Ecotoxicology, 10, 373±388, 2001
# 2001 Kluwer Academic Publishers. Manufactured in The Netherlands.
Effects of Endocrine Disruptors on Prosobranch Snails
(Mollusca: Gastropoda) in the Laboratory. Part III: Cyproterone
Acetate and Vinclozolin as Antiandrogens
MICHAELA TILLMANN, ULRIKE SCHULTE-OEHLMANNy, MARTINA DUFT,
È RG OEHLMANN y
BERND MARKERT AND JO
Internationales Hochschulinstitut Zittau, Lehrstuhl Umweltverfahrenstechnik,
Fachgruppe Human- und OÈkotoxikologie, Markt 23, D-02763 Zittau, Germany
Received 2 December 2000; Accepted 26 January 2001; Revised March 21 2001
Abstract. The effects of suspected endocrine disrupting chemicals on freshwater and marine prosobranch
species were analysed in laboratory experiments. In this last of three publications, the responses of the fresh water
snail Marisa cornuarietis and of two marine prosobranchs (Nucella lapillus, Nassarius (Hinia) reticulatus) to the
antiandrogenic model compounds cyproterone acetate (CPA) and vinclozolin (VZ) are presented. The snails were
exposed to nominal CPA concentrations of 1.25 mg/L alone and simultaneously to a potent synthetic estrogen
(ethinylestradiol), androgen (methyltestosterone) or an indirectly acting xeno-androgen (tributyltin) in
experiments with adult specimens and in a life cycle test for 12 months. Marisa and Nucella were furthermore
exposed to nominal concentrations of 0.03±1.0 mg VZ/L for up to 5 months. The antiandrogens induced a number
of biological responses in all three species. The length of the penis and of accessory male sex organs (e.g., penis
sheath, prostate) were significantly reduced. For Marisa, this effect occurred only in sexually immature specimens
and was reversible as the males attained puberty. Typical androgen-mediated responses (imposex development,
delayed spermatogenesis, tubulus necrosis of the testis with orchitis and Leydig cell hyperplasia) were partially or
totally suppressed by a simultaneous administration of CPA. In the two marine species even adult, sexually mature
males responded to antiandrogens with a reduction of the male sex organs and an advancement of the sexual repose
phase. The results for CPA and VZ are compared with the effects of an exposure to xeno-estrogens (bisphenol A,
octylphenol) and xeno-androgens (triphenyltin, tributyltin) in the same species. Each group of endocrine disruptors
induces a characteristic set of toxicological effects in prosobranch snails which can be used as endpoints in an
organismic invertebrate test for the identification of endocrine mimetic test compounds. Estrogens cause primarily
an induction of superfemales resulting in an increased female mortality by the enhancement of spawning mass and
egg production. The main effects of androgens are a virilization of females by imposex development and a marked
decrease of the fecundity. Compared with estrogens and androgens, the antiandrogen responses seem to be less
drastic and might haveÐin contrast to the two other disruptor classesÐno biologically significant effects at the
population level.
Keywords: endocrine disruptors; antiandrogens; cyproterone acetate; vinclozolin; snails
To whom correspondence should be addressed:
J. W. Goethe-UniversitaÈt Frankfurt, Zoologisches Institut,
Siesmayerstraûe 70, D-60054 Frankfurt/M., Germany
Tel.: ‡49-69-798-24738; Fax: ‡49-69-798-24748;
E-mail: [email protected].
y
Present address: J. W. Goethe-UniversitaÈt Frankfurt,
È kologie und, Siesmayerstraûe 70, D-60054
Abteilung O
Frankfurt/M., Germany
374 Tillmann et al.
Introduction
It has been shown for several xenobiotics in the
environment that they are capable of inducing adverse
effects in animals and humans by interfering with the
normal endocrine function of the organism. The
reported effects of these compoundsÐreferred to as
endocrine disruptorsÐinclude decreased sperm
counts, increased cases of breast, testicular and other
forms of reproductive cancers, genital abnormalities
(e.g., hypospadia, cryptorchidism), premature puberty
in females, and increased cases of endometriosis in
humans (Gist, 1998). Most wildlife studies on the
effects of hormone-mimetic industrial chemicals were
focussed on vertebrates despite the fact that invertebrates represent more than 95% of the known animal
species (deFur et al., 1999). The limited number of
examples for endocrine disruption in invertebrates is
partially due to the fact that their hormonal systems
are comparably poorly understood and that therefore
deleterious endocrine changes following an exposure
to these compounds may easily be missed or simply
be unmeasurable at present. A review of the literature
has recently gathered an extended number of field
investigations and laboratory studies with some
evidence that endocrine disruption has probably
occurred in invertebrates (deFur et al., 1999). One
of the best documented examples are the masculinising effects of tributyltin (TBT) compounds in about
150 species of prosobranch molluscs. In these
invertebrates an apparently trivial biochemical
changeÐthe inhibition of the aromatase (Bettin
et al., 1996) as one of the key enzymes of the steroid
metabolismÐresults in drastic effects up to the
population and community levels by a final sterilization of females. According to Matthiessen and Gibbs
(1998) there is no reason to suppose that such far
reaching changes are in any sense unique. The main
endocrine effects of TBT in these molluscs are the
induction of imposex, an additional formation of male
sex characters like penis and/or vas deferens on
females (Gibbs et al., 1987; Oehlmann, 1994) and of
intersex which is characterized by a modification or
supplanting of female by male sex organs (Bauer et al.,
1995; Bauer et al., 1997).
This publication is the final one in a series of three
which investigates effects of compounds suspected to
act as endocrine modulators on freshwater and
marine prosobranch species in the laboratory. Most
of the results were obtained during a research project
for the German Federal Environmental Agency
(project code 297 65 001/04) between September
1997 and May 2001. The objective was to develop an
organismic invertebrate test system for the simultaneous identification of either (anti)androgen- or
estrogen-mimicking chemicals. Recently, gonochoristic prosobranchs were rated as the most promising
candidates for this purpose next to insects and
crustaceans (deFur et al., 1999). While the first two
papers were dedicated to xeno-estrogens (Oehlmann
et al., 2000) and xeno-androgens (Schulte-Oehlmann
et al., 2000), this publication is focussed on the
effects of two antiandrogens, cyproterone acetate
(CPA) and vinclozolin (VZ) with a comparison of the
effects for all three groups of compounds.
The synthetic steroid CPA (6-chloro-17acetoxy-1,2-methylenepregna-4,6-diene-3,20-dione;
CAS 427-51-0; Androcur1, Cyprostat1) has found a
widespread use in human therapy as a steroidal
antiandrogen which reduces free androgen binding
sites by competitively blocking the androgen receptors. Additionally, CPA exhibits a low gestagenic
activity in mammalian species (Forth et al., 1990). As
an antiandrogen, it is used primarily to treat advanced
prostate cancer (PCTCG, 2000), hirsutism (Pazos
et al., 1999; Tartagni et al., 2000), polycystic ovary
syndrome (Rittmaster, 1999), acne (Beylot et al.,
1998; Gollnick et al., 1999), Tourette's syndrome
(Izmir and Dursun, 1999), and sex-offending behaviours (Eriksson and Eriksson, 1998; Zonana and
Norko, 1999).
In 1993, more than one million women in
Germany, the United Kingdom, France and Italy
used Diane-351 (Krebs et al., 1997), a drug which
contains CPA in combination with ethinylestradiol.
Although no information is available on the worldwide production or environmental fate, including the
occurrence in the environment, it is likely that CPA
(or its metabolites) may be present in trace amounts
in sewage and possibly even in surface waters due to
the broad pharmacological use.
CPA is not only a very potent antagonist of the
androgen receptor (Vingaard et al., 1999), but has
been shown to exhibit a considerable hepatic toxicity
(Tucker et al., 1996), to induce DNA repair synthesis
and to form DNA adducts in the liver at low doses,
which are in the therapeutic range used in women
(Topinka et al., 1996; Krebs et al., 1997).
VZ (3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,
3-oxazolidine-2,4-dione; CAS 50471-44-8; e.g.,
Endocrine Effects on Prosobranchs. III: Antiandrogens
Ronilan1, Konker R1) is a chlorinated fungicide
used predominantly for treatment of grains, grapes,
ornamental plants and turfgrass (Makynen et al.,
2000). The annual application in the EU is estimated
to be several hundred metric tonnes (Readman et al.,
1997). VZ had not been considered to be particularly
hazardous to vertebrates (US-EPA, 1997); however,
recent data indicate that the pesticide exhibits
antiandrogenic activity in vivo in mammals (Kelce
et al., 1997). Specifically, two VZ metabolites
(M1 ˆ 2-{[(3,5-dichlorophenyl)-carbamoyl]oxy}-2methyl-3-butenoic acid; and M2 ˆ 3 0 ,5 0 -dichloro-2hydroxy-2-methylbut-3-enanilide) act as antagonists
of the mammalian androgen receptor (Kelce et al.,
1994, 1998; Vingaard et al., 1999). Although the
fungicide was previously ranked as essentially nongenotoxic, recent reports indicate that it induces
chromosome damage (micronuclei induction) in the
bone marrow cells of treated mice (Hrelia, 1996;
Hrelia et al., 1996).
There are only limited data available on the
occurrence and fate of VZ in the environment. In
BBA standard soils, the half life was 35±45 days
(BBA, 1997) Ueoka et al. (1997) found no measurable leaching of the fungicide following an application at the maximum recommended rates to soils. VZ
concentrations were very low throughout the soil core
profiles (0.05±1.4% applied dose) suggesting that the
compound was unstable with more than 98% degradation in 10 days. Nevertheless, VZ has occasionally
been detected in surface waters with maximum
concentrations of 0.5 mg/L (GuÈlden et al., 1997;
Readman et al., 1997) and also in a survey of
drinking waters from more than 200 German waterworks with maximum concentrations of 0.1 mg/L
(Iwan, 1988). There are no reported data concerning
the bioconcentration or metabolism of VZ by nonmammalian species, despite the fact that it has been
a registered crop-use pesticide for over 20 years.
Makynen et al. (2000) report that VZ does not have
a large bioconcentration potential in fish with
BCF values of 62 and 167 in males and females,
respectively.
Materials and methods
The experiments were performed with three gonochoristic prosobranch species, the freshwater ramshorn snail, Marisa cornuarietis (Mesogastropoda:
375
Ampullariidae), the marine dogwhelk Nucella lapillus (Neogastropoda: Muricidae), and the netted whelk
Nassarius (Hinia) reticulatus (Neogastropoda: Buccinidae). Marisa specimens came from the breeding
stock in our own laboratory which was built up with
specimens obtained from the breeding stock of
Aquazoo DuÈsseldorf (Germany) in 1991. The two
marine species were collected in Brittany (France)
at MeÂan MeÂlen (N. lapillus) and PleÂneuf Val AndreÂ
(H. reticulata).
The three prosobranch species were exposed to the
test compound cyproterone acetate (CPA, Sigma
Chemicals, Germany, article no. C3412), Marisa
and Nucella to VZ (kindly supplied by BASF AG
Limburgerhof, Germany, batch no. N195) via water.
These experiments were conducted as 24 h (weekends: 48 h) semi-static renewal systems in 60 litre
glass aquaria filled with tap water (for Marisa) or
artificial seawater (for Nucella and Nassarius,
salinity 35½) and provided with an Eheim power
filter. All tests were performed under constant
conditions with a temperature of 22 1 C for the
freshwater and 14 1 C for the marine snails; the
light dark rhythm was adjusted to 12 : 12 h.
Five different series of exposure experiments were
conducted with the test compounds CPA and VZ:
1. Marisa LC- (life cycle) test with CPA: Eggs
produced by adult ramshorn snails in groups
exposed to ethinylestradiol (EE2) or methyltestosterone (MT) at nominal concentration ranges
from 0.1±1.0 mg/L (including a solvent control)
were further exposed to 0.5 mg MT/L, with and
without 1.25 mg/l CPA, and to 0.5 mg EE2/L,
with and without 1.25 mg/l CPA, including a
solvent control (ethanol; concentration: 12.5 mg/
L) over a period of 12 months until the hatched
F1 specimens were 1 year old. They reached
sexual maturity in their eighth month. Thirty
specimens from each group were collected for
analysis at an age of 3, 6, 9 and 12 months.
2. Adult Marisa test with CPA: Sexually mature
ramshorn snails of comparable age were exposed
to nominal concentrations of 0.2 mg tributyltin as
Sn (TBT-Sn)/L, with and without 1.25 mg/l
CPA for 6 months, including a solvent control
(ethanol; concentration: 12.5 mg/L). Thirty specimens from each group were collected for
analysis at the beginning of the experiment
and at bimonthly intervals.
376 Tillmann et al.
3. Nucella and Nassarius test with CPA: Sexually
mature dogwhelks and netted whelks were exposed
to nominal concentrations of 0.05 mg TBT-Sn/L,
with and without 1.25 mg/l CPA, and to
1.25 mg/l CPA alone for 5 months, including a
solvent control (glacial acetic acid; concentration: 10 mg/L). Thirty specimens from each
group were collected for analysis at the beginning of the experiment and at monthly intervals
with an additional sample 2 weeks after starting
the test.
4. Juvenile Marisa test with VZ: Sexually immature ramshorn snails of comparable age (3 months)
were exposed to nominal concentrations of 0.03,
0.1, 0.3 and 1.0 mg VZ/L for 5 months, including
a solvent control (ethanol; concentration:
12.5 mg/L). Thirty specimens from each group
were collected for analysis at the beginning of
the experiment and at monthly intervals.
5. Nucella test with VZ: Sexually mature dogwhelks were exposed to nominal concentrations
of 0.03, 0.3 and 1.0 mg VZ/L for 3 months,
including a solvent control (glacial acetic acid;
concentration: 10 mg/L). Thirty specimens from
each group were collected for analysis at the
beginning of the experiment and at monthly
intervals.
All specimens were narcotized prior to analysis
(2.5% MgCl2 in distilled water for Marisa, 7%
MgCl2 for Nucella and Nassarius). The individual
shell and aperture height were measured to the
nearest 0.1 mm before the shell was cracked and the
snail was removed. The presence, normal appearance,
and extension (to the nearest 0.1 mm) of all sex
organs was checked, as well as the occurrence of
oocytes and sperm in the genital system and of visible
excrescences on genital and other organs with a
dissection microscope. Additionally, the VDSI (vas
deferens sequence index ˆ mean value of imposex
stages in a sample with values of 0 to 3 in M.
cornuarietis, 0 to 6 in N. lapillus and 0 to 4 in H.
reticulata) was calculated (for details see (Oehlmann
et al., 1991; Stroben et al., 1992; Schulte-Oehlmann
et al., 1995).
Furthermore, a histopathological analysis of the
gonads was performed for the Marisa LC-test with
CPA (all experimental groups) and the juvenile
Marisa test with VZ (only control and 1 mg VZ/L).
Six male and six female specimens from each sample
were fixed in Carnoy's and Bouin's fluid, respectively, and then preserved in ethanol. After embedding in paraplast, serial sections (5±7 mm) were made
and stained with haemalum-chromotrope. The sections were analysed using an image analysis system
(Optimas 5.2, Optimas Cooperation) coupled with an
Olympus microscope (BX 50).
Standard statistical analyses of the results (e.g.,
analyses of covariance (ANCOVA) with multiple
comparison of samples according to Tukey, H-test
(Kruskal-Wallis test with multiple comparison of
samples according to Nemenyi), 2 test, and Weir
test for classified values were performed according to
Weber (1972), LozaÂn (1992) using the computer
programme StatEasy for Windows NT.
Results and discussion
Marisa LC-test with CPA
During the LC-test with Marisa cornuarietis, effects
of nominal concentrations of 0.5 mg MT/L or 0.5 mg
EE2/L, either with or without 1.25 mg/l CPA including a solvent control (ethanol), were assessed ex ovo
for 12 months. An exposure to MT or EE2 without the
antiandrogen resulted in a time dependent enhancement of imposex intensities in females. This effect
was not statistically significant before the ninth
month of the experiment, although higher VDSI
values than in the control group were already
measured after 3 months for EE2 and after 6 months
for EE2 and MT (Fig. 1a). The finding that not only
an exposure to the synthetic androgen, but also to the
estrogen promotes imposex in ramshorn snails at
nominal concentrations above 0.1 mg/L, seems to be
paradoxical. Nevertheless, it was confirmed in further series of laboratory experiments with adult
M. cornuarietis (concentration range: 0.1±1.0 mg/L)
and Nucella lapillus (concentration range:
0.05±0.25 mg/L). These results will be presented and
discussed elsewhere (Schulte-Oehlmann et al., in
prep.). In the LC-test with Marisa, a simultaneous
application of CPA and EE2 led to a complete
suppression of imposex development, indicating
that imposex as an EE2 response is androgenmediated: The external administration of high concentrations of synthetic estrogens causes an increase
of endogenous testosterone concentrations, probably
Endocrine Effects on Prosobranchs. III: Antiandrogens
Figure 1. Effects of CPA exposure in Marisa cornuarietis.
(a) Development of the VDSI in the life cycle-test F1 generation.
Exposure groups, () solvent control, (+) 0.5 mg MT/L, ( ) 0.5 mg
EE2/L, (~) 0.5 mg MT plus 1.25 mg CPA/L, (&) 0.5 mg EE2 plus
1.25 mg CPA/L. (b) Mean length ( standard deviation; n: 10±18)
of penis sheath in F1 males during the life cycle-test at an age of 3
and 6 months. Exposure groups by bars from left to right: (&)
control; ( ) 0.5 mg MT/L; (&) 0.5 mg EE2/L; ( ) 0.5 mg.
MT plus 1.25 mg CPA/L; ( ) 0.5 mg EE2 plus 1.25 mg CPA/L. (c)
Development of the VDSI in adult snails. Exposure groups: ()
solvent control, ( ) 0.2 mg TBT-Sn/L, (~) 0.2 mg TBT-Sn plus
1.25 mg CPA/L. Asterisks indicate statistical significant differences
to control (in (a) and (c) Weir test for classified values, in (b) H
test): $, p < 0.05; $$, p < 0.01.
377
due to a negative feedback of these high estrogen
titres on the aromatase activity. The enhancement of
testosterone concentrations in the tissue is then
responsible for imposex development in EE2 exposed
snails (Schulte-Oehlmann et al., in prep.). The
applied concentrations of the competitive antiandrogen CPA in the Marisa LC-test were sufficient to
suppress the (indirect) EE2 effect on imposex development completely, but were too low to suppress the
(direct) MT effects in the experimental group, which
was exposed to MT and CPA simultaneously
(Fig. 1a).
CPA acted as an antiandrogen not only in female
specimens by the suppression of imposex development under simultaneous exposure to high EE2
concentrations, but had furthermore an adverse impact
on the extension of the pallial sex organs in juvenile,
sexually immature males (prostate gland, penis, penis
sheath, penis pouch), as indicated for the penis sheath
in Fig. 1b. The length of these male sex organs did not
differ significantly between the solvent control and the
two experimental groups, which were exposed to
either 0.5 mg MT/L or 0.5 mg EE2/L without CPA at an
age of 3 months, but was significantly reduced in those
groups, which received additionally 1.25 mg CPA/L.
At an age of 6 months these sex organs were
significantly larger in all experimental groups except
for the solely MT-treated males (Fig. 1b). Two months
later the males become sexually mature and at an age
of 9 and 12 months no significant length differences
were found between the groups. This indicates that
CPA suppresses the growth of the accessory male sex
organs in immature male ramshorn snails but also that
the effect does not persist until puberty in Marisa.
The effectiveness of CPA in reducing the size of
the copulatory organs in immature male ramshorn
snails is demonstrated by a direct comparison of four
specimens in Fig. 2. At an age of 3 months, it is
virtually impossible to distinguish between an MTtreated female exhibiting the imposex stage 3 (Fig.
2b) and a male, which received CPA additionally to
MT or EE2 (Fig. 2d) if only the extension of the
accessory male organs at the roof of the mantle cavity
are considered. In contrast to this similarity between
masculinized females and antiandrogen-treated
males, the differences to a normal female (Fig. 2a)
and male (Fig. 2c) from the control group are
striking.
The histopathological analysis of the gonads
revealed that in 66.7% of the males from the solely
378 Tillmann et al.
Figure 2. Marisa cornuarietis. Photographs of the mantle cavity roof, taken with identical magnification: (a) normal female (control group;
imposex stage 0); (b) imposex female (stage 3; methyltestosterone treatment); (c) normal male (control group); (d) male with reduced sex
organs (simultaneous MT and CPA treatment). Abbreviations: an, anus; g, gill; p, penis; pp, penis pouch; pr, prostate gland; ps, penis sheath; v,
vagina.
MT-treated group spermatogenesis was delayed when
compared to the control. This effect was statistically
significant ( 2 test, p < 0.05). No differences were
found between the control and the other three
experimental groups (MT plus CPA, EE2, EE2 plus
CPA), indicating that this androgenic response could
be prevented by CPA completely. A second observation was that in post-pubertal males from the 0.5 mg
MT/L and 0.5 mg EE2/L groups a tubulus necrosis
occurred in the testis which was followed by a
massive infiltration of phagocytic active hemocytes
as an inflammatory reaction (Fig. 3c and d), an effect
which can be referred to as an orchitis (B. Watermann,
pers. comm.). Parallel to the tubulus necrosis and
orchitis, an extensive proliferation (hyperplasia) of
those interstitial testis cells occurred (Fig. 3e) which
are referred to in the older literature as ``Leydig
cells'' (Frank, 1914; Ronkin, 1952; Taylor and
Anderson, 1969; for review: Voltzow, 1994) although
it is at least doubtful whether or not these cells are
Endocrine Effects on Prosobranchs. III: Antiandrogens
endocrine active. Tubulus necrosis, orchitis and
Leydig cell hyperplasia were not observed in the
control specimens or in the snails which received
CPA additionally to the synthetic androgen or
estrogen (Fig. 3a and b), indicating that also these
effects are androgen-mediated because they can be
reverted by CPA completely. The incidences of these
effects were 16.6% and 100% in the MT-treated
and 0% and 33.3% in the EE2-treated males at an age
of 9 and 12 months, respectively. The differences to
the control were statistically significant at the end
of the experiment ( 2 test; p < 0.01 for MT and
p < 0.05 for EE2).
Adult Marisa test with CPA
During this laboratory test series, the ramshorn snails
were exposed to a nominal concentration of 0.2 mg
TBT-Sn/L with and without 1.25 mg CPA/L. The
exclusively TBT-treated females of Marisa cornuarietis developed obvious imposex characteriztics with
a significant increase of the VDSI already after two
months (Weir test, p < 0.01) and a further quasilinear increase in the following months (Fig. 1c). In
the group, which received CPA simultaneously to
TBT, CPA was able to reduce imposex development
but not to block the TBT-induced increase of the
imposex intensities completely. Consequently, the
VDSI values in this experimental group exhibit a
slight increase throughout the duration of the experiment, which was not significantly different from the
control before month 6, but significantly different
from the solely TBT-exposed group during the entire
test (Weir test, p < 0.05). It can be assumed that CPA
would have been more effective in preventing the
development of imposex if higher concentrations of
the antiandrogen or lower concentrations of TBT
were applied. Bettin et al. (1996) conducted comparable experiments with two marine species, the
dogwhelk Nucella lapillus and the netted whelk
Nassarius (Hinia) reticulatus. They report that a
concentration of 1.25 mg CPA/L could suppress
imposex development in Nucella completely at a
TBT concentration of 0.05 mg as Sn/L, whereas in
female Nassarius the increase of the VDSI is only
reduced but not completely blocked. The distinct
reaction of both snails was attributed to a speciesspecific content of androgens and to a different
androgen sensitivity of their tissues.
379
Nucella and Nassarius test with CPA
The CPA laboratory test series with Nucella lapillus
and Nassarius (Hinia) reticulatus resembles this
earlier experiment performed by Bettin et al. (1996)
with respect to the experimental design, including
test duration and nominal concentrations of the test
compounds. The original findings of Bettin et al.
(1996) that CPA is capable of suppressing TBTinduced imposex development completely in Nucella
and to reduce the increase of imposex intensities in
TBT-exposed Nassarius females in the applied
concentration range (0.05 mg TBT-Sn/L and 1.25 mg
CPA/L) was confirmed. However, the main reason to
repeat the experiment was to assess the potential
effects of CPA on males in both prosobranch species.
In the experiment with Nucella lapillus, CPA
exposure resulted in a reduction of the penis length in
males, irrespective of an additional administration of
TBT (Fig. 4a). This effect was statistically significant
already after 4 weeks and could be observed until the
end of the experiment after 5 months, when the mean
penis length in both groups was reduced by 18.6%
(CPA without TBT) and 17.0% (CPA with TBT)
compared to the control. TBT without an additional
administration of CPA did not affect the extension of
the male copulatory organ. The slight drop in the penis
length of the control group is due to the fact that the
experiment started in March at the peak of the
reproductive cycle of dogwhelks with a consequent
reduction of the copulatory organ as males attain their
sexual repose (Stroben et al., 1996).
In general, these findings can be confirmed for the
netted whelk with some minor differences (Fig. 4c).
Exclusively TBT-treated Nassarius males were able to
sustain a slightly longer copulatory organ during the
sexual repose phase compared to the control although
this difference was not statistically significant.
Furthermore, the reduction of the penis length was
less marked in the group which received TBT and CPA
simultaneously than in the solely CPA-treated males
during the first 3 months of the experiment. In the last
two months, the extension of the penis in both CPAgroups was almost identical with a mean reduction of
18.3% and 15.6% compared to the control for CPA
alone and CPA plus TBT, respectively.
CPA does not only exhibit a negative impact on
the extension of male sex organs in the two marine
snails but also on spermatogenesis as indicated by the
relative numbers of males with a sperm-filled seminal
380 Tillmann et al.
Figure 3. Marisa cornuarietis. Histological photographs of testis in a control male (a,b) and from the 0.5 mg MT/L group (c±e). (a) Overview of
unaffected testis; (b) detail of (a) with intact testis tubules and spermatogenesis; (c) overview of a testis with tubulus necrosis, hemocyte
infiltration (orchitis; white arrows) and Leydig cell hyperplasia (black arrows); (d), (e) details of (c). Abbreviation: nt, necrosis of tubulus
epithelium.
Endocrine Effects on Prosobranchs. III: Antiandrogens
381
Figure 4. Effects of CPA exposure in Nucella lapillus (a, b) and Nassarius (Hinia) reticulatus (c). (a, c) Development of mean penis length
( standard deviation; n = 10±23 for Nucella, n = 9±19 for Nassarius) in adult males. Exposure groups: () solvent control, ( ) 0.05 mg TBTSn/L, (&) 1.25 mg CPA/L, (~) 0.05 mg TBT-Sn plus 1.25 mg CPA/L. (b) Relative numbers of males with sperm-filled vesiculae seminalis.
Exposure groups by bars from left to right: (&) control; ( ) 0.05 mg TBT-Sn/L; (&) 1.25 mg CPA/L; ( ) 0.05 mg TBT-Sn plus 1.25 mg
CPA/L. Asterisks indicate statistical significant differences to control (in (a) and (c) H test, in (b) 2 test): $, p < 0.05; $$, p < 0.01.
382 Tillmann et al.
vesicle during the experiment with Nucella lapillus
(Fig. 4b). The first males without sperm in this
section of the genital tract (acting as a reservoir)
occurred in the two CPA-treated groups already after
2 weeks, despite the fact that the animals were in the
active phase of the reproductive cycle. The first
control animals attained their sexual repose not
before May (end of month 2 in the experiment), but
still 84.6% of them had ripe sperm in their vesicle
while the corresponding values were 33.3% and
30.4% for the CPA and CPA plus TBT groups,
respectively (significantly different at p < 0.01, 2
test). Comparable results were found during the
Nassarius experiment which was conducted in
parallel. It indicates that CPA induces an earlier
sexual repose in males of both marine prosobranch
species, although it has to be considered that the
nominal test concentration of 1.25 mg CPA/L was far
beyond any environmental significance. For the two
experimental series with VZ an environmentally
relevant nominal concentration range between 0.03
and 1.0 mg/L was applied for both species, Marisa
cornuarietis and Nucella lapillus.
Juvenile Marisa test with VZ
At the beginning of the experiment the ramshorn
snails were 3 months old and sexually immature. At
the end of the test, 5 months later, the first males in
the control group had attained sexual maturity but
none of the specimens in the VZ-treated groups,
irrespective of the applied concentration. The test
compound did not show any effects in the females
during the experiment but it has to be considered that
no spawning occurred and fecundity parameters
could therefore not be analysed. In males a slight
but statistically significant decrease of the extension
of the male accessory sex organs, especially for the
penis and penis sheath was found compared to the
control. This response could only be detected during
the first 2 to 3 months of the experimentÐwhich
resembles the earlier observation for CPA in
MarisaÐand only for the two lowest concentrations
(0.03 and 0.1 mg VZ/L; (Fig. 5)). No differences in
the length of the penis or penis sheath were found
during the last two months of the test.
Literature reports show that endocrine disruptors
(e.g., diethylstilbestrol, bisphenol A, octylphenol)
may induce a response at low doses, while high
doses can have no or the opposite effect (Nagel et al.,
1997; vom Saal et al., 1997; 1998). Although this
hypothesis was called in question as the results of
these studies were not reproducibly established by
some other laboratories, an US EPA expert panel has
recently confirmed these exclusively low-dose effects
of endocrine-mimicking chemicals (Kaiser, 2000).
Nucella test with VZ
The 3 months VZ experiment with adult Nucella
lapillus gave comparable results (like the CPA test) in
spite of the much lower nominal concentrations for
the fungicide of between 0.03 and 1.0 mg/L. Not only
the mean length of the penis (Fig. 6a) and prostate
gland (Fig. 6b) were significantly reduced when
compared to the control but also the relative numbers
of males with ripe sperm stored in the seminal vesicle
(Fig. 6c) indicating that VZ causes an earlier sexual
repose of male dogwhelks. There is only little
evidence that the responses are concentration dependent as the highest VZ concentration of 1.0 mg/L had
comparable effects as the lowest with 0.03 mg/L.
These experiments show that antiandrogens
induce a characteristic pattern of endpoints in
exposed prosobranch snails. In all three species, the
extension of the male copulatory organ and of other
accessory sex organs are reduced, like the penis
sheath and prostate gland. For Marisa, this effect can
only be found in sexually immature specimens and is
reversible when the males attain puberty. The direct
effects of potent androgens (e.g., MT) or androgenmediated responses to synthetic estrogens (e.g., EE2),
for example the development of imposex, delayed
spermatogenesis in sub-adult snails or tubulus necrosis of the testis with orchitis and Leydig cell
hyperplasia can be partially or totally suppressed by
a simultaneous administration of antiandrogens. In
the two marine species even adult, sexually mature
males respond to antiandrogens with a reduction of
the male sex organs and an earlier sexual repose.
Both factors might have negative implications for the
reproductive success in the field as the male penis is
inserted into the bursa copulatrix of the female during
copulation, which is positioned in the distal section of
the pallial oviduct. Therefore, a minimum extension
of the penis is necessary for a successful transfer of
sperm. Although it should be pointed out that it was
not possible to investigate a potential influence of
Endocrine Effects on Prosobranchs. III: Antiandrogens
383
Figure 5. Effects of VZ exposure in Marisa cornuarietis. Mean length ( standard deviation; n: 11±23) of penis (a) and penis sheath (b) in
immature males. Exposure groups by bars from left to right: (&) control; ( ) 0.03, ( ) 0.1, ( ) 0.3, and (&) 1.0 mg VZ/L. Asterisks indicate
statistical significant differences to control (H test): $, p < 0.05.
CPA or VZ on fecundity parameters in females
during the experiments, it seems that the adverse
impact of antiandrogens is much less severe compared to the effects of xeno-estrogens and xenoandrogens in the same prosobranch species (see
below).
A comparison of our own results with reports in
the literature is difficult because only a few studies
have investigated the hormone-mimetic effects of
CPA, VZ or other antiandrogens in invertebrates so
far. LeBlanc and McLachlan (1999) evaluated the
effects of CPA on growth, moulting, sexual differentiation, and reproduction in Daphnia magna.
Exposure at sub-lethal concentrations reduced growth
but had no effect on moulting or other developmental
and maturation parameters. CPA reduced the number
of offspring in parthenogenetically reproducing
daphnids, but the authors attribute this effect to the
smaller size of CPA-exposed specimens and their
inability to accommodate a brood of more than
approximately 10 eggs. Therefore, it seems to be at
least doubtful whether this is an endocrine-related
effect in daphnids.
In contrast to this single study on antiandrogen
effects in invertebrates, there are numerous publications investigating the responses of CPA and VZ in
in vitro tests or tests with vertebrates. Especially the
in vivo studies show that the two antiandrogens
exhibit comparable effects to our experiments with
prosobranch snails like a size or weight decrease in
accessory male sex glands, delayed male puberty or
decreasing sperm counts. Vingaard et al. (1999)
384 Tillmann et al.
found in a reporter gene assay a 30- to 60-fold
induction of luciferase activity by the synthetic
androgen receptor agonist, R1881 at a concentration
of 10 nM. A number of antiandrogenic compounds,
including CPA and VZ, inhibited the response to
0.1 nM R1881. Ashby and Lefevre (2000) tested a
range of chemicals in the peripubertal male rat assay
to evaluate their activity as endocrine disruptors,
including VZ and CPA. Both compounds affected a
number of analysed endpoints, like changes in the
weights of testes, epididymides, seminal vesicles and
prostate. Gray and Kelce (1998) found additionally a
delayed puberty and an alteration of sex differentiation in VZ-treated male rates. These results were
confirmed by Wolf et al. (2000). If the fungicide was
administered orally to pregnant rats, the male offspring was characterized by a female anogenital
distance at birth, cleft phallus with hypospadia and
nipple development at 2 weeks of age (Gray et al.,
1994). The authors report that after puberty, most of
the male offspring was unable to attain intromission
even though they all mounted sexually receptive
females and even if they appeared to achieve
intromission, they failed to ejaculate normally, as
no sperm were found in the uterus after matings.
Male rabbits, which were VZ exposed in-utero or
during infancy, developed severe ethological changes
according to Veeramachaneni (2000). Several treated
animals, but no control animal, failed to show any
sexual interest in the female or failed to ejaculate
indicating the potential of the fungicide to alter
sexual behaviour permanently in adult life.
An epidemiological study of 67 men who were
occupationally exposed to VZ for 1 to 13 years and
of 52 controls provided no evidence of hormonal
responses induced by the fungicide. Furthermore, no
evidence of liver injury, prostate changes, cataract
formation, or haemolytic anaemia was found (Zober
et al., 1995).
An antiandrogenic action of VZ on non-mammalian vertebrates is questionable on the basis of the
results of a limited number of reports in the literature.
Makynen et al. (2000) found a slight increase in the
serum estradiol concentration of male fathead minnows (Pimephales promelas) exposed to 700 mg VZ/
L and a marked reduction in gonadal condition of
female fish from this treatment. But as the compound
and their two active metabolites M1 and M2 failed to
compete for high-affinity, low-capacity testosterone
binding sites in fathead minnow brain and ovary
cytosolic fractions, the authors conclude that these
chemicals might not act as antiandrogens in this fish
species. Crain et al. (1997) report that VZ had no
apparent effect on male or female alligators.
The ecological relevance of VZ effects, which
have been described in in vivo laboratory studies, was
tested by Caslin and Wolff (1999) in field experiments with gray tailed voles (Microtus canicaudus).
Although the mean lengths of testes and seminal
vesicles were smaller, levels of plasma testosterone
and proportion of pregnant females were lower in
treatment than in control animals, these differences
were not sufficient to affect juvenile recruitment, sex
ratio, or population growth. The authors conclude
that their results revealed no biologically significant
effects of the fungicide at the population level under
the conditions of their experiments, but multiple
applications or a higher application rate of a VZ
product may have negative effects on male reproductive development and demography in wild populations.
Comparison of effects of endocrine-mimetic test
compounds in prosobranchs
It was one of the main objectives of this research
project to develop an organismic invertebrate test
system for the identification of either estrogen-,
androgen-, and antiandrogen-mimicking chemicals.
This can only be achieved if each of these groups of
compounds induces a characteristic pattern of toxicological effects during the exposure period so that a
certain test substance can be assigned to one of these
classes of endocrine disruptors. The different adverse
effects, which were found during the exposure
experiments with the three prosobranch snail species
and which can be used as endpoints for the test, are
summarized in Table 1.
Especially the use of the first three mentioned
effects in the table as endpoints in a biotest with
prosobranch snails allows an unambiguous identification of estrogenic (by the induction of superfemales), androgenic (by the induction of imposex)
or antiandrogenic properties (by the suppression of
imposex development under simultaneous exposure
to androgens) of test compounds.
The present study for CPA and VZ as antiandrogenic
agents, the results for the two xeno-estrogens bisphenol
A and octylphenol (Oehlmann et al., 2000) and for the
Endocrine Effects on Prosobranchs. III: Antiandrogens
385
Figure 6. Effects of VZ exposure in Nucella lapillus. Development of mean penis (a) and prostate (b) length ( standard deviation;
n ˆ 15±23) in adult males. Exposure groups: () solvent control, ( ) 0.03 mg VZ/L, (&) 0.3 mg VZ/L, (~) 1.0 mg VZ/L. (b) Relative numbers of
males with sperm-filled vesiculae seminalis. Exposure groups by bars from left to right: (&) control; ( ) 0.03, (&) 0.3, and ( ) 1.0 mg VZ/L.
Asterisks indicate statistical significant differences to control (in (a) and (b) H test, in (c) 2 test): $, p < 0.05; $$, p < 0.01.
386 Tillmann et al.
Table 1. Comparison of adverse effects following an exposure to xeno-estrogens (according to Oehlmann et al., 2000), -androgens (according
to Schulte-Oehlmann et al., 2000) and -antiandrogens during the experiments with Marisa cornuarietis, Nucella lapillus and Nassarius
(Hinia) reticulatus
Xeno-estrogens
(e.g., bisphenol A,
octylphenol)
Effect
Imposex development
Suppression of imposex development
Induction of ``superfemales''
Reduced female fecundity
Oogenesis impairment
Reduced male sex organs
Advanced male sexual repose
Spermatogenesis impairment
‡, effect observable;
Xeno-androgens
(e.g., triphenyltin,
tributyltin)
‡
‡
‡
‡
‡
‡
‡
‡
Antiandrogens
(e.g., cyproterone
acetate, vinclozolin)
‡
n.d.
n.d.
‡
‡
n.d.
, no effect; n.d., not determined.
aromatase inhibitors triphenyltin and tributyltin
(Schulte-Oehlmann et al., 2000) show clearly, that
each group of endocrine disruptors induces a different
set of toxicological effects in prosobranch snails. Xenoestrogens cause primarily an induction of superfemales
resulting in an increased female mortality by the
enhancement of spawning mass and egg production.
The main effects of direct acting (i.e., androgen receptor
mediated like methyltestosterone) and indirect acting
xeno-androgens (like the two mentioned organotin
compounds via an inhibition of aromatase activity)
are a virilization of females by imposex development
and a marked decrease of the fecundity. Compared with
estrogens and androgens, the antiandrogen responses
seem to be less drastic and might haveÐin contrast to
the two other disruptor classesÐno biologically significant effects at the population level. In general it can
be concluded that not only vertebrates but also
invertebrates are sensitive to endocrine disruption at
environmentally relevant concentrations of hormonemimetic compounds. The example of the ramshorn
snail M. cornuarietis shows that prosobranchs in
particular offer a most promising organismic invertebrate test system for the identification of endocrine
disruptors.
Acknowledgements
The experiments with Marisa cornuarietis were
financially supported by the Federal Environmental
Agency, Berlin (R&D project 297 65 001/04).
We gratefully acknowledge the excellent technical
assistance of Constanze Stark, Ulrike Schneider and
Christina Schmidt during the experiments and sample
analyses. Furthermore, we would like to thank Dr.
Burkard Watermann, LimnoMar (Hamburg) for discussions and advice on the gonad histopathology and
two anonymous reviewers for their helpful comments. We also appreciate the cooperation and
assistance of BASF AG, Limburgerhof (Germany)
and especially of Prof. Dr. C. KuÈnast for the generous
supply of VZ.
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