EFFECTS OF EXPOSURE TO LOW LEVELS OF WATER-BORNE 17β-ESTRADIOL ON NEST HOLDING ABILITY AND SPERM QUALITY IN FATHEAD MINNOWS Heiko L. Schoenfuss Dalma Martinovic Peter W. Sorensen ABSTRACT
This study assessed the effects of exposure to low
disruption in fishes (Purdom et al., 1994; Harries et al.,
levels of estradiol (E2), as they have been found in
1997; Routledge et al., 1998). Numerous studies have
some treated sewage effluents, on nest holding ability
assessed the effects of E2 and associated xenoestrogens
and sperm quality, two measures likely related to
on wild fish (Folmar et al., 1996; Jobling et al., 1998;
reproductive success in fathead minnows (Pimephales
Lee et al., 2000; Folmar et al., 2001), fish that were
promelas), a species with paternal nest care. Male
caged (Purdom et al., 1994; Harries et al., 1996;
fathead minnows were exposed for 21 days to either 50
Harries et al., 1997; Routledge et al., 1998; Rodgers-
ng/L (E2) in 25 µl/L ethanol, an E2 concentration
Gray et al., 2001), and on fish in the laboratory
found in the effluent of some British and Canadian
(Kramer et al., 1998; Panter et al., 1998; Miles-
sewage treatment plants (STP), or to a 25 µl/L ethanol
Richardson et al., 1999; Korte et al., 2000; Bjerselius et
control (aqueous flow-through exposure). After
al., 2001). All of these studies found induction of the
exposure, groups of six males from one treatment were
yolk protein vitellogenin (VTG) in exposed male fish,
placed into an observation aquarium with females and
as well as other effects such as gonadal abnormalities
a limited number of spawning sites and allowed to
(Gimeno et al., 1998; Jobling et al., 1998; Miles-
spawn for four days. Plasma vitellogenin (VTG)
Richardson et al., 1999; Rodgers-Gray et al., 2001),
concentrations, the ability to acquire and defend a nest
changes in behavior (Bayley et al., 1999; Bjerselius et
site, and sperm quality were compared between E2
al., 2001), and sperm production (Gimeno et al., 1998;
exposed and control males. E2 exposure resulted in a
Bjerselius et al., 2001). However, little is known about
significant increase in VTG concentrations in E2
the reproductive effects of exposure to E2 at these low
exposed males, but did not affect nest holding ability or
concentrations. In a previous study (Schoenfusset al.,
sperm quality. These preliminary results indicate little
in press), we found that 50 ng/L E2 exposure resulted
effect of 21-day exposure to levels of E2 that match the
in minor reductions in spawning behavior and sperm
highest reported E2 loads in STP effluent, and parallel
production in male goldfish (Carassius auratus), which
our findings from a previous study using goldfish.
have no parental care. Based on these results, we
However, caution is warranted in extrapolating these
designed a new experiment to determine whether
results to populations of wild fish, as other factors such
exposure to similar E2 concentrations would be more
as the overall estrogenic potency of effluent, the
detrimental to fish with greater paternal involvement in
duration and timing of exposure to estrogenic
reproduction. E2 exposure concentrations were chosen
compounds, and the competition between fish for
to match the highest E2 concentrations reported in
spawning opportunities need to be estimated.
sewage effluent in Great Britain (Desbrow et al., 1998;
Rodgers-Grayet al., 2000) and Canada (Ternes et al.,
1999). In this study we exposed male fathead minnows
(Pimephales promelas), a species with paternal nest
The steroid 17ß-estradiol (E2) is an important hormone
care, to low levels of E2 and assessed the effects of
in endocrine pathways of fishes (Arcand-Hoy &
exposure on the fishes ability to hold nest sites and to
Benson 1998) and occurs frequently in effluent
discharge of municipal sewage treatment plants (STP)
(Desbrow et al., 1998; Ternes et al., 1999; Rodgers-
The fathead minnow is widely used in toxicological
Gray et al., 2000) where it can cause endocrine
studies (Ankley et al., 1998) and reproduces in the
laboratory. Fathead minnows breed year round with
females and allowed to spawn. Male behavior was
the male guarding a cavernous nest site until eggs
observed, after which plasma VTG concentrations, and
hatch. The ability to defend a nest is determined by the
individual’s social status in which dominant males,
identifiable by their tubercles, dorsal pad, and bright
banding pattern, establish successful breeding
territories (Cole & Smith, 1987). Smith (1974)
For each trial, 16 spermiating fathead minnows were
suggested that these morphological characteristics are
netted from a stock aquarium and distributed evenly
associated with elevated androgen levels. The male
among four 20 L aquaria. Aquaria were aerated,
defends the nest site continuously until hatching as
shielded on three sides, and fish were maintained
eggs are quickly eaten by other fathead minnows in the
otherwise as in the stock aquaria. Males were held for
absence of the nest-guarding male (Sargent, 1989).
21 days in either (i) 50 ng/L E2 dissolved in 25 µl/L
Estrogen exposure could increase circulating E2 levels
ethanol, or in (ii) a 25 µl/L ethanol control.
in male fish and suppress androgen levels, by altering
Experiments with guppies (Poecilia reticulata) and
neuroendocrine feedback loops (Trudeau et al., 1993;
goldfish (Bayley et al., 1999; Schoenfuss et al., in
Jobling et al., 1996; Ankley et al., 1998).
press) found no effects of ethanol exposure on
spawning behavior and sperm production. Both
We expect nest holding ability to be a sensitive
treatments were administered via continuous flow-
indicator of reproductive success as the opportunity to
through delivery (280 ml/min). A peristaltic pump
spawn is conditional upon possession of a nest site
continuously supplied two aquaria with an E2 stock
(Unger, 1983; Huntingford & Torricelli, 1993). We
solution, while the other two aquaria received ethanol
also expect sperm production to serve as a sensitive
at concentrations similar to the one used to prepare the
and relevant measure of the effects of estrogen
E2 stock solution. E2 was extracted weekly from E2
exposure as sperm production is important in externally
exposure aquaria and bi-weekly from the control
fertilizing fish (Petersen & Warner, 1998). A reduction
aquaria. Water samples were concentrated using C18
in sperm quantity (which normally increases in
SepPaks (Waters Corporation, MA), and than
association with interactions with ovulating females
measured with an E2 ELISA test kit (Cayman
(Stacey & Sorensen, 1986; Dulka et al., 1987;
Chemicals, MI). Aquarium concentrations of E2 were
DeFraipont & Sorensen, 1993)) or quality can lead to
approximately 50 percent of the 50 ng/L nominal
reduced fertilization rates (Nakatsuru & Kramer, 1982;
concentration at 24 ± 3.6 ng/L E2 (mean ± standard
Zheng et al., 1997; Suquet et al., 1998; Cosson et al.,
error). E2 concentrations in the ethanol control aquaria
2000). By exposing fathead minnows to E2 we
addressed two questions. (1) Is sperm quality in
fathead minnows compromised by E2 exposure? (2)
Are E2 exposed male fathead minnows able to
After 21 days, groups of six male fish from the same
treatment were moved from the exposure aquaria into
160 L observation aquaria (day one). Each observation
MATERIALS & METHODS
aquarium contained six mature female fathead
minnows and three nest sites (3” PVC pipe sections) to
ensure that only nest sites are limiting reproductive
success. In preliminary studies performed with
Fish for this study were bred and reared at the
unexposed males in a similar setting, males routinely
University of Minnesota. Fish were held in 28°C water
occupied all nest sites within 24 hours. Males were
(flow-through, 300 ml/min) from an in-house well, at a
marked using a combination of the same three latex
constant photoperiod (16 h:8 h light:dark, 0800 lights
colors that were injected subcutaneously. A pre -
on), and were fed frozen brine shrimp (San Francisco
experiment found no effects of latex injection on the
Bay Company, CA) twice daily ad libitum.
reproductive ability of male fathead minnows.
Male fathead minnows were observed twice each
morning (days two through five) for five-minute
An experiment was conducted to assess the effects of
intervals. During the observation period the nest
E2 exposure on nest holding ability and sperm quality
holding male in each of the three nest cavities was
in male fathead minnows. In four trials, males were
identified. Each nest defended by a male fathead
exposed for 21 days to either E2 or an ethanol control.
minnow was scored as a “nest holdin g event” for the
After exposure, groups of males were placed with
treatment. Data from all four replicates were pooled
resulting in a total of 48 “nest holding events” per
a Fisher’s Exact test. All calculations were performed
aquarium and treatment (three nests x four observation
with the Prism 2.0 statistical package for the Macintosh
On the fifth day, fish were anesthetized with 0.1%
phenoxyethanol (Sigma, St. Louis, MO), placed
upside-down into a grooved sponge, and then milt
Plasma VTG concentrations in E2 exposed male
(sperm and seminal fluid) was drawn into a capillary
fathead minnows were significantly higher than in
tube by applying light abdominal pressure following
control males (p<0.01, Mann-Whitney U test; Table 1).
established protocols (Stacey & Sorensen, 1986). Care
Despite VTG induction in exposed males, sperm
was taken not to contaminate the sample with feces or
quality did not differ between E2 exposed and control
urine. For storage, 1 µl of milt was transferred into
males and the MPrV was virtually identical (p>0.05,
100 µL of isoosmotic sperm extender following
Mann Whitney U test, Table 1). The ability to hold
published protocols (Chao et al., 1987). For the sperm
nest sites also did not differ between E2 exposed and
analysis, 10 µl of sperm extender solution was
control fish. The number of nest holding events was
suspended in 100 µl distilled water (1:2000 final
similar for E2 exposed and control male fathead
dilution) and vortexed. The drop in osmotic pressure
minnows (p>0.05, Fisher’s Exact test; Table 1). E2
activated the sperm sample (Billard et al., 1995), which
exposed male fathead minnows were holding nest sites
was then analyzed using a Hamilton-Thorn mo tility
during all 48 observations, while control males held
analyzer (Danvers, MA). Mean Progressive Velocity
nests during 44 out of 48 observations. As males were
(MPrV), a measure of sperm velocity along its path
color-coded it was also possible to identify changes in
was used to characterize sperm quality (Kime et al.,
the identity of the nest holder from day to day.
1996). Each sperm sample was analyzed three times
However, very few incidents in which the nest holding
and values were averaged to determine the MPrV for
male changed from one day to the next were observed
each fish. MPrV was calculated only for those fish
Our preliminary results indicate that three-week
After completion of sperm analysis, blood was
exposure to 50 ng/L E2, a concentration exceeding E2
collected from the caudal vasculature, stored on ice,
loads in the effluent of at least two of the few STP
and centrifuged at 10,000 rpm for one minute. Plasma
effluents that have been analyzed for E2 loads,
was transferred into aprotinine coated microcentrifuge
consistently induced VTG production in mature male
tubes and stored at –20°C for later analysis. Plasma
fathead minnows. The reproductive consequences
VTG concentrations were determined using a fathead
associated with this physiological effect appear minor
minnow-specific ELISA following published protocols
under the experimental conditions presented in this
(Parks et al., 1999). For the statistical analysis VTG
study. These results match our earlier findings in
concentrations below detection limit were reported as
goldfish, for which ten week exposure to E2 at similar
concentrations results in VTG induction but had
relatively minor effects on spawning behavior and
sperm production (Schoenfuss et al., in press). Ours is
the first study to evaluate the effects of aqueous
Sperm quality and plasma vitellogenin concentrations
exposure to E2 at concentrations that simulate E2 loads
were analyzed using a nonparametric Mann-Whitney U
in some STP effluents on nest holding ability and
test as some of the data were not normally distributed.
The proportional nest holding data were analyzed using
Plasma vitellogenin (VTG) concentrations, sperm quality, and nest holding ability in male fathead minnows exposed to E2 or a control.
The E2 exposure protocol used in this study is notable
(2001) saw an increase in sperm numbers in guppies
because the absolute exposure concentration in this
exposed to 30 ng/L E2. In this experiment we were
study (24 ng/L) is below the highest E2 loads reported
unable to measure sperm quantity as the small size of
for STP effluent. Although the E2 concentration in this
the fathead minnow renders it impossible to strip
study was higher than effluent E2 concentration
consistently all milt while avoiding contamination by
reported for U.S. STP effluents, some studies have
reported E2 concentrations in STP effluent as high as
88 ng/L in Great Britain (Desbrow et al., 1998,
Although we did not find any impairment of nest
Rodgers-Gray et al., 2000) and 64 ng/L in Canada
holding ability and sperm quality in male fathead
(Ternes et al., 1999). Thus, our exposure protocol
minnows exposed to E2, we remain cautious about
represents a high, but still realistic exposure scenario.
extrapolating these laboratory results to populations of
Furthermore, E2 is but one of many estrogenic
wild fish. Indeed, ongoing studies in our laboratory
compounds that have been found in STP effluent, and
suggest that intra -species interactions might be an
the overall estrogenic potency of the effluent could be
important component in evaluating the effects of E2
much greater than simulated in this experiment.
exposure. Furthermore, STP effluent consists of
complex mixtures of chemicals, many of which have
The plasma VTG concentrations found in E2 exposed
been shown to be estrogenic to fish and their combined
male fathead minnows in this study match those
action may exceed our exposure concentrations. Our
reported by Parks et al., (1999), in fathead minnows
understanding of the effects of STP effluent on fish
exposed to E2 via injection. Despite VTG induction,
populations is still rudimentary and further studies will
the fish appeared healthy and active and in each
be necessary to determine the effects of estrogen and
replicate spawning behavior could be observed within
xenoestrogen exposure on all life stages of fish.
24 h in both treatments. Nest holding ability of E2
Answering these questions will allow us to determine
exposed fathead minnows was also not affected by E2
the effects of effluent exposure on wild fish
exposure, with E2 exposed males holding all nest sites
in all replicates of the experiment. The ability to
defend a nest site is crucial for the reproductive success
of male fathead minnows as eggs in any unguarded
nest are quickly eaten by other fathead minnows
(Sargent, 1989). Our results are similar to those
The authors acknowledge funding from the National
reported in two studies on guppies that did not find
Sea Grant College Program. We thank Gary Ankley
reproductive impairment at 30 ng/L E2 exposure (Toft
and the staff at the Duluth EPA laboratory for help
& Baatrup, 2001), and saw no behavioral impairment
with setting up a fathead minnow breeding colony and
at 100 ng/L E2 exposure (Bayley et al., 1999). In
the VTG assay. We are grateful to Mats Troedsson
contrast, a previous study conducted in our laboratory
(School of Veterinary Medicine, University of
(Schoenfuss et al. in press) found a significant decline
Minnesota) for making the computer assisted motility
in reproductive behavior in male goldfish exposed to
analyzer available to us, and Kate Loseth for providing
50 ng/L E2 for ten weeks. Bjerselius et al., (2001),
also found behavioral impairment at E2 exposure
concentrations exceeding 1 µg/L E2, but that E2
exposure level is far above any recorded for STP
Heiko L. Schoenfuss, Dalma Martinovic, and Peter
MPrV, a measure of sperm quality did not vary
W. Sorensen, Department of Fisheries, Wildlife, and
between E2 exposed and control fish. Sperm quality
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