7.78

10.2

7.0

18

32

24.2

24.4

Lake 114

1-5

f

5

5

46-50

















Lake 260

1-5

m

5

5

21-25

7.37

7.58

10.4

6.9

22

43

24.0

24.5

Lake 260

1-5

f

5

5

51-55

















Day 0 Initial
Fish

1-5

m

5

5

46-50

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Day 0 Initial
Fish

1-5

f

5

5

41-45

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

71

-------
Table 25. Results for the Adult FHM Gene Expression test with 2 ELA Lake sediment samples.
Conducted 5/29/02. Treatments included MHRW control, MHRW + DMSO, 5 ng/1 EE2
positive control sample and sediment samples from Lake 114 and 260. Duration was 48H.
Study #2-20.



pH

DO

Conductivity

Temp

Cone

Rep#

sex

exp

live

Tube#

I

F

I

F

I

F

I

F

MHRW

1-5

m

5

5

11-15

8.28

8.00

8.2

7.4

308

329

24.1

24.6

MHRW

1-5

f

5

5

36-40

















Cont +
DMSO

1-5

m

5

5

16-20

8.16

8.01

8.2

7.6

306

328

24.5

24.4

Cont +
DMSO

1-5

f

5

5

41-45

















5 ng/1 EE2

1-5

m

5

5

21-25

8.12

7.85

8.0

6.5

306

328

24.5

24.3

5 ng/1 EE2

1-5

f

5

5

46-50

















Lake 114
sediment

1-5

m

5

5

26-30

6.93

7.28

5.8

4.0

242

241

24.6

24.5

Lake 114
sediment

1-5

f

5

5

51-55

















Lake 260
sediment

1-5

m

5

5

31-35

6.68

7.11

2.9

3.9

225

211

24.5

24.4

Lake 260
sediment

1-5

f

5

5

56-60

















Day 0 Initial
Fish

1-5

m

5

5

1-5

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Day 0 Initial
Fish

1-5

f

5

5

6-10

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

** Sample 1, Lake 260, no eggs.

72

-------
Table 26. Results from the P. promelas (FHM) embryo/larval test using elutriate samples
prepared from the Canadian ELA Lakes 114 and 260 Sediments. Conducted 6/13/02. Test used
50 ml of test solution, 40 FHM eggs/replicate, MHRW control, 5 ng/1 EE2 positive control and 2
ELA samples. Duration was 5 days. Study #2-25.



pH

DO

Conductivity

Temp

Cone.

Rep #

% Sur

CV

P

value

Tube#

I

F

I

F

I

F

I

F

MHRW

1-4

94.4

1.3

n/a

1-4

8.28

8.17

8.6

8.4

316

368

24.0

24.3

Cont +
DMSO

1-4

95.0

0

.686

5-8

8.16

7.97

8.2

8.2

313

324

24.0

24.6

5 ng/1
EE2

1-4

93.1

4.6

.595

9-12

8.16

7.93

8.3

8.2

315

330

24.1

24.7

ELA
Lake 114

1-4

88.1

6.3

.070

13-16

7.05

7.68

9.3

8.2

228

241

25.0

24.8

ELA
Lake 260

1-4

93.8

6.3

1.00

14-20

6.19

7.33

9.6

8.2

202

212

24.9

24.9

73

-------
Table 27. Results for the Adult FHM Gene Expression test with Canadian ELA samples.
Conducted 6/20/02. Treatments included MHRW control, MHRW + DMSO, 5 ng/1 EE2
positive control sample and a two Canadian ELA samples. Duration was 48H. Study #2-27.



pH

DO

Conductivity

Temp

Cone

Rep#

sex

exp

live

Tube#

I

F

I

F

I

F

I

F

MHRW

1-5

m

5

5

36-40

7.77

7.86

8.7

8.4

322

328

24.3

24.6

MHRW

1-5

f

5

5

11-15

















Cont + DMSO

1-5

m

5

5

41-45

7.86

7.79

00
00

8.3

322

327

24.1

24.8

Cont + DMSO

1-5

f

5

5

16-20

















5 ng/1 EE2

1-5

m

5

5

46-50

7.87

7.81

00
00

6.2

319

329

24.0

25.1

5 ng/1 EE2

1-5

f

5

5

21-25

















Lake 114

1-5

m

5

5

51-55

5.88

7.15

9.1

8.0

13

34

24.7

25.0

Lake 114

1-5

f

5

5

26-30

















Lake 260

1-5

m

5

5

56-60

6.57

7.17

9.1

7.9

21

37

24.6

25.1

Lake 260

1-5

f

5

5

31-35

















Day 0 Initial
Fish

1-5

m

5

5

6-10

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Day 0 Initial
Fish

1-5

f

5

5

1-5

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

74

-------
Table 28. Results for the Adult FHM Gene Expression test with 2 ELA Lake sediment samples
conducted 7/24/02. Treatments included MHRW control, MHRW + DMSO, 5 ng/1 EE2 positive
control sample and sediment samples from Lake 114 and 260. Duration was 48 hours.

Study #2-37.



pH

DO

Conductivity

Temp

Cone

Rep#

sex

exp

live

Tube#

I

F

I

F

I

F

I

F

MHRW

1-5

m

5

5

36-40

8.14

8.01

8.3

8.0

349

339

24.8

24.5

MHRW

1-5

f

5

5

11-15

















Cont +
DMSO

1-5

m

5

5

41-45

8.08

8.00

8.4

8.0

318

327

24.3

24.7

Cont +
DMSO

1-5

f

5

5

16-20

















5 ng/1
EE2

1-5

m

5

5

46-50

8.09

7.96

8.5

8.0

318

324

24.5

24.9

5 ng/1
EE2

1-5

f

5

5

21-25

















Lake 114
sediment

1-5

m

5

5

51-55

7.08

7.62

7.8

7.8

238

223

24.4

24.8

Lake 114
sediment

1-5

f

5

5

31-35

















Lake 260
sediment

1-5

m

5

5

56-60

6.45

7.08

6.3

7.4

221

214

24.5

24.7

Lake 260
sediment

1-5

f

5

5

26-31

















Day 0
Initial
Fish

1-5

m

5

5

1-5

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Day 0
Initial
Fish

1-5

f

5

5

6-10*

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

*Fish in tube #10 appeared to be a male, not a female.

75

-------
Table 29. Results from the P. promelas (FHM) embryo/larval test using elutriate samples
prepared from the Canadian ELA Lakes 114 and 260 Sediments. Conducted 8/09/02. Test used
50 ml of test solution, 40 FHM eggs/replicate, MHRW control, 5 ng/1 EE2 positive control and 2
ELA samples. Duration was 5 days. Study #2-40.



PH

DO

Conductivity

Temp

Cone.

Rep #

%
Sur

CV

t-test
P

value

Tube#

I

F

I

F

I

F

I

F

MHRW

1-5

98.8

1.1

n/a

1-5

8.35

8.08

8.4

8.0

327

335

24.1

24.7

Cont +
DMSO

1-5

93.6

3.8

0.013

6-10

8.28

8.01

8.7

8.0

327

331

24.1

24.6

5 ng/1 EE2

1-5

97.4

1.4

0.108

11-15

8.28

8.04

8.7

8.1

327

340

24.0

24.6

Lake 114

1-5

96.8

4.0

0.302

16-20

7.13

7.14

00
00

8.1

251

268

24.1

24.5

Lake 260

1-5

98.2

2.1

0.580

21-25

6.16

6.21

7.5

7.7

228

239

24.1

24.5

Bold/underline indicates sample determined to have survival statistically different from the
control, t-test, alpha = 0.05.

76

-------
Table 30. Results for the Adult FHM Gene Expression test with 2 ELA Lake samples.
Conducted 10/27/02. Treatments included MHRW control, MHRW + DMSO, 5 ng/1 EE2
positive control sample and samples from Lakes 239 and 260. Duration was 24 hours.
Study #2-56.



pH

DO

Conductivity

Temp

Cone

Rep#

sex

exp

live

Tube#

I

F

I

F

I

F

I

F

MHRW

1-5

m

5

5

1-5

7.98

8.09

7.9

7.6

315

364

24.1

24.7

MHRW

1-5

f

5

5

26-30

















Cnt + DMSO

1-5

m

5

5

6-10

7.96

7.66

8.0

7.6

312

343

24.3

24.5

Cnt + DMSO

1-5

f

5

5

31-35

















5 ng/1 EE2

1-5

m

5

5

11-15

8.01

7.86

8.0

7.6

310

333

24.0

24.6

5 ng/1 EE2

1-5

f

5

5

36-40

















Lake 239

1-5

m

5

5

16-20

6.80

7.50

9.2

7.4

28

52

24.5

25.1

Lake 239

1-5

f

5

4*

42-45

















Lake 260

1-5

m

5

5

21-25

6.78

7.52

9.4

7.5

22

60

24.6

24.9

Lake 260

1-5

f

5

5

46-50

















Day 0 Initial
Fish

1-5

m

5

5

A

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Day 0 Initial
Fish

1-5

f

5

5

A

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

*Female fish in rep # 1 dead. No tube 41.

A Refer to initial fish used in Study 2-55 for information on baseline fish data.

77

-------
Table 31. Results for the Adult FHM Gene Expression test with 2 ELA Lake sediment samples
Conducted 12/16/02. Treatments included MHRW control, MHRW + DMSO, 5 ng/1 EE2
positive control sample and sediment samples from Lakes 239 and 260. Duration was 48H.
Study #2-64.



pH

DO

Conductivity

Temp

Cone

Rep

ii

sex

exp

live

Tube#

I

F

I

F

I

F

I

F

MHRW

1-5

m

5

5

11-15

8.17

7.5
6

8.3

7.4

335

345

24.4

24.4

MHRW

1-5

f

5

4*

41-44

















Cont + DMSO

1-5

m

5

5

16-20

8.12

7.6
8

8.1

7.5

333

350

24.1

24.7

Cont + DMSO

1-5

f

5

5

46-50

















5 ng/1 EE2

1-5

m

5

5

21-25

8.07

7.5
8

8.0

7.0

330

346

24.1

25.2

5 ng/1 EE2

1-5

f

5

5

51-55

















Lake 239
sediment

1-5

m

5

5

31-35

7.23

5.8
6

8.4

6.9

312

238

24.0

24.7

Lake 239
sediment

1-5

f

5

5

61-65

















Lake 260
sediment

1-5

m

5

5

36-40

7.43

6.6
5

7.6

7.0

307

288

24.0

24.9

Lake 260
sediment

1-5

f

5

5

66-70

















Day 0 Initial Fish

1-5

m

5

5

1-5

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Day 0 Initial Fish

1-5

f

5

5

6-10

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

*Female fish in rep # 5 dead.

\Jo tube 45.

78

-------
Table 32. Results from the P. promelas (FHM) embryo/larval test using elutriate samples
prepared from the Canadian ELA Lakes 239 and 260 Sediments. Conducted 12/18/02. Test
used 50 ml of test solution, 40 FHM eggs/replicate, MHRW control, 10 ng/1 EE2 positive control
and 2 ELA samples. Duration was 5 days. Study #2-65.



PH

DO

Conductivity

Temp

Cone.

Rep

#

% Sur

CV

t-test
pvalu

e

Tube#

I

F

I

F

I

F

I

F

MHRW

1-5

97.0

2.2

n/a

1-5

7.74

7.94

8.2

7.7

324

347

25.1

24.3

Cont + DMSO

1-5

95.5

4.7

0.516

6-10

7.89

8.04

8.2

7.4

321

350

25.1

24.4

10 ng/1 EE2

1-5

97.0

2.8

1.000

11-15

7.91

8.00

8.2

7.5

321

342

25.0

24.4

Lake 239

1-5

98.0

2.8

0.548

16-20

7.27

7.64

7.6

7.1

231

240

25.1

24.8

Lake 260

1-5

94.0

4.0

0.160

21-25

6.55

7.48

8.2

7.3

224

238

25.1

24.5

Bold/underline indicates sample determined to have survival statistically different from the
control, t-test, alpha = 0.05.

Table 33. Summary of P. promelas survival data for Canadian ELA Elutriate toxicity tests.
Elutriates were prepared using ELA sediments and MHRW. FHM fry were 36 to 48 hours old at
the start of the test. Test duration was 48-H, with a test solution renewal at 24-H. CB indicates
sediment sample collected at the Center Buoy of the Lake. SS indicates sediment sample
collected from the near shore surface sediment. Study # 3-37.

SITE
ID

Collection
Date

Mean
% Survl

S.D.

c.v. %

t-test

MHRW + DMSO

n/a

99.5

0.0112

1.1

n/a

5 ng/1 EE2

n/a

99.5

0.0112

1.1

1

Lake 114 CB

9/21/03

99.5

0.0112

1.1

1

Lake 260 SS

9/21/03

96.7

0.0271

2.8

0.062

Lake 260 CB*

9/21/03

8.5

0.163

191.8

0.008

* Sample determined to be statistically different from MHRW for survival using t-test, alpha =
0.05.

79

-------
TABLE 34. Initial routine chemistries for P. promelas tests. Study # 3-43.



pH

D.O. (ppm)

Conductivity

( S)

Temp (°C)

sxs

24 hr

48 hr

24 hr

48 hr

24 hr

48 hr

24 hr

48 hr

(-) Cnt.

7.96

8.30

8.7

8.6

332

336

24.1

24.6

(+) Cnt

7.91

8.27

8.5

8.5

332

336

24.1

24.6

S+0 ug/1

7.79

8.18

00
00

8.0

325

320

24.1

26.2

S+l ug/1

7.79

8.18

00
00

8.3

336

329

24.1

25.6

S+50 ug/1

7.80

8.16

00
00

8.0

334

330

24.1

26.2

S+500 ug/1

7.81

8.17

8.7

7.9

336

330

24.1

26.2

239+0 ug/1

5.66

6.19

7.8

8.6

179

175

24.1

25.8

239+1 ug/1

5.57

6.01

7.3

8.6

176

173

24.1

25.8

239+50 ug/1

5.62

5.98

7.1

8.6

175

171

24.1

25.7

239+500 ug/1

5.57

5.95

7.6

8.7

174

170

24.1

25.3

260+0 ug/1

5.37

5.86

7.1

8.1

173

166

24.1

25.2

260+1 ug/1

5.31

5.66

7.0

8.0

173

170

24.1

25.1

260+50 ug/1

5.33

5.67

6.6

7.8

174

170

24.1

25.3

260+500 ug/1

5.27

5.64

6.8

7.7

174

171

24.1

25.3

80

-------
TABLE 35. Final routine chemistries from P. promelas toxicity tests. Study # 3-43.



pH

D.O. (ppm)

Conductivity ( S)

Temp (°C)

sxs

24 hr

48 hr

24 hr

48 hr

24 hr

48 hr

24 hr

48 hr

(-) Cnt.

8.07

8.00

7.7

7.9

337

339

24.4

24.8

(+) Cnt

8.27

7.81

7.7

7.9

336

346

24.8

24.8

S +0 ug/1

8.18

8.12

7.8

7.8

329

336

24.8

24.6

S +1 ug/1

8.13

8.14

7.5

7.8

344

355

24.4

24.6

S +50 ug/1

8.10

8.10

7.7

7.8

333

345

24.7

25.0

S +500 ug/1

8.07

8.10

7.6

7.8

337

346

24.3

24.7

239+0 ug/1

7.29

7.29

7.4

8.2

186

192

24.8

24.9

239+1 ug/1

7.30

7.31

7.7

8.2

188

186

24.9

24.8

239+50 ug/1

7.28

7.32

7.9

8.2

176

188

24.6

24.4

239+500 ug/1

7.32

7.38

7.9

8.2

181

185

24.7

24.7

260+0 ug/1

6.78

6.87

8.0

8.2

175

181

24.7

24.7

260+1 ug/1

6.75

6.81

7.9

8.0

184

182

24.9

25.1

260+50 ug/1

6.79

6.67

7.6

8.0

170

187

24.7

24.9

260+500 ug/1

6.78

6.84

7.9

8.0

179

177

25.0

24.7

81

-------
Table 36. Summary of P. promelas survival data for Canadian ELA Elutriate toxicity tests.
Elutriates were prepared using ELA sediments and MHRW. FHM fry were 24 to 48 hours old at
the start of the test. Test duration was 48-H, with a test solution renewal at 24 hours and a 25°C
test temperature. Study # 3-43.

SITE

Collection

Mean

C T\

c.v.

ID

Date

% Survl

o.ll.

%

MHRW + DMSO

N/A

100.0

0

0

MHRW + 10 ng/1 EE2

N/A

99.0

0.014

1.38

Sand + 0 DMSO

N/A

100.0

0

0

Sand + lug/1 DMSO

N/A

100.0

0

0

Sand + 50 ug/1 DMSO

N/A

100.0

0

0

Sand + 500 ug/1 DMSO

N/A

99.5

0.011

1.12

239 + 0 DMSO

10/30/03

100.0

0

0

239 + 1 ug/1 DMSO

10/30/03

99.0

0.014

1.38

239 + 50 ug/1 DMSO

10/30/03

99.5

0.011

1.12

239 + 500 ug/1 DMSO

10/30/03

100.0

0

0

260 + 0 DMSO

10/30/03

100.0

0

0

260 + 1 ug/1 DMSO

10/30/03

100.0

0

0

260 + 50 ug/1 DMSO A

10/30/03

94.5

0.057

6.03

260 + 500 ug/1 DMSO

10/30/03

100.0

0

0

A Indicates this concentration originally contained no fry. Restarted this
concentration the following day.

82

-------