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Environmental Toxicology and Chemistry, Vol. 17, No. 1, pp. 49–57, 1998
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Annual Review
FISH REPRODUCTION: AN ECOLOGICALLY RELEVANT
INDICATOR OF ENDOCRINE DISRUPTION

LISA D. ARCAND-HOY and WILLIAM H. BENSON*

Environmental Toxicology Research Program, Environmental and Community Health Research, School of Pharmacy,
The University of Mississippi, University, Mississippi 38677, USA

(Received 17 April 1997; Accepted 21 July 1997)

Abstract—Endocrine-disrupting compounds have the potential to alter hormone pathways that regulate reproductive processes.
With the exception of endocrine effects leading to reproductive impairment and population declines in a few wildlife species (e.g.,
Great Lake [USA] bald eagles, Lake Apopka [Florida, USA] American alligators), the ecological implications of exposure to
endocrine disrupting compounds has not been adequately investigated. For example, male fish exposed to estrogenic compounds
show induced production of vitellogenin, an egg yolk precursor, but the biological significance of elevated vitellogenin levels is
speculative. The development of techniques to predict and more accurately assess the ecological relevance of exposure to endocrine-
disrupting compounds is needed. In this review, we focus on fish reproduction as an ecologically relevant indicator of endocrine
disruption, specifically estrogenic activity. The following will provide a brief review of gonochoristic reproductive and endocrine
physiology, as well as outline some of the commonly used techniques to screen for estrogenic activity in fish. Last, a proposed
model reproductive assay using Japanese medaka (Oryzias latipes) is presented.

Keywords—Endocrine disruption Estrogenic activity Reproductive toxicity Japanese medaka Ecological relevance

INTRODUCTION and a decline in Lake Apopka (Florida, USA) American al-

Concern for the successful development and reproduction
of human and wildlife populations has been heightened by the
published literature focused on endocrine-disrupting com-
pounds. Broadly defined, endocrine-disrupting compounds are
natural or man-made agents present in the environment that
interfere in some way with normal endocrine function. This
diverse group of chemicals can include environmental estro-
gens or exoestrogens, substances that elicit an estrogenic re-
sponse by mimicking the action of endogenous estradiol. A
number of industrial, municipal, agricultural, and natural com-
pounds have been shown or are suspected to be estrogenic [1–
5]. Such compounds include the biodegradation products of
alkylphenol polyethoxylates, polychlorinated biphenyls, and
select pesticides (dichlorodiphenyltrichloroethane [DDT],
chlordecone, methoxychlor), as well as natural and synthetic
estrogens (17b-estradiol, ethinyl estradiol).
Endocrine-disrupting compounds have the potential to per-
turb sensitive hormone pathways that regulate reproductive
functions. In fish, this may result in decreased fertility and egg
production in females, or lead to reduced gonad size or fem-
inization of genetic male fish. It is known that male fish ex-
posed to estrogenic compounds show induced production of
vitellogenin, but the organismal significance of elevated vi-
tellogenin levels has only been, for the most part, speculative.
Considerable discussion and debate also has occurred con-
cerning the concentration of endocrine-disrupting compounds
and whether they are sufficiently elevated to exert whole an-
imal effects. With the exception of a few well-documented
findings such as developmental effects in wildlife associated
with exposure to polyhalogenated aromatic compounds [6,7]
* To whom correspondence may be addressed (whbenson@
olemiss.edu).
ligators coincident with a chemical spill [8,9], the ecological
implications of organismal exposure to endocrine-disrupting
compounds have not been established.
Developmental and reproductive toxicity may occur during
larval development or the juvenile or adult stage depending
upon the species examined. Exposure at an early life stage
may lead to alterations in key developmental processes (e.g.,
sexual differentiation), as well as increased susceptibility to
chemical insult as adults. Exposure at maturity could also dis-
rupt normal reproductive parameters. A number of ways exist
in which compounds can interfere with endocrine function. To
date, most studies have examined estrogen receptor-mediated
responses, with less attention given to changes as a result of
interference with hypothalamic–pituitary function. Thyroid
hormones are also under hypothalamic–pituitary control and
may play a significant role in reproductive processes. Thyroid
hormones in fish are often elevated during gonadal develop-
ment and latter stage reproductive processes (e.g., gameto-
genesis, ovulation, spermiation). Communication systems also
exist between the endocrine and immune systems. Exposure
to endocrine disrupting compounds has been hypothesized to
result in immunosuppression or altered immune cellular func-
tion, which in turn could lead to adverse reproductive effects.
Other steroid hormones that regulate reproductive processes
include androgens and progestins. These hormones are essen-
tial to reproductive processes in male and female fish, but a
thorough discussion of their role in fish reproduction is beyond
the scope of this review. Also not included in this review are
the many nonendocrine-mediated effects (e.g., oxidative stress,
bioenergetics) that could play a role in reproduction.
Many of the mechanisms leading to disruption of endocrine
function in fish are unknown. Presently, development of tech-

49

50 Environ. Toxicol. Chem. 17, 1998
niques to assess and predict the organismal cost of exposure
to endocrine disrupting compounds is needed. For example,
examination of reproductive effects associated with exposure
to endocrine disrupters would provide information on the
whole organism that could be used to more accurately predict
ecological effects. Investigations that focus on a relationship
between exposure and population-level effects (e.g., repro-
ductive success) will provide relevant information to further
understand the importance and relevance of biological indi-
cators of exposure to endocrine-disrupting compounds. Fur-
thermore, development is needed of approaches specifically
designed to examine organismal exposure during critical de-
velopmental windows controlled by specific hormonal axes
and to identify and quantify the associated adverse reproduc-
tive effects. Although it is recognized that it will be difficult
to relate indicators of exposure to detrimental changes in pop-
ulation dynamics, the development of such studies and bio-
assays are needed to provide relevant data regarding the eco-
logical significance of exposure to endocrine-disrupting com-
pounds. Thus, developed techniques that aid establishment of
the ecological significance of endocrine disruption could be
applied to ecological risk assessments.
The goal of this review is to present the state-of-the-science
of endocrine disruption as it pertains to fish reproduction with
focus on assessing the ecological ramifications of the issue.
Because reproductive physiology and strategies vary greatly
among fishes, the following provides a brief review of gon-
ochoristic reproductive and endocrine physiology, as well as
outlines some of the currently available screening techniques
and methods used to assess developmental and reproductive
toxicity in teleosts. Last, a proposed model reproductive assay
using Japanese medaka (Oryzias latipes) is presented.
OVERVIEW OF REPRODUCTIVE
ENDOCRINOLOGY IN FISH
Hypothalamic–pituitary–gonadal axis
The hypothalamus controls the synthesis and release of
hormones and chemical messengers resulting from neural stim-
ulation from the central nervous system (Fig. 1). Messenger
substances that are synthesized in the hypothalamic nerve cells
control the synthesis and release of pituitary hormones. Pi-
tuitary hormones include adrenocorticotropin, prolactin,
growth hormone, thyroid-stimulating hormone, and gonado-
tropin (GtH). Gonadotropin is the hormone principally in-
volved in reproductive processes. Gonadotropin-releasing hor-
mone is released from the hypothalamus and stimulates the
release of gonadotropins from the pituitary. Two forms of
gonadotropin have been isolated in fish [10,11]: GtH I and
GtH II, which are analogous to mammalian follicle-stimulating
hormone (FSH) and luteinizing hormone (LH), respectively
[12]. Gonadotropin I is involved in gametogenesis and ste-
roidogenesis, whereas GtH II is involved in the final maturation
stages of gametogenesis. The gonadotropins are responsible
for stimulating the synthesis of sex steroids (i.e., androgens,
estrogens, and progestins), which in turn act on the target
tissues to regulate gametogenesis, reproduction, sexual phe-
notype, and behavioral characteristics. This hypothalamus–pi-
tuitary–gonadal axis is ultimately controlled by feedback sys-
tems. For example, estrogen produced by the ovary can have
a positive or negative effect on the hypothalamus, pituitary,
or the gonad itself, depending upon the concentration of hor-
mone currently needed to meet the physiologic and reproduc-
tive needs of the fish. Alterations in steroid production could
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L.D. Arcand-Hoy and W.H. Benson
Fig. 1. Interrelationship of immune–neuro–endocrine system axes.
Gonadotropic hormones, thyroid hormones, and adrenocorticotropic
hormones (ACTH) are under the control of the hypothalamus and
pituitary. The gonadotropins (GtH I, GtH II) both have an effect on
the synthesis of sex steroids (E2), which in turn regulate reproduction,
phenotype, and behavior. The conversion of T4 to T3 may be inhibited
by elevated plasma E2 and cortisol (bold lines). Thyroxine may en-
hance the effect of gonadotropin-mediated events such as oocyte de-
velopment and maturation (bold lines). TSH, thyroid-stimulating hor-
mone; GtH, gonadotropin; GtH I, gonadotropin I; GtH II, gonadotro-
pin II; ATCH, adrenocorticosterone; E2, estradiol; T4, thyroxine; T3,
triiodiothyronine.
ultimately affect feedback pathways, or vice versa, which
could lead to an impairment in reproductive processes.
In the majority of fish studied, the development of the oo-
genesis is controlled by GtH I. Circulating GtH I increases
during early oocyte development and binds to receptors on the
thecal and granulosa layer of the follicle. The thecal cells
synthesize testosterone, and allow aromatization to result in
the formation of estradiol in the granulosa layer prior to plasma
secretion [12,13]. Subsequently, plasma estradiol will bind to
estrogen receptors and trigger a series of steps resulting in the
production of vitellogenin, a precursor to egg yolk protein
produced in the liver. Vitellogenin is released from the liver
into the blood and binds to receptors on the oocyte, which
incorporate the protein. As the development of the oocyte
continues, levels of GtH I begin to decrease and are replaced
by increasing levels of GtH II. Receptors for GtH II are found
predominantly on the granulosa cells and binding stimulates
the synthesis and release of progestins [12]. Progestins play a
role in gamete maturation and stimulate ovulation.
In male fish, GtH I is typically elevated throughout sper-
matogenesis and decreases at time of spawning, whereas GtH
II is typically low throughout the growth process and is ele-
vated at spawning [14]. The gonadotropins stimulate prolif-
eration of spermagonia as well as the synthesis of androgens
required for gametogenesis and development of secondary sex-
ual characteristics. Androgen synthesis typically takes place
in the Leydig cells. The type of androgen synthesized is de-
pendent upon the species and developmental stage but may
include testosterone, 11-ketotestosterone, and/or androstene-
dione [12]. A decline in androgen levels and a sharp increase
in progestins during spawning is primarily attributed to ele-
vated levels of GtH II. A high level of progestins with a decline
in androgen level are needed for spermiation. The reader is

Reproduction: A relevant indicator of endocrine disruption
referred to review articles by Nagahama [14], Nagahama et
al. [15], and Jobling [16].
In most fish, sex determination is under genetic control:
sex is determined by sex chromosomes after fertilization. In
some fish, autosomes (pairs of chromosomes with sex-modi-
fying genes) determine the sex, but this is less common [16].
Sexual differentiation in fish is believed to be similar to mam-
malian systems whereby the presence or absence of a testis-
determining factor directs male or female differentiation [16].
After ovarian or testicular development, secondary sex char-
acteristics are expressed by means of hormonal control. How-
ever, it has long been known in aquaculture that exposure to
estrogens or androgens at select developmental stages can lead
to the development of phenotypically male or female popu-
lations. For example, genetic males will possess female gonads
and secondary sex characteristics and genetic females will
resemble and act as male fish. The creation of phenotypically
reversed sexes suggests a potential for same genetic sex mat-
ings. That is, a genetic male fish could mate with a pheno-
typically altered genetic male (phenotypic female), thereby
producing an all genetically male population. The same holds
true for genetic female–phenotypically altered genetic female
matings. This type of same sex mating leading to the produc-
tion of monosex tilapia populations has been exploited for
aquaculture purposes [17]. The question remains as to whether
a similar scenario can exist in wild fish populations, and if so
what are the potential ecological implications. Although it is
realized that in some fish exposure to steroids at a critical life
stage can reverse the sex from its genetic predisposition, re-
productive success of morphologically altered species has not
been adequately investigated [18]. Examples of sex reversal
and intersex conditions with exposure to estrogenic com-
pounds are presented below.
As previously stated, reproductive endocrinology is gov-
erned by a complex set of intricate pathways that require com-
munication and interaction along system axes. Therefore, it is
not surprising that hormones and other physiologic systems
not typically associated with reproduction may play a role.
For example, study thyroid hormones and immunosuppression
are two areas of research that have received a great deal of
attention with regard to fish health. As researchers begin to
investigate the organismal cost of endocrine disruption, an
understanding of other mechanisms that could play a role in
regulation of reproduction is of great interest.
Thyroid hormones. Similar to the steroid hormones, thyroid
hormones are under control of the hypothalamus and pituitary.
Thyroid-stimulating hormone acts on the thyroid gland and
signals the synthesis and release of thyroid hormone. To date,
thyroid hormone in fish has been characterized as thyroxine
(T4), which is metabolized to triiodothyronine (T3) by means
of enzymatic deiodination by iodothyronine 59-monodeiodi-
nase, type 1 (59-ID1). Triiodothyronine appears to be more
biologically active, having a greater affinity for receptor sites,
than T4 [13]. The mechanistic action of T3 in fish is largely
unexplored, but mammalian studies suggest that T3 binds with
nuclear receptors, creating a T3–receptor complex that in turn
binds to a thyroid response element and initiates DNA tran-
scription [13]. A major role of thyroid hormone in fish is
regulation of growth and development. However, in a large
majority of teleost species studied an association exists be-
tween the thyroid hormones and reproduction. In most fish
studied, thyroid activity increased during early gonadal de-
velopment, remained elevated during the reproductive cycle,
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Environ. Toxicol. Chem. 17, 1998 51
and decreased after spawning [13]. This relationship does not
directly indicate that thyroid hormones influence reproductive
function, but does suggest that there is a potential for such
interactions.
A number of studies have suggested that thyroid hormones
may play a role in reproductive processes (Fig. 1). More spe-
cifically, thyroid hormones are believed to play a role in oocyte
growth and maturation. For example, Cyr and Eales [13] have
demonstrated that T3 acts synergistically with gonadotropin
to stimulate steroidogenesis in some female fish. The inves-
tigators reported that T3 enhanced estradiol secretion during
vitellogenesis, a gonadotropin-mediated process. Similar find-
ings were reported by Sullivan et al. [19] who showed that T3
enhanced gonadotropin-stimulated ovulation in rainbow trout.
In addition, receptors for T3 have been found in ovarian tissue
of some teleost species [20]. Sullivan et al. [19] suggested that
thyroid hormones could influence the pituitary or ovary to
enhance secretion of gonadotropins or gonadotropin-releasing
hormone. Furthermore, Cyr and Eales [13] suggested structural
similarities between thyroid-stimulating hormone and gonad-
otropins. A role for T3 in male fish has also been reported
whereby the inhibition of thyroid hormone resulted in an in-
hibition of testicular growth in developing male fish [13].
Many studies pertaining to thyroid hormones have been
directed to obtaining a greater understanding of mechanistic
actions. By comparison, relatively little research has been in-
vested in examining the effect of thyroid hormones as a result
of xenobiotic exposure. Ruby et al. [21] examined plasma
estradiol, T3, and T4 in rainbow trout following exposure to
cyanide. Results showed that plasma estradiol and T3 levels
were lower in cyanide-treated fish. No difference was reported
in plasma T4 levels. The investigators suggested that although
cyanide inhibited the conversion from T4 to T3, the lowered
estradiol levels may have been a result of chemical interaction
along the hypothalamic–pituitary–ovarian axis. Cyr and Eales
[13] also reported on the influence of estradiol on T4 and T3
levels. The investigators suggested that estradiol may depress
the clearance of T4, as well as the conversion of T4 to T3
[13]. A study by Zhou et al. [22] suggested that in rats me-
thoxychlor may disrupt the T4 to T3 conversion by binding
to 59-ID1. Thyroid hyperplasia in salmon and herring gull
populations have also been reported in select regions of the
Great Lakes (USA) [23]. The goiter condition was suggested
to possibly be a result of the inability of the organism to
produce T3.
The thyroidal status of fish can be measured using a number
of techniques. Perhaps the most reliable and useful assays
include the measure of T3 and T4, metabolism of thyroid
hormone deiodinase activity, and thyroid receptor levels [13].
Continued examination of the above parameters in relation to
reproductive function in fish exposed to compounds that in-
terfere with thyroidal processes (perhaps estrogenic com-
pounds) in the laboratory will ultimately provide valuable in-
formation concerning the relationship between reproduction
and thyroid hormones.
Immunomodulators. A number of factors can influence the
immune response in fish, including species, age, reproductive
or social status, stress, diet, and water temperature [24]. Chem-
icals present in the environment may also act as immuno-
modulators and suppress or stimulate the immune response in
fish. The literature has shown that wild fish exposed to pol-
lutants are often more susceptible to disease. Winter flounder
inhabiting waters near a pulp and paper mill showed a lowered

52 Environ. Toxicol. Chem. 17, 1998
Table 1. Select estrogenic and anti-estrogenic compounds in the environmenta
Compound Concn.
Estrone 1.4 6 0.15 to 76 6 10.3 ng/L
Estradiol 2.7 6 0.1 to 48 6 6.0 ng/L
Estradiol/estrone 48.0–54.0 ng/L
19.0 ng/L
Ethinyl estradiol nd to 7.0 6 3.7 ng/L
Nonylphenol 0.15–2.80 mg/L
Octaphenol 0.04–0.28 mg/L
Phthalates 3.20 mg/L
p,p9-DDE 5.21 mg/g
5.80 mg/g
TCDD (TEQ) 29.30 mg/g
Total PCBs 14.18 mg/g
a DDE 5 dichlorodiphenyldichloroethylene; TCDD 5 tetrachlorodibenzo-p-dioxin; TEQ 5 toxic equivalent; PCBs 5 polychlorinated biphenyls.
b Range of concentrations from seven sewage treatment works.
c Range of concentrations from four sewage treatment works.
d Measure of dissolved compound only.
e From United States textile mills.
f Herring gulls collected from Hamilton Harbour, Ontario, Canada.
g Alligator eggs from Lake Apopka, FL, USA.
number of lymphocytes and reduced blood hemoglobin, as well
as a high incidence of parasitic infections and external lesions
when compared to reference site fish [25]. Arkoosh et al.
[26,27] demonstrated that fish collected from a polychlorinated
biphenyl (PCB)-contaminated region exhibited a lowered sec-
ondary immune response, indicating that chemical contami-
nants interfered with B-lymphocytes. In addition, laboratory
fish exposed to Aroclor 1254 had an increased susceptibility
to bacterial and/or viral infections, such as fin rot disease [28].
Immunomodulators can also affect other physiological sys-
tems, but for the most part, mechanistic pathways of action
are unknown. Recently, exposure to endocrine disrupting com-
pounds was suggested to possibly lead to immunosuppression
or altered function or production of key immunologic cells,
which may lead to developmental and reproductive effects
[29]. Although the immune system and its relation to repro-
ductive function has received considerable attention, the effect
of endocrine disrupters on immune function has been largely
unexplored [30].
Chronic stress is mediated by means of the nervous and
endocrine system and often leads to increased disease suscep-
tibility. In particular, cortisol, the major stress hormone under
hypothalamic control, has a direct effect on immune and dis-
ease resistance [31]. Cortisol is believed to be responsible for
mediating immune suppression in chronically stressed fish,
which may ultimately lead to a decline in fish growth and
reproductive activities. Coho salmon [32] and channel catfish
[33] administered cortisol demonstrated an increased suscep-
tibility to disease. In fish, cortisol has been shown to have an
effect on leukocyte distribution and growth [31]. Investigators
have also examined the thyroid status of fish under elevated
levels of cortisol. Vijayan and Leatherland [34] demonstrated
that elevated cortisol levels in coho salmon resulted in lowered
plasma T3 levels. This scenario of immuno–thyroidal inter-
action is illustrated in Figure 1. The apparent association of
immune system responses with thyroid hormone metabolism
demonstrates the importance for studying the interaction of
endocrine-related systems.
Complexity of endocrine regulation—system interrela-
tionships. The endocrine regulation of reproduction is com-
plex, and may involve a number of intricate pathways and
hormones other than the sex steroids. Figure 1 illustrates the
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L.D. Arcand-Hoy and W.H. Benson
Medium and reference
Sewage effluent [2]b
Sewage effluent [2]b
Chicken manure [37]
Surface water runoff [37]
Sewage effluent [2]b
Sewage effluent [2]cd
Sewage effluent [2]cd
Textile mill effluent [50]e
Herring gull eggs [51]f
Alligator eggs [52]g
Herring gull eggs [51]f
Herring gull eggs [51]f
role of the hypothalamus, pituitary, and gonadal steroids (es-
trogen) in reproductive processes, as well as the potential in-
terrelationships between thyroid hormones and immune system
functions. A large database of scientific research addressing
reproductive processes has been compiled that focuses on the
pituitary hormones and synthesis of gonadal sex steroids. The
mechanistic role of thyroidal and immunologic processes in
reproductive output is largely unknown and presents a signif-
icant area of research in which to contribute to our current
understanding of the endocrine disruption paradigm. When
examining other systems, such as thyroid and immunologic
functions, it will be important that mechanistic studies be con-
structed in an attempt to link molecular and biochemical end-
points to higher order biological responses.
ASSESSMENT OF ESTROGENIC ACTIVITY
A number of chemicals in the environment have been
shown, or are suspected to be, estrogenic. Such compounds
include polyhalogenated aromatic compounds, biodegradation
products of alkylphenol polyethoxylates, select pesticides, as
well as natural and synthetic estrogens (Table 1). The question
often raised is whether endocrine-disrupting compounds are
present in the environment in sufficiently high enough con-
centrations to elicit adverse reproductive effects. This question
is derived, in part, from findings demonstrating that a number
of compounds with endocrine-disrupting properties have sig-
nificantly lower potencies than 17b-estradiol. Arnold et al. [35]
reported that environmental endocrine disrupters can be 1/50
to 1/10,000 times less potent than estradiol. In addition, the
ecological relevance of exposure to these compounds is largely
unknown. In contrast to the low-potency environmental estro-
gens, natural and synthetic estrogens possess a high affinity
for the estrogen receptor, but have been somewhat ignored as
a contaminant of concern because of perceived low environ-
mental concentrations. Recently, investigators in the United
Kingdom identified estradiol and estrone, as well as the syn-
thetic estrogen used in oral contraceptive formulations, ethinyl
estradiol, as the active estrogenic component of sewage efflu-
ent [2]. An additional source of synthetic and natural estrogens
to the aquatic environment may be those associated with an-
imal waste [36]. Shore and Hall [37] reported concentrations
of estradiol/estrone in agricultural laden areas of the Chesa-

Reproduction: A relevant indicator of endocrine disruption
peake Bay watershed. The persistent and bioaccumulative
properties of the lower potency endocrine disrupters, coupled
with their widespread use, certainly warrants their continued
investigation. In addition, continued attention should be di-
rected towards the examination of potent natural and synthetic
estrogens in aquatic systems. Natural and synthetic estrogens,
specifically ethinyl estradiol, have been widely used in aqua-
culture to create monosex populations. To date, the reproduc-
tive capabilities of phenotypically altered species have not
been adequately evaluated.
Despite the lack of ecologically relevant data regarding
organismal exposure to endocrine-disrupting compounds,
studies exist that demonstrate a strong causal relationship be-
tween organismal exposure to endocrine-disrupting chemicals,
reproductive impairment, and subsequent population level ef-
fects. Persistent and bioacummulative chemical compounds,
some of which have been banned from production in the United
States (e.g., PCBs and DDT), have been associated with de-
velopmental and reproductive effects in some wildlife species.
A number of studies have demonstrate that fish-eating birds
of the Great Lakes have been adversely affected by exposure
to organochlorine compounds [38]. For example, bald eagles
inhabiting contaminated regions of the Great Lakes had high
concentrations of dichlorodiphenyldichloroethylene (DDE)
and PCBs in eggs and plasma that were linked to eggshell
thinning and population declines [39]. Decreased fertility, de-
creased hatchability, reduced numbers of viable offspring, al-
tered sexual behaviors, and altered hormone levels have been
reported in these and other fish-eating birds. Along with DDE
and PCBs, dieldrin was also detected in the eggs of bald eagles.
Interestingly enough, the investigators concluded that the sub-
lethal levels of dieldrin were not likely to result in bald eagle
population-level effects. This is significant, as environmental
exposure of fish to endocrine-disrupting chemicals is primarily
associated with sublethal levels. However, it has been sug-
gested that dieldrin concentrations in the eggs may play a role
in terms of influencing adult survival [40].
Perhaps one of the most complete assessments of the effects
of endocrine-disrupting chemicals in wildlife species is that
of the American alligators inhabiting contaminated Lake
Apopka Guillette et al. [8,9] reported a decline in juvenile
American alligators in Lake Apopka coincident with a 1980
chemical spill, of most notably, DDE and its metabolites. Sub-
sequently, populations of juvenile alligators declined by 90%
in the 4 years after the spill, and have not yet fully recovered.
Alligators from this lake showed abnormal gonadal develop-
ment and altered plasma sex hormone concentrations [8]. For
example, the young alligators collected from Lake Apopka
had abnormal ovarian development and poorly differentiated
testes [8]. Of these alligators, females had higher levels of
estradiol and males had reduced testosterone concentrations
when compared to alligators collected from a reference site.
The research conducted on Lake Apopka strongly supports the
hypothesis that endocrine disruption has lead to population-
level effects in Lake Apopka American alligators.
Endocrine disruption in wild fish has been demonstrated in
a population of white suckers exposed to bleached kraft mill
effluent (BKME) along the north shore of Lake Superior, Can-
ada [41,42]. The exposed fish showed abnormal gonadal de-
velopment, delayed age to maturity, and altered steroid levels
[42]. Van Der Kraak et al. [43] also reported reduced secretion
of gonadotropin and depressed ovarian steroid synthesis in
these fish. Female white suckers had a smaller number of eggs
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Environ. Toxicol. Chem. 17, 1998 53
at maturity and males had a lower expression of secondary
sex characteristics [42]. Gonads of both male and female fish
were smaller at the BKME site as well. Although females
exposed to BKME had fewer and smaller eggs, no difference
in fecundity was found when compared to reference site white
suckers. McMaster et al. [42] reported no effect on hatch-
ability, size of larvae, or larval survival. To date, the population
dynamics of this white sucker population have not been in-
vestigated.
Estrogenic screening
The adverse reproductive effects found in fish and wildlife
species and the emergence of newly found chemicals with
estrogenic activity has resulted in the revision of environ-
mental statutes. For example, the Safe Drinking Water Estro-
genic Substances Screening Program Act was introduced in
the United States House of Representatives in 1996 in order
to determine whether humans may be exposed to and affected
by compounds with endocrine-disrupting activity. In addition,
the Food Quality Protection Act of 1996 was signed into law
and mandated the development of a program to screen chem-
icals for endocrine-disrupting effects. Under this law, the U.S.
Environmental Protection Agency (U.S. EPA) was mandated
to develop a comprehensive endocrine disrupter screening and
testing program.
Assays commonly used to screen compounds for estrogenic
activity and thus potential endocrine-disrupting effects typi-
cally include radio ligand receptor binding assays, cell culture
techniques, and measurement of estrogen-mediated protein
production. These assays are based upon estrogen receptor-
mediated mechanisms and are designed to test whether a chem-
ical is estrogenic and in some instances measure the potency
of the compound. The above assays were not designed to assess
reproductive effects, but rather to screen for compounds that
have the potential to elicit reproductive toxicity. A rapid and
efficient screening technique is warranted as there are a large
number of compounds to test in single chemical and multiple
chemical exposures. Compounds that show estrogenic activity
may then be ranked according to potency and subsequently
tested for their ability to elicit reproductive toxicity.
Receptor binding assays are used to measure the binding
affinity (potency) of environmental estrogens to the estrogen
receptor. This assay has been performed using estrogen re-
ceptors isolated from uterine and liver tissue of rodents and
fish, respectively, as well as in yeast cell lines transfected with
a human estrogen receptor. More recently, Nimrod and Benson
[44] have used estrogen receptor–radio ligand assays to dem-
onstrate up- or down-regulation of estrogen receptors in the
liver of fish exposed in vivo to estradiol and select estrogenic
compounds. Although it is this type of assay is unlikely to be
utilized as a screening tool, receptor number is important as
alteration in receptor regulation may sensitize or desensitize
the organismal response to estrogenic compounds.
Future screening and testing programs probably will en-
compass a battery of both in vitro and in vivo techniques. The
use of a suite of assays is required to demonstrate that a mea-
sure of potential estrogenic activity is linked to a biological
response. For example, just because a compound is able to
bind to the estrogen receptor does not necessarily suggest that
it can stimulate estrogenic activity. The production of vitel-
logenin in fish has been used as an in vivo indicator of exposure
to estrogenic compounds in field and in laboratory studies.
More recently, fish primary hepatocyte cultures have been used

54 Environ. Toxicol. Chem. 17, 1998
Fig. 2. Model to assess reproductive impairment in Japanese medaka (Oryzias latipes) following exposure at the larval stage (A) with possible
reexposure at maturity (B). A subset of exposed fish may be sacrificed for examination of biological indicators (e.g., vitellogenin, steroid hormone
production, receptor number). The experiment may be terminated at this stage with gonadal histologic analysis, or larvae of the F0 generation
maybe grown to maturity and assessment continued through the F1 generation (C), or limited to an adult exposure (B).
to measure vitellogenin induction after dosing with suspected
environmental estrogens [45]. The primary hepatocyte assay
provides a quick in vitro technique that may be used to screen
a larger number of compounds. This assay may be used in
conjunction with the receptor binding assay to determine if
binding to the receptor elicits an estrogenic response. Previ-
ously, the proliferation of the MCF-7 human breast cancer cell
line was used as a screening tool for estrogenic compounds
[5].
Assessment of plasma hormones has been used to indicate
endocrine disruption, but many of these technique are often
less specific with respect to their mode of action and are more
likely to be used in assessing the reproductive effects of en-
docrine disruption. For example, plasma hormone levels (e.g.,
gonadotropin, sex steroid hormones, thyroid hormones) can
be measured in treated fish and compared to levels in control
fish. Investigators have also been able to stimulate or inhibit
key processes in the brain–gonadal axis to learn where en-
docrine disruption may be occurring. Alterations to hormones
and system processes could provide key information with re-
gards to reproductive impairment. These types of data provide
evidence of endocrine alterations that may be related to re-
productive parameters such as fecundity, and thus may play a
role in providing data that can be used to more accurately
predict the ecological relevance of endocrine-disrupting com-
pounds.
REPRODUCTIVE ASSESSMENTS
Ecological Effects Test Guidelines have been developed by
the U.S. EPA’s Office of Prevention, Pesticides and Toxic Sub-
stances to examine the potential adverse effects of pesticides
and toxic substances. Specific guidelines were designed to
meet the testing mandates set forth by the Federal Insecticide,
Fungicide and Rodenticide Act and the Toxic Substances Con-
trol Act. Such guidelines to assess whole organism responses
15528618, 1998, 1, Downloaded from https://setac.onlinelibrary.wiley.com/doi/10.1002/etc.5620170108 by Statens Beredning, Wiley Online Library on [21/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
L.D. Arcand-Hoy and W.H. Benson
include the fish life cycle toxicity test as well as the fish early
life stage toxicity test [46,47]. The life cycle toxicity test re-
quires full life cycle exposure, followed by measurements of
reproductive endpoints such as time of spawning, egg number,
fertility, and fecundity, as well as locomotion, behavioral,
physiologic, and pathologic effects [46]. Although, full life
cycle exposures are often critical to simulate environmental
exposures, the methodologies are time and labor intensive.
The fish early life stage toxicity test involves exposure at
time of fertilization through hatch and first feeding [47]. In
this short-term assay, many of the reproductive parameters
typically measured in adults are exchanged for successful
hatch, survival, growth, and developmental and behavioral ab-
normalities. To examination of early life stage endpoints is
important, but it is also of utmost importance to examine what
effect sublethal exposures at an early life stage may have on
adults. This type of test method leaves room for multigener-
ational studies as larval fish can be grown to maturity with
subsequent monitoring of the reproductive success of the sec-
ond generation. This may be done in a more cost- and time-
efficient manner that does not require full life cycle exposure,
but rather exposure at critical windows of developmental and
reproductive processes.
Reproductive model using Japanese medaka
The Japanese medaka is an excellent model organism that
can be induced to breed daily in response to a prescribed
photoperiod, temperature, and food regime. This small aquar-
ium species requires relatively little husbandry and is fully
mature within 6 to 8 weeks of hatch. The rapid time to mat-
uration makes this species an attractive model organism for
studying reproductive and development toxicity in F0 as well
as subsequent generations. Figure 2 outlines a proposed model
to measure the developmental and reproductive toxicity of
endocrine-disrupting chemicals in Japanese medaka. By ex-

Reproduction: A relevant indicator of endocrine disruption
amining the effects of exposure at early life and/or adult stage,
the reproductive model combines the effort of the U.S. EPA’s
life cycle toxicity test and the early life stage toxicity test.
Investigative pathways and key exposure stages are referred
to in Figure 2.
The proposed medaka reproduction assay is based upon
exposure during key developmental events and is sufficiently
flexible in design to evaluate developmental and reproductive
toxicity in larval and/or adult fish. Larval medaka may be
exposed during a critical window of time when developmental
processes are most susceptible to chemical insult (Fig. 2A).
Unpublished data from our laboratory indicate that 6 to 10
days posthatch represents such a critical period. During ex-
posure larval fish are monitored daily for mortality and feeding
behavior. Upon termination of exposure, larval fish are trans-
ferred to aquaria containing dilution water only and grown to
maturity.
The next phase of the medaka reproduction model may
involve a second exposure of the adult fish to the same chem-
ical compound utilized during the initial larval exposure (Fig.
2B). Exposure at the adult stage is important because it eval-
uates the effect of secondary chemical insult on reproductive
processes. In addition to assessing reproductive output by mea-
suring time of first spawn, fecundity, clutch size, and egg
hatchability, a subset of fish can be sacrificed daily and vi-
tellogenin as well as other indicators of chemical exposure
may be measured. Therefore, at the end of chemical exposure
and reproductive monitoring, information on adult fecundity
can be related to a biological indicator of exposure (Fig. 2).
The Japanese medaka reproductive model permits sufficient
flexibility to examine other biological indicators (e.g., steroid
levels, receptor number), which will permit association with
reproductive endpoints (Fig. 2).
Linking biological indicators to reproductive assessment
endpoints is considered critical to a fundamental understanding
of the biological significance of a number of reported endo-
crine-associated events. Induced production of vitellogenin in
male fish is well known to be a biological indicator for ex-
posure to estrogenic compounds; however, few investigations
have attempted to examine the significance of such production.
For example, Jobling et al. [4] demonstrated that elevated vi-
tellogenin levels in developing male fish were associated with
decreased testicular growth. The decrease in testicular growth
was only evident in developing male fish; testicular growth
was not affected in mature or regressed fish. The investigators
also reported that the estrogenic potency of the chemical was
directly related to the inhibition of testicular growth in de-
veloping male fish. Likewise, the medaka reproduction model
provides a means to measure an estrogenic response, which
can then be related back to developmental and reproductive
assessments.
As outlined in Figure 2, following adult exposure and mea-
surement of reproductive parameters, a number of options for
further evaluation are available to the investigator depending
upon the research objectives and time/cost restraints of the
investigation. One option is to grow the offspring to maturity
and follow the identical experimental design for the F1 gen-
eration (Fig. 2C). This may or may not involve exposure at
the larval stage. It is also important to note that larval exposure
need not be an integral part of the proposed model. Investi-
gators may chose to examine the relationship between biolog-
ical indicators and reproductive endpoints using only fish ex-
posed as adults.
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Environ. Toxicol. Chem. 17, 1998 55
Developmental effects measured at the commencement of
the reproductive assessment by means of histopathologic anal-
ysis may provide a wealth of information with regards to the
nature of reproductive impairments. Thus far, data collected
include the measure of a biological indicator (e.g., vitellogenin,
steroid levels, receptor number) along with reproductive pa-
rameters such as time of first spawn, fecundity, clutch size,
and egg hatchability. Although histology is often labor inten-
sive, it provides an important component to the reproductive
assessment data previously collected on the same population
of fish. Not only does histologic information assist in deter-
mining the state of reproductive development of individual
male or female fish, but it confirms the gonadal phenotype of
the fish, which would have been initially sexed by examining
external phenotype.
Histologic analysis of gonadal tissue has been used to ex-
plain reproductive impairment in several wildlife species. Ex-
amination of gonadal tissue from the American alligators of
Lake Apopka provided valuable information regarding egg
production in this species. For example, Guillette et al. [8]
found that female alligators from a contaminant-laden lake
showed polyovular ovarian follicles and polynuclear oocytes.
In addition to these histologic findings, extreme differences in
levels of circulating sex steroids as well as reduced egg via-
bility were found when compared to alligators from a reference
lake. Male alligators from Lake Apopka also showed poorly
differentiated seminiferous tubules.
A number of studies have reported a variety of develop-
mental effects associated with gonadal differentiation in fish
following exposure to natural, synthetic, and/or some envi-
ronmental estrogens. Most notable developmental changes in-
clude complete sex reversal where the phenotype is different
from the genetic composition, and intersex conditions, where-
by gonochoristic species exist with both testicular and ovarian
tissue (ova-testis). Gimeno et al. [48] exposed genetic male
carp during a period of sexual differentiation to 4-tert-pen-
tylphenol (TPP) and observed oviducts in all male fish exposed
to TPP for 60 d. Gray and Metcalfe [49] also demonstrated
an intersex condition in medaka exposed to 4-nonylphenol
from hatch to 3 months of age. In fish exposed to 50 mg/L
and 100 mg/L of nonylphenol, 50 and 86% of the male fish
developed ova-testis, respectively. The ratio of male to female
fish was one to two, compared to two to one in the control
population. Gimeno et al. [48] also found that exposure of
male carp to estradiol for 90 d produced phenotypic females
with no testicular tissue. Sex reversal by means of adminis-
tration of synthetic sex hormones has been used to produce
monosex populations for aquaculture purposes. However, the
majority of studies with monosex populations have failed to
examine the reproductive capabilities of these morphologically
altered species. Should these conditions be detected in medaka
exposed at the larval and/or adult stages in the proposed model,
a number of reproductive endpoints could be used to assess
the reproductive potential of such fish.
CONCLUSIONS
An assessment of the current state-of-the-science has dem-
onstrated that endocrine-disrupting compounds are present in
the aquatic environment in sufficient concentrations to elicit
responses normally under the control of endocrine hormones.
These compounds can act along the hypothalamus–pituitary–
gonadal axis that governs reproductive processes in fish. Lab-
oratory studies have demonstrated that exposure of fish to

56 Environ. Toxicol. Chem. 17, 1998
estrogenic compounds can lead to phenotypic alteration, as
well as reduced fertility, egg size, and viable offspring in sex-
ually mature fish. Field studies with white suckers exposed to
BKME also reported adverse reproductive effects, such as de-
layed age to maturity, reduced gonad size, smaller eggs, and
altered hormone levels [41,42]. However, despite the above
alterations in hormone levels and egg size, no effect was found
on fecundity and survival of the offspring. Thus, with the
exception of a few well-documented studies (e.g., decline in
bald eagles and American alligator populations with exposure
to DDE), the ecological implications of exposure to endocrine-
disrupting compounds remain unclear. In view of the above
points, research focused on examining the ecologically rele-
vant consequences of alterations in endocrine function merits
further consideration.
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