HELGOL~NDER MEERESUNTERSUCHUNGEN
Helgol~nder Meeresunters. 50, 299-317 (1996)
TBT-induced imposex in marine neogastropods is
mediated by an increasing androgen level
C. B e t t i n 1, J. O e h l m a n n 2 & E. S t r o b e n 1
11nstitut ffir S p e z i e l l e Zoologie und Vergleichende Embryologie, Universit~t Mfinster;
Hfifferstrafle i, D-48149 Mfinster, G e r m a n y
z Lehrstuhl ffir U m w e l t v e r f a h r e n s t e c h n i k , Internationales Hochschulinstitut (IHI) Zittau;
M a r k t 23, D-02763 Zittau, G e r m a n y
ABSTRACT: Tributyltin (TBT) exposure at different concentrations (5, 50, and 100 ng TBT as Sn/1)
induces a concentration- a n d t i m e - d e p e n d e n t imposex (= pseudohermaphroditism) development in
female Nucella lapillus and Hinia reticulata. In both species the average imposex stage, termed as
vas deferens s e q u e n c e (VDS) index, and the average female penis length increases with increasing
TBT concentration and duration of TBT exposure. Testosterone added at a concentration of 500 ng/1
induces a faster a n d more intensive imposex development compared to that induced by the TBT
concentrations used in the present experiments. Radioimmunological determination of endogenous
steroid content reveals increasing testosterone titres in female gastropods exposed to TBT which
correlate with the TBT concentration used and the duration of the experiment. The most marked and
highest increase of the endogenous testosterone level is exhibited by females of both species
exposed to testosterone. Simultaneous exposure to TBT and to the antiandrogen cyproterone acetate
which suppresses imposex development completely in N. lapillus and reduces imposex development
strongly in H. reticulata proves that the imposex-inducing effects of TBT are m e d i a t e d by an
increasing a n d r o g e n level a n d are not caused directly by the organotin compound itself. Furthermore, TBT-induced imposex development can be suppressed in both snails by adding estrogens to
the aqueous medium. These observations suggest that TBT causes an inhibition of the cytochrome
P-450 d e p e n d e n t aromatase system which catalyses the aromatization of androgens to estrogens.
The increase of the a n d r o g e n content or the shift of the androgen-estrogen balance in favour of
androgens induces the development of pseudohermaphroditism in marine prosobranchs. Artificial
inhibition of the cytochrome P-450 d e p e n d e n t aromatase system using SH 489 (1-methyl-1,4androstadiene-3,17-dione) as a steroidal aromatase inhibitor and flavone as a nonsteroidal aromatase inhibitor induces imposex development in N. lapfllus as well as in H. reticulata.
INTRODUCTION
T h e u s e of t r i b u t y l t i n (TBT) c o m p o u n d s as b i o c i d e s i n a n t i f o u l i n g p a i n t s a n d w o o d
p r e s e r v a t i v e s l e a d s to c o n t a m i n a t i o n of m a r i n e a n d f r e s h w a t e r e n v i r o n m e n t s . T B T h a s
b e e n s h o w n to b e h i g h l y toxic to a n u m b e r of a q u a t i c a n i m a l s i n c l u d i n g fish (Hall &
P i n k n e y , 1985; M a r t i n e t al., 1989; F e n t , 1991; F e n t & M e i e r , 1992), m u s s e l s , o y s t e r s
( L a u g h l i n e t al., 1986; T h a i n , 1986; T h a i n & W a l d o c k , 1986; L a n g s t o n e t al., 1987; C h a g o t
e t al., 1990) a n d c r u s t a c e a n s ( E v a n s & L a u g h l i n , 1984; C a r d w e l l & S h e l d o n , 1986). In
m a r i n e p r o s o b r a n c h snails, T B T i n d u c e s r e p r o d u c t i v e a b n o r m a l i t i e s a n d s t e r i l i z a t i o n of
f e m a l e a n i m a l s . T h i s p h e n o m e n o n , t e r m e d as p s e u d o h e r m a p h r o d i t i s m ( J e n n e r , 1979) or
i m p o s e x (Smith, 1971), is c h a r a c t e r i z e d b y t h e d e v e l o p m e n t of a d d i t i o n a l m a l e s e x o r g a n s
9 Biologische Anstalt Helgoland, H a m b u r g
300
C. Bettin, J. O e h l m a n n & J. Stroben
(penis a n d / o r vas deferens a n d prostate tissue) on females. A first description of i m p o s e x
w a s given b y Blaber (1970) for Nucella lapillus; he d e s c r i b e d females with a penis*like
o u t g r o w t h b e h i n d the right ocular tentacle. M e a n w h i l e . TBT-induced p s e u d o h e r m aphroditism is a w i d e s p r e a d p h e n o m e n o n within the Prosobranchia a n d has b e e n
r e c o r d e d in 118 species b e l o n g i n g to 63 g e n e r a (Fioroni et al. 1991p. Since 1970, mainly
Ocenebra emnacea (F6ral. 1974, 1976a-c, 1980; O e h l m a n n et al., 1992), IIyanassa
obsoleta (Smith, 1971, 1980. 1981a-d; Bryan et al. 1989) a n d especially N. lapillus (F6ral,
1980; Gibbs et al., 1987. 1988; O e h l m a n n et al., 1991) and Hinia reticulata (Stroben et al.,
1992a,b) h a v e b e e n u s e d as indicator species for TBT biomonitoring. B e c a u s e of its high
TBT sensitivity, N. lapillus has d e c l i n e d m m a n y a r e a s of Europe over the p a s t 10 to 15
y e a r s (Gibbs & Bryan, 1986, 1987; O e h l m a n n et al., 1991).
The intensity of i m p o s e x d e v e l o p m e n t is c h a r a c t e r i z e d b y a n e w classification system
(Fig. 1) which is b a s e d on the description of Blaber (1970) a n d d i s t i n g u i s h e s 6 s t a g e s of
i m p o s e x e x p r e s s i o n with three different types (a,b,c) in the first three s t a g e s a n d in stage
5, a n d two t y p e s in stage 4 a n d 6 (Oehfmann et al., 1992; Stroben et al., 1992a). This
i m p o s e x classification, which is g e n e r a l l y valid for the i m p o s e x description in all prosob r a n c h species, d e p e n d s on the p r e s e n c e of a penis, the extension of the vas d e f e r e n s a n d
an intact v a g i n a l opening. Stage 0 refers to a normal female without a n y male
characteristics. The first symptoms of p s e u d o h e r m a p h r o d i t i s m as d e s c r i b e d in s t a g e 1 are
a small penis without a p e n i s duct b e h i n d the right tentacle (type a), a short distal vas
d e f e r e n s - s e c t i o n also b e h i n d the right tentacle (type b) or a short p r o x i m a l v a s deferenssection b e g i n n i n g at the v a g i n a l o p e n i n g (type c). D e p e n d i n g on the e n v i r o n m e n t a l TBT
pollution a n d the sensitivity of e a c h g a s t r o p o d species, the m o r p h o l o g i c a l e x p r e s s i o n of
m a l e sex characteristics increases continuously. Stage 4, which r e p r e s e n t s t h e last fertile
s t a g e of imposex, is c h a r a c t e r i z e d b y a penis with a penis duct a n d a c o m p l e t e vas
deferens either r u n n i n g from the penis over the bottom of the m a n t l e cavity up to the
v a g i n a l o p e n i n g or the vas deferens p a s s e s the v a g i n a l o p e n i n g a n d runs into the ventral
c h a n n e l of the capsule g l a n d (type 4 *). In stage 5, the v a g i n a is either r e p l a c e d b y a small
prostate g l a n d (type a) or the v a g i n a l o p e n i n g is o c c l u d e d by proliferating vas deferens
tissue (type b). In t y p e c, the o n t o g e n e t i c closure of the pallial oviduct is incomplete;
either the b u r s a copulatrix alone or, additionally, the capsule g l a n d are split v e n t r a l l y for
up to two-thirds of its length. From this stage onwards, T B T - i n d u c e d i m p o s e x d e v e l o p m e n t l e a d s to reproductive failure a n d to sterility b e c a u s e copulation is i m p o s s i b l e a n d
e g g - c a p s u l e s can n e i t h e r b e r e l e a s e d in t y p e a a n d b nor b e p r o d u c e d in t y p e c. In stage
6, the l u m i n a of the capsule g l a n d a n d its v e s t i b u l u m are filled with an a c c u m u l a t i o n of
abortive e g g capsules, p r o v o k i n g an intensive swelling of the capsule g l a n d and, finally,
often a r u p t u r e causing the d e a t h of the animal. As a c o n s e q u e n c e of h i g h TBT e x p o s u r e
in early life stages, a sex c h a n g e m i g h t occur in the d o g w h e l k N. lapillus (Gibbs et al.,
1988; O e h l m a n n et al., 1991) a n d in Ocinebrina aciculata (Fioroni et al., 1991; O e h l m a n n
et al., 1996), w h i c h is c h a r a c t e r i z e d b y a s u p p r e s s e d oogenesis a n d a c o m m e n c e d
spermatogenesis.
A l t h o u g h the m o r p h o l o g i c a l aspects of T B T - i n d u c e d p s e u d o h e r m a p h r o d i t i s m have
b e e n i n v e s t i g a t e d intensively in m a n y p r o s o b r a n c h species b y different r e s e a r c h groups
(Fioroni et al., 1991), the d e t a i l e d b i o c h e m i c a l m e c h a n i s m of this p h e n o m e n o n has
r e m a i n e d obscure. F6ral & Le Gall (1983) d e m o n s t r a t e d in f e m a l e Ocenebra e r / n a c e a an
alteration of the n e u r o s e c r e t a of the p e d a l g a n g l i a after TBT-exposure w h i c h induces
TBT-induced imposex in snails
301
L~ te
,
S
0
cg
1
i,/
L
~
\/i
4 ~",d
J
sex
change
Fig. 1. General scheme of imposex evolution in prosobranchs. Abbreviations: ac, aborted capsules;
cg, capsule gland; gp, genital papilla; obc, open bursa copulatrix; ocg, open capsule gland;
ocv, occlusion of the vulva; p, penis; pd, pems duct; pr, prostate; te, tentacle; vd, vas deferens;
vdp, vas deferens passage into capsule gland; vds, vas deferens section
302
C. Bettin, J. O e h l m a n n & J. Stroben
d e v e l o p m e n t of a penis a n d vas deferens-sections. Our own radioimmunological determinations of sexual hormones in unaffected a n d affected females suggest the i n v o l v e m e n t
of steroids in the expression of imposex (Stroben et al., 1991). Pure females display the
lowest testosterone content, whereas a d v a n c e d imposex stages have the h i g h e s t testosterone content. Similar results in the d o g w h e l k were o b t a i n e d by Spooner et al. (1991).
These findings gave occasion for further experiments to investigate the i n f l u e n c e of TBT
on the steroid metabolism in H. reticulata a n d N. lapillus.
MATERIALS AND METHODS
All experiments were conducted with Nucella lapillus and Hinia reticulata. Because
of the w i d e s p r e a d coastal TBT pollution, it is virtually impossible to find p o p u l a t i o n s of
both species in Europe which are totally unaffected by imposex. The e x p e r i m e n t a l
specimens were collected in low-polluted areas of the French coast, in M 6 a n M61en (N.
lapillus) a n d Pointe de Pl6neuf (H. reticulata) where about 50 to 90 % of the females
already exhibit imposex d e v e l o p m e n t that was, however, still at a m o d e r a t e stage.
For the laboratory experiments, a 24- to 48-h static r e n e w a l system in 80-1itre glass
aquaria provided with Eheim power filters a n d artificial seawater (salinity 35 %0) was
used. The n a t u r a l t e m p e r a t u r e fluctuations at Roscoff (Brittany, France; 8.5 ~ in winter,
16 ~ in summer) were imitated. The experiments with application of different TBT
concentrations a n d testosterone were r u n u n d e r a constant photoperiod of 12:12 (L:D),
whereas all other experiments were carried out u n d e r n a t u r a l light conditions according
to season. For every experiment, 200 to 250 snails of each species were kept in two
connected aquaria, a n d a tide simulation system was employed.
E x p e r i m e n t 1. To e x a m i n e the c o n c e n t r a t i o n - d e p e n d e n t relationship b e t w e e n
TBT exposure a n d imposex development, N. lapillus a n d H. reticulata were exposed for 6
months to TBT at concentrations of 5, 50, or 100 n g as Sn/1.
E x p e r i m e n t 2. To investigate an i m p o s e x - i n d u c i n g capability of androgens,
the gastropods were exposed in this e x p e r i m e n t for 5 months to testosterone at a
concentration of 500 ng/1.
E x p e r i m e n t 3. To prove w h e t h e r the i m p o s e x - i n d u c i n g effects of TBT are
m e d i a t e d by androgens, the snails were simultaneously exposed for 5 m o n t h s to a TBT
concentration of 50 n g as Snfl with a n d without 1.25 mg/1 cyproterone acetate. The
a n t i a n d r o g e n cyproterone acetate reduces free a n d r o g e n b i n d i n g sites b y competitively
blocking the a n d r o g e n receptors without causing a n d r o g e n effects.
E x p e r i m e n t 4. To study the influence of estrogens in T B T - i n d u c e d p s e u d o h e r maphroditism, N. lapiflus a n d H. reticulata were kept for 5 months in s e a w a t e r which
contained 50 n g TBT as Sn/1 with a n d without 1 [tg/1 of a n estrone a n d 1713-estradiol
mixture (1:1).
E x p e r i m e n t 5. In this experiment, the effects of the specific a r o m a t a s e inhibitor
SH 489 a n d of the non-specific aromatase inhibitor flavone on both gastropods were
studied. SI--I489 (1-methyl-1,4-androstadiene-3,17-dione) used at a c o n c e n t r a t i o n of 0.3
mg/1 for 5 months b e l o n g s to the group of c o m p o u n d s which cause a competitive a n d
irreversible inhibition of the cytochrome P-450 d e p e n d e n t aromatization of a n d r o g e n s to
estrogens (Henderson, 1987). Flavone was u s e d at a concentration of 2.7 mg/1 seawater.
The analyses were conducted at m o n t h l y intervals (sample size 30 specimens) with
TBT~
imposex in snails
303
a n additional sample 2 weeks after starting the experiments. The s p e c i m e n s were
narcotized in a solution of 7 % MgC12 in distilled water. After cracking the shell with a
vice, the external properties of the genital tract i n c l u d i n g vas deferens extension and
penis length were m e a s u r e d to the n e a r e s t 0.1 mm. For imposex classification the snails
were sexed; the penis a n d vas deferens extension were m e a s u r e d a n d the imposex stage
according to Stroben et al. (1992a) determined. As imposex indices, the VDS (vas
deferens sequence) index as the average imposex stage of a population a n d the average
female penis l e n g t h were calculated.
Reagents
All solvents used for extraction a n d purification were p.a. grade and were obtained
either from Merck, G e r m a n y or J. T. Baker, Netherlands. Cyproterone acetate a n d SH
489 (1-methyl-l,4-androstadiene-3,17-dione) were generously supplied by Schering,
Germany. Flavone ( 2 - p h e n y l - 4 H - l - b e n z o p y r a n - 4 - o n e ) was purchased from Sigma, Germany. Tritiated steroids employed for the estimation of extraction recovery were purchased from bio Merieux, France. E n d o g e n o u s steroids of the snails were m e a s u r e d after
extraction by radioimmunoassays with 125j labelled tracers using the Clinical Assay T M
(Sorin Biomedica, Italy) for the d e t e r m i n a t i o n of 1713-estradiol and the Double Antibody
Assay (Diagnostic Products Corporation, USA) for the estimation of testosterone. To
approximate the matrix effects of the m e a s u r e m e n t , the standards of the 17B-estradiol
assay which were dissolved in h u m a n serum were replaced by our own standards in
borate buffer r a n g i n g from 0.25 to 25 pg 17~~
per tube. Radioimmunological
d e t e r m i n a t i o n of the steroids was carried out in duplicate according to the instructions of
the manufacturers. Since the testosterone antiserum reacts not only with testosterone but
also with dihydrotestosterone (34 % cross-reactivity), this a n d r o g e n was also registered
d u r i n g the m e a s u r e m e n t . The cross-reactivity to other naturally occurring steroids was
less t h a n 3.8 % for the testosterone a n t i s e r u m a n d 0.6 % for the 1713-estradiol antiserum.
Steroid extraction
Single w e i g h t e d specimens were h o m o g e n i z e d in 2 ml ethanol and frozen for at least
24 h. H o m o g e n a t e s were extracted at first with 8 ml of diethyl ether a n d in two further
extractions with 10 ml diethyl ether:ethanol (4:1, v/v) for 10 min. The solvent extracts
were c o m b i n e d a n d evaporated u n d e r a stream of compressed air in a w a t e r b a t h at 37 ~
After b e i n g redissolved in 2 ml 80 % methanol, the residues were washed twice for 5 min
with 5 ml p e t r o l e u m ether to remove the lipid fraction. The w a s h e d m e t h a n o l fractions
were evaporated to dryness and redissolved in 1 ml borate buffer for radioimmunological
determination. Serial dilutions of extracts gave o p t i m u m parallehsm to the standard
curves of the r a d i o i m m u n o a s s a y s used a n d indicated the suitability of the extraction
m e t h o d which can be used without extracting interfering substances. In order to estimate
the extraction efficiency, h o m o g e n a t e s were extracted after addition of either 3H labelled
testosterone or aH labelled 17~-estradiol. A n ahquot of each sample was c o u n t e d after
extraction in a h q u i d scintillation counter. Calculated recovery values were 62.2 ___5.04 %
for testosterone (n -- 9), a n d 84.3 __ 5.84 % for 17~-estradiol (n = 9). The p e t r o l e u m ether
phase used to remove the lipids c o n t a i n e d 3 . 1 2 •
of testosterone and
5.03 • 1.03 % of 17[~-estradiol.
304
C. Bettin, J. O e h l m a n n & J. Stroben
Organotin analysis
TBT a n d dibutyltin (DBT) c o m p o u n d s were d e t e r m i n e d according to S t r o b e n et al.
(1992a). O n e to five specimens were h o m o g e n i z e d in stoppered tubes a n d 10 ml of
c o n c e n t r a t e d HC1 was added. After s h a k i n g for 30 min. the tissues were extracted with 10
ml h e x a n e on a n automatic shaker for 30 rain a n d then centrifuged TBT as Sn plus DBT
as Sn were m e a s u r e d in the h e x a n e extract using a Perkin-Elmer HGA-500 attached to a
Perkin-Elmer 5000 AAS (wave l e n g t h 224.6 nm.. slit 0.7 nm; injection v o l u m e 25 ~tl). A
b a c k g r o u n d correction was used. TBT as Sn was d e t e r m i n e d in the h e x a n e extract after
s h a k i n g with 3 ml 1N N a O H for 3 min. Internal standardization (standard addition with
spiked samples) was employed. Certified reference material (CRM: PACS-1, delivered
by the National Research Council of Canada] was analysed additionally. O u r results were
within the s t a n d a r d deviation of the certified values of the CRM. Recovery factors were
92.3 ___9.4 % for DBT a n d 91.4 ___8.4 % for TBT. The detection limit (3a) in a single
sample was 7.4 n g DBT as Sn a n d 8.8 n g TBT as Sn.
RESULTS AND DISCUSSION
Exposure to TBT at different concentrations (5, 50, a n d 100 ng TBT as Sn/1) triggers a
concentration- a n d t i m e - d e p e n d e n t imposex d e v e l o p m e n t both in female Nucella Idpil]us a n d in female Hinia reticulata (Fig. 2). In both species, the average i m p o s e x stage of a
sample, t e r m e d as vas deferens s e q u e n c e (VDS) index, a n d the a v e r a g e female penis
l e n g t h increases with increasing TBT concentration a n d duration of TBT exposure.
Testosterone a d d e d at a concentration of 500 ng/1 induces in the a b s e n c e of TBT a faster
a n d more i n t e n s i v e imposex d e v e l o p m e n t compared to the applied TBT c o n c e n t r a t i o n s of
5 to 100 n g TBT as Sn/1.
In comparison with the controls, female gastropods of the TBT exposure groups
present i n c r e a s i n g e n d o g e n o u s testosterone titres which correlate with the TBT concentration a n d the duration of the e x p e r i m e n t (Fig. 3). After a 2 - m o n t h exposure, the
testosterone values for the highest TBT concentration in N. lapillus are significantly
different from those of the control; a n d after four months of exposure, the v a l u e s for the 50
a n d 100 n g TBT as Sn/1 exposure groups differ from those of the u n t r e a t e d animals. In
H. reticulata, differences of the testosterone titre from that of the control a p p e a r after
4 months exposure in all three TBT-treated groups. After 6 months, both species exhibit a
significantly higher testosterone content in all TBT exposure groups c o m p a r e d to the
c o n t r o l A m a r k e d a n d very high increase of the e n d o g e n o u s testosterone level is
m e a s u r e d in females of both species exposed to testosterone. The h i g h testosterone
values w h i c h are significantly different from those of the control group at all m o n t h l y
intervals a c c o u n t for the a d v a n c e d imposex d e v e l o p m e n t of this e x p e r i m e n t a l group
c o m p a r e d to all TBT-exposed animals a n d indicate the capability of the snails to
a c c u m u l a t e steroids from the a q u e o u s e n v i r o n m e n t . A comparison of the e n d o g e n o u s
testosterone content a n d the different imposex stages of both species in the TBT
e x p e r i m e n t s demonstrates that the more a d v a n c e d imposex stages exhibit higher testosterone levels in the tissue compared to those in pure females (imposex stage 0; cf. Figure
4). All values in N. lapillus a n d all values, with the exception of stage 0 versus stage 1, in
H. retictrlata are significantly different from each other.
-
0.0
0.5
1.0-
1.5-
2.0-
2.5-
3.0
3.5-
1.5
2.0
2.5-
3,0
3,5
4.0
2
month
3
4
0
1
2
month
3
4
J
average female penis length in mm
1
VDS I n d e x
I
i
5
A,
5
6
d
b
0.5
1.0
1.5-
2.0-
2.5
3.0-
1.5-
1.7
1.9
2.1
2.3
2.5
2.7
2.9
3.1
1
j
9
2
--
3
9
month
--
4
0
1
2
../
/
3
month
/
4
a v e r a g e f e m a l e penis length In mm
0
VDS I n d e x
5
5
#
6
6
9 500 n g testosterone/1
Fig. 2. T h e effect of TBT a n d testosterone e x p o s u r e on the d e v e l o p m e n t of the VDS index (a, b) a n d the p e n i s l e n g t h (c, d) in female Nucella
lapillus (a, c) a n d female Hinia reticulata (b, d). 9 control; 9 5 n g TBT as Sn/1; 9 50 ng TBT as Sn/1; 9 100 n g TBT as Sn/l; 9 t e s t o s t e r o n e control;
C
a
r
O
r
r
r
306
C. B e t t i n , J. O e h l m a n n
a
& J. S t r o b e n
t e s t o s t e r o n e titre ( p g / g w e t wt.) In f e m a l e s (n = 6)
2200
20001800 1600 140012001000 800 600 400 200 -
2
4
6
m o n t h s a f t e r start o f e x p e r i m e n t
b
t e s t o s t e r o n e tltre ( p g / g w e t wt.) In f e m a l e s (n = 6)
2400
22002000180016001400
1200
1000
600 600400200 0
i
1
2
4
S
m o n t h s a f t e r start o f e x p e r i m e n t
Fig. 3. The effect of TBT exposure on the e n d o g e n o u s testosterone titres in female NucelIa lapillus
(a) and female Hinia reticulata (b). 9 control; [] 5 ng TBT as Sn/1; [] 50 ng TBT as Sn/1; [] 100 ng TBT
as Sn/1. " Significantly different at p <0.01 versus control by Student's t-test (n = 6)
P r e v i o u s r e s u l t s s u g g e s t t h e i n v o l v e m e n t of a n d r o g e n s i n t h e i m p o s e x d e v e l o p m e n t
i n m a r i n e n e o g a s t r o p o d s , b u t g i v e n o e v i d e n c e t h a t t h e e f f e c t s of T B T a r e i n d u c e d e i t h e r
d i r e c t l y i n t h e o r g a n i s m or a r e m e d i a t e d b y a n i n c r e a s i n g a n d r o g e n l e v e l . T o g a i n t h i s
proof, t h e s n a i l s w e r e s i m u l t a n e o u s l y e x p o s e d to a T B T c o n c e n t r a t i o n of 50 r i g a s Sn/1 a n d
to t h e a n t i a n d r o g e n c y p r o t e r o n e a c e t a t e w h i c h is a c o m p e t i t i v e i n h i b i t o r of a n d r o g e n
r e c e p t o r s (Pig. 5). I m p o s e x d e v e l o p m e n t is c o m p l e t e l y s u p p r e s s e d i n /'4. lapillus. In
T B T - i n d u c e d i m p o s e x in snails
a
307
testosterone tltre (pg/g wet wt.)
2OOO
1600160014001200
1000
8OO
6OO
4002000
0
I
Imposex stage
b
testosterone tltre (pg/g wet wt.)
2000
1800
1600
1400
1200
1000
800
600
400
iiiiiii
200
0
0
2
3
4
Imposex stage
Fig. 4. Testosterone titres in the different i m p o s e x s t a g e s of female Nucella lapillus (a) a n d female
Hinia reticulata (b). T h e values for the single s t a g e s are with the exception of stage 0 versus stage 1
in/-/, reticulata significantly different from e a c h other (Student's t-test, p < 0.01, in N, lapillus stage 0:
n = 13; stage 1: n = 16; stage 2: n = 53; stage 3: n = 53; stage 4: n = 105;
in H. reticulata stage 0: n = 29; stage 1: n = 25; stage 2: n = 27; stage 3: n = 138; stage 4: n = 36)
f e m a l e H. r e t i c u l a t a , p e n i s g r o w t h is a l s o e n t i r e l y s u p p r e s s e d , w h e r e a s t h e i n c r e a s e of t h e
V D S i n d e x is o n l y r e d u c e d b u t n o t c o m p l e t e l y b l o c k e d .
T h e d i s t i n c t r e a c t i o n of b o t h s n a i l s m a y b e a t t r i b u t e d to a s p e c i e s - s p e c i f i c c o n t e n t of
a n d r o g e n s a n d to a d i f f e r e n t a n d r o g e n s e n s i t i v i t y of v a r i o u s t i s s u e s i n e a c h s p e c i e s .
3-
4
0
1
2
month
3
0
1
2
month
3
average female penis length In mm
1,0
1.5
2.0-
2.5
3.0
3.5
4.0-
VDS Index
4
4
5
5
d
b
4
0
1
2
month
'L'"
3
0
I
2
month
3
average female penis length In mm
1.0
1.5
2.0
2,5
3.0
'3.5
VDS Index
4
4
5
5
Fig. 5. T h e effect of TBT e x p o s u r e w i t h a n d w i t h o u t a d d i t i o n of t h e a n t i a n d r o g e n c y p r o t e r o n e a c e t a t e o n t h e d e v e l o p m e n t of t h e VDS i n d e x (a, b)
a n d t h e p e n i s l e n g t h fc, d) in f e m a l e Nucella lapillus (a, c) a n d f e m a l e Hinia reticulata Ib, d). 9 control., 9 50 n g TBT a s Sn/l; 9 1.25 m g c y p r o t e r o n e
acetate/l; 9 50 n g TBT as Sn/1 p l u s 1.25 m g c y p r o t e r o n e a c e t a t e / l
C
a
0
~q
9
N
Co
C~
O0
TBT-induced imposex in snails
309
Although the m e a s u r e d testosterone levels are almost in the same range, the concentration of the e n d o g e n o u s a n d r o s t e n e d i o n e may possibly be higher in H. reticulata than in
iV. lapi]lus. Androstenedione, one of the male sex hormones, can be aromatized to
estrogens by molluscs (Le Guellec et al., 1987) a n d was detected beside progesterone,
testosterone, 17B-estradiol, estrone a n d estriol in H. reticulata by HPLC a n d radioimm u n o a s s a y (Bettin, u n p u b l i s h e d results). A n d r o g e n action in a n d r o g e n - s e n s i t i v e tissues
requires the presence of a n d r o g e n receptors to mediate transcriptional activation. A
different regional distribution a n d intensity of a n d r o g e n receptors a m o n g the target
tissues m a y lead to a different reaction, although the tissues are exposed to the same
a n d r o g e n concentration. In both alternatives, higher a n d r o g e n content and/or higher
a n d r o g e n sensitivity of special tissues, the a d d e d a m o u n t of cyproterone acetate was not
sufficient in H. reticulata to block all a n d r o g e n receptors. The lack of a n d r o g e n receptors
in distinct tissues might be the reason for low or missing imposex d e v e l o p m e n t in some
gonochoristic prosobranch species, although their n a t u r a l e n v i r o n m e n t suffers from high
TBT pollution.
Mitochondrion
~, C~-Side-chainCleavage
I Pregnen~176 I
Microsome
S
Pregnenolone ]
~lr 3]3-Hydroxysteroid-Dehydrogenase
I PrOgesterOne ]
~lr 17cr
17c~-Hydroxyprogesterone I
~r C,~:o-Lyase
dependent Aromatase
\
[
Estrone
I
1
J 1713-Estradiol ]
/
Fig. 6. Scheme of biosynthesis of steroid hormones with possible target of TBT
310
C. Bettin, J. O e h l m a n n & J. Stroben
The results of this e x p e r i m e n t prove that the i m p o s e x - i n d u c i n g effects of TBT are
m e d i a t e d by a n increasing a n d r o g e n level a n d are not caused directly by the organotin
c o m p o u n d itself.
The p r e s e n t studies suggest that TBT disturbs the biosynthesis of steroid hormones
b y i n h i b i t i n g the aromatization of the a n d r o g e n s a n d r o s t e n e d i o n e and testosterone to the
estrogens estrone a n d 17fi-estradiol (Fig. 6). Steroid hormones, which have b e e n detected
i n various organs of mollusc species, control both the d e v e l o p m e n t a n d the function of
gonads a n d accessory sex glands as well as the sexual b e h a v i o u r during the reproductive
cycle (cf. Joosse, 1984; Joosse & Geraerts, 1983). Investigations on the g o n a d tissue of
some molluscs have d e m o n s t r a t e d that steroidogenesis takes place by following a
p a t h w a y very similar to that found in vertebrates (De Longcamp et al., 1974; Lupo di
Prisco & Dessi'Fulgheri, 1975). In vertebrates a n d m a n y invertebrates, i n c l u d i n g molluscs, a multifunctional oxygenase (MFO) system exists which catalyses the conversion of
a n d r o g e n s to estrogens (Kirchin et al., 1988; Livingstone et al., 1989, 1990; P a y n e et al.,
1991). The same MFO system is believed to be responsible for the TBT d e b u t y l a t i o n (Lee,
1985, 1986). We p r e s u m e that TBT leads to a competitive inhibition of the cytochrome
P-450 d e p e n d e n t aromatase a n d c o n s e q u e n t l y to an increase in the a n d r o g e n level.
It could b e expected that a n increase of the a n d r o g e n content a n d / o r a c h a n g e of the
a n d r o g e n - e s t r o g e n balance in favour of the a n d r o g e n s i n d u c e s the d e v e l o p m e n t of
pseudohermaphroditism. To verify this hypothesis, the gastropods were simultaneously
exposed to TBT at a concentration of 50 ng as Sn/1 a n d to an estrogen mixture c o n t a i n i n g
500 ng/1 171~-estradiol a n d 500 ng/1 estrone (Fig. 7). The a d d e d estrogens s u p p r e s s TBTi n d u c e d imposex d e v e l o p m e n t in both gastropod species. In N. lapillus an i n c r e a s e of the
VDS index a n d in H. reticulata an increase of the female penis length are completely
prevented, w h e r e a s the female penis growth in the dogwhelk a n d the increase of the
VDS index in I--Iinia are strongly reduced.
In Ix[. iapillus no significant c h a n g e was m e a s u r e d in the e n d o g e n o u s 17~-estradiol
content d u r i n g exposure to TBT at a concentration from 5 to 50 ng as Sn/1, w h e r e a s in
I-I. reticulata rising 17~-estradiol levels were found in all TBT exposure groups compared
to the control group after 4 months (Fig. 8). For the d e v e l o p m e n t of p s e u d o h e r m a p h r o d i Table 1. Effect of testosterone exposure (500 ng/1) on the TBT (tributyltin) and DBT (dibutyltin) body
burden in female Nucella lapillus and female Hinia reticulata
Body burden
in N. lapillus
TBT
DBT
(~tg as Sn/kg dry wt.)
Controla
Controlb
Testosterone
Exposure b
35.80
12.70
121.47
values at the beginning of experiment
b values 3 months after start of experiment
cnd, not detectable
13.50
42.13
nd c
Body burden
in H. reticulata
TBT
DBT
(~g as Sn/kg dry wt.)
44.20
16.48
52.29
132.00
37.78
nd c
0.0
0.5
1.0
1.5
2.0
2.5
3,0
3.5
1.0
1.5
2.0
2,5
3.0
3.5
4.0
1
2
month
3
0
1
f B - -
--
i
-B
2
--
--
month
--
3
average female penis length In mm
0
VDS Index
4
4
/
/
j
5
5
d
b
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
1.0
1.5
2.0
2.5
3.0
3.5
1
2
month
3
0
1
2
month
3
average f e m a l e penis length In mm
0
VDS I n d e x
4
I
4
5
5
Fig. 7. T h e effects of TBT e x p o s u r e w i t h a n d w i t h o u t addition of e s t r o g e n s on t h e d e v e l o p m e n t of t h e VDS i n d e x (a, b) a n d t h e p e n i s l e n g t h (c, d)
in f e m a l e Nucella lapillus (a, c) a n d f e m a l e Hinia reticulata (b, d). 9 control; 9 50 n g TBT as Sn/l; 9 500 n g 17~-estradiol/1 a n d 500 n g estrone/1;
9 50 n g TBT as Sn/1 p l u s 500 n g 17~-estradiol/1 a n d 500 n g estrone/1
C
a
09
r
t~
r
i
C. Bettin, J. Oehlmann & J. Stroben
312
a
17B-estradloltltre (pg/g wet wt.) in females (n = 6)
4so |
400 -]
350 -~
300 -]
,oo1
i
150 -~
100 -]
0
b
i
I
F
l
2
4
months after start of experiment
E
6
160 117R'ee~adl~ (Pg/g wet wt.) In females(n = 6)
140 ~
100
120 -~
~
t
.oI
t
i
6O
4O
2O
0
~
2
~
4
6
months after start of experiment
Fig. 8. The effect of TBT exposure on the endogenous 17~-estradiol titres in female Nucella lapillus
(a) and female Hinia reticulata (b). 9 control; [] 5 ng TBT as Sn/1; [] 50 ng TBT as Sn/1;
[] 100 ng TBT as Sn/1
tism, a s p e c i a l t h r e s h o l d v a l u e of a n d r o g e n s or a n a l t e r a t i o n of the a n d r o g e n - e s t r o g e n
r e l a t i o n m i g h t b e i m p o r t a n t . In N. lapfllus, t h e ratio of t e s t o s t e r o n e to i T ~ - e s t r a d i o l
i n c r e a s e s f r o m 6: i in u n a f f e c t e d a n i m a l s to 17:1 in snails e x p o s e d for 6 m o n t h s to 100 n g
TBT as Sn/1 (Fig. 9). In H. reticulata, a s i g n i f i c a n t c h a n g e in t h e t e s t o s t e r o n e - 1 7 ~ - e s t r a d i o l
ratio is not o b s e r v e d . In this m o l l u s c s p e c i e s , t h e m a i n steroids m a y b e a n d r o s t e n e d i o n e
a n d estrone, w h i c h w e r e not s e l e c t e d for m o n i t o r i n g in this study.
T B T - i n d u c e d i m p o s e x in s n a i l s
313
teetosterone/176-estradlol ratio In females (n = 6)
a
18
16
14
12
10
8
6
4
2
0
1
2
4
6
months after start of experiment
b
testosterone/17B-estradiol ratio In females (n = S)
18
16
10a ~//~ !
14
12
/
s
4
2
1
2
4
6
months after start of experiment
Fig. 9. The effect of TBT exposure on the ratio of testosterone to 17~-estradiol in female IVucella
lapillus (a) and female I-tinia reticulata (b). 9 control; [] 5 n g TBT as Sn/1; [] 50 n g TBT as Sn/1;
[] 100 n g TBT as Sn/1
F u r t h e r e v i d e n c e of a c o m p e t i t i v e i n h i b i t i o n of t h e c y t o c h r o m e P - 4 5 0 d e p e n d e n t
a r o m a t a s e s y s t e m b y t h e o r g a n o t i n c o m p o u n d w a s p r o v i d e d b y t h e d e t e r m i n a t i o n of t h e
e n d o g e n o u s c o n t e n t of T B T a n d its d e b u t y l a t i o n p r o d u c t D B T i n t h o s e s n a i l s e x p o s e d to
t e s t o s t e r o n e a t a c o n c e n t r a t i o n of 500 ng/1. D u r i n g t h i s e x p e r i m e n t , N. lapillus a n d
H. reticulata s h o w a n i n c r e a s e of e n d o g e n o u s T B T c o n t e n t a n d a d e c r e a s e of t h e
d e b u t y l a t i o n p r o d u c t DBT, w h e r e a s t h e c o n t r o l s s h o w a c o n t i n u o u s T B T d e b u t y l a t i o n . N o
-
0.0
0.5
1.0-
1.5-
2.0-
2,5
3.0-
1.0
1.5
2.0
2,5
3,0
3.5
4.0
month
2
0
1
month
2
j /
/ f
average female penis length In mm
1
3
3
d
b
0.0
0.5
1.0
1.5
2.0
2.5
3.0-
3.5
4.0-
1.0
1.5
2.0
2,5
3.0
3.5
1
2
month
3
~Ir
' ' ~ _
~e ~ I
0
~
I
2
month
3
-'-------"
4
-4)--
--
--
4
~ .--e-- -- _
average female penis length in mm
0
VDS Index
5
Fig. I0. Effect of the a r o m a t a s e inhibitor SH 489 ( 1 - m e t h y l - l , 4 - a n d r o s t a d i e n e - 3 , 1 7 - d i o n e ) on the d e v e l o p m e n t of the VDS i n d e x (a, b) a n d the
penis l e n g t h (c, d} in female Nucella lapillus (a, c) a n d female Hinia reticulata (b, d) c o m p a r e d to TBT exposure. 9 control; 9 50 n g TBT as Sn/li
9 0.3 m g SH 489/1
C
a
VOS index
r
O
r
9
n
,,~
C,O
T B T - i n d u c e d im p o sex in snails
315
DBT could b e d e t e c t e d in animals of either snail species that h ad b e e n e x p o s e d for three
months to testosterone; this indicates that TBT d e g r a d a t i o n had c e a s e d (Table 1).
Th e low TBT body b u r d e n in the e x p e r i m e n t a l control snails is due to the backg r o u n d TBT pollution of their original biotope. TBT accumulation in the gastropods is also
c a u s e d by TBT pollution of the supplied food (mussels and barnacles containing
12.8 __ 9.3 ~g TBT as S n / k g dry wt.; n = 8). But in n ei t h er mollusc species is the low
a c c u m u l a t e d e n d o g e n o u s TBT concentration able to i n d u c e a d v a n c e d i m p o s e x stages.
T h e results of th e s e e x p e r i m e n t s s u g g e s t that due to the different concentrations of both
substrates w h i c h are m e t a b o l i z e d by the cytochrome P-450 d e p e n d e n t a r o m a t a s e - a
h i g h e n d o g e n o u s testosterone and a low e n d o g e n o u s TBT content - the b a l a n c e of
m e t a b o l i s m is d i s p l a c e d in the direction of testosterone aromatization with the effect of
d e c r e a s i n g TBT debutylation.
If TBT inhibits the aromatization of androgens, specific aromatase inhibitors should
also be able to i n d u c e imposex. In a further experiment, the effects of the specific
steroidal a r o m a t a s e inhibitor SH 489 (1-methyl-l,4-androstadiene-3,17-dione) w e r e comp a r e d to a solvent control and to a TBT exposure group with 50 ng TBT as Sn/l (Fig. 10).
As ex p ect ed , the specific aromatase inhibitor shows the same i m p o s e x - i n d u c i n g effect as
a TBT-exposure. Both N. lapillus and H. reticulata d e m o n s t r a t e increasing VDS indices,
and in both snails the a v e r a g e female penis l e n g t h is significantly g r eat er than in the
control. I m p o s e x - i n d u c i n g effects w e r e also obtained in both mollusc species by using
flavone in a co n c e n tr a ti o n of 2.7 mg/1 s e a w a t e r as a non steroidal aromatase inhibitor
(Ibrahim & Abul-Hajj, 1990). Because of the lethal effects of the non specific ar o m at ase
inhibitor flavone, t h e s e e x p e r i m e n t s h a v e to be s u s p e n d e d after three months exposure
(data not shown).
The p r e s e n t studies suggest that TBT inhibits aromatization by c o m p e t i n g with
a n d r o s t e n e d i o n e a n d testosterone for the substrate b i n d i n g sites of the cytochrome P-450
d e p e n d e n t aromatase. The resulting increase of a n d r o g e n s w h i ch causes an alteration of
a n d r o g e n - e s t r o g e n b a l a n c e induces the d e v e l o p m e n t of p s e u d o h e r m a p h r o d i t i s m in
m a r i n e prosobranchs. Further b io c h e m i c a l studies on the structural elucidation of the
aro m at as e active sites m a y be helpful in d e l i n e a t i n g the m e c h a n i s m of action of the TBT
a r o m a t a s e inhibition.
Acknowledgements. The authors thank Schering AG (Berlin) for financial support and Prof. F. H.
Kemper, Prof. H.-P. Bertram and Dr. C. Mfiller for the possibility to carry out the organotin analysis at
the Environmental Specimen Bank for Human Tissue, Mtinster.
LITERATURE CITED
Blaber, S. J. M., 1970. The occurrence of a penis-like outgrowth behind the right tentacle in spent
females of Nucefla lapfllus {L.). - Proc. malac. Soc. Lond. 39, 231-233.
Bryan, G. W., Gibbs, P. E., Huggett, R. J., Curtis, L. A., Bailey, D. S. & Dauer, D. M., 1989. Effects of
tributyltin pollution on the mud snail, Ilyanassa obsoleta, from York River and Sarah Creek,
Chesapeake Bay. - Mar. Pollut. Bull. 20, 458-462.
Cardwell, R. D. & Sheldon, A. W., 1986. A risk assessment concerning the fate and effects of
tributyltins on the aquatic environment. In: Oceans '86 Conference Record. Vol. 4; Organotin
Symposium, Washington, D.C. IEEE Service Center, Piscataway, N.J., 1117-1129.
Chagot, D., Alzieu, C., Sanjuan, J. & Grizel, H., 1990. Sublethal and histopathological effects of trace
316
C. Bettin, J. O e h l m a n n & J. S t r o b e n
levels of tributyltin fluoride on adult o y s t e r s Crassostrea gigas. - A q u a t living Resoun 3,
121-130.
De Longcamp, D., Lubet, P. & Drosdowsky, M., 1974. The in vitro biosynthesis of steroids: by the
gonad of the mussel (Mytilus edulis). - Gem comp. EndocnnoL 22, 1~6--127.
Evans, D. W. & Laughlin, R. B., 1984. Accumulation of bis(tributyltin) oxide by the mud crab,
R h i t h r o p a n o p e u s harrasii. - C h e m o s p h e r e I3, 213-219,
Fent, K., 1991. Bioconcentration and elimination of tributyltin chloride by embryos and larvae of
minnows P h o x i n u s p h o x i n u s l - A q u a t . Toxicol. 20, 147-158.
Fent, K. & Meier, W., 1992. Tributyltin-induced effects on early life stages of minnows P h o x i n u s
p h o x i n u s . - Archs environ: Contain. Toxicol. 22, 428-438.
F4ral, C., 1974. Etude comparee des populations d ' O c e n e b r a erinacea (L.) de Granville et d'Arcachon. - Haliotis 4, 123-134.
F~ral, C., 1976a. Etude statistique de la pr6sence d'un tractus genital m&le externe chez les femelles
d'un Mollusque Gast~ropode gonochorique: O c e n e b r a erinacea (L.). - Cah. Biol. mar. 17, 61-76.
Feral, C., 1976b. Analyse experimental de la morphogenese et du cycle du penis chez les femelles
d' O c e n e b r a erinacea, espece g o n o c h o r i q u e . - Haliotis 6, 267-271.
F4ral, C., 1976c. R6partition geographique des femelles a tractus genital male externe chez
O c e n e b r a erinacea (L,), espece gonochorique. - Haliotis 7, 29-30.
F4ral, C., I980. Variations dans l'evolution du tractus genital male externe des femelles de trois
Gasteropodes Prosobranches gonochoriques de stations atlantiques. - Cah. Biol. mar. 21,
479-491.
F4ral, C. & Le Gall, S., 1983. The influence of a pollutant factor (tributyltin) on the neuroendocrine
mechanism responsible for the occurrence of a penis in the females of O c e n e b r a erinacea. In:
Molluscan neuro-endocrinology. Ed. by J. Lever & H. H. Boer. North-Holland Publ. Comp.,
Amsterdam, 173-175.
Fioroni, P., Oehlmann, J. & Stroben, E., 1991. The pseudohermaphroditism of prosobranchs;
morphological aspects. - Zool. Anz. 226, 1-26.
Gibbs, P. E. & Bryan, G. W., 1986. Reproductive failure in populations of the dog-whelk, N u c e l l a
lapfllus, caused by imposex induced by tributyltin from antifouling paints. - J. mar. biol. Ass. U.K.
66, 767-777.
Gibbs, P. E. & Bryan, G. W., 1987. TBT paints and the demise of the dog-whelk, N u c e l l a lapillus
(Gastropoda). In: Oceans '87 Conference Record. Vol. 4: Organotin Symposium, Halifax, Nova
Scotia. ]EEE Service Center, Piscataway, N.J., I482-1487.
Gibbs, P. E., Bryan, G. W., Pascoe, P. L. & Burt, G. R., 1987. The use of the dog-whelk, N u c e l l a
lapillus, as an indicator of tributyltin (TBT) contamination. - J. mar. bioL Ass. U.K. 67, 507-523.
Gibbs, P. E., Pascoe, P. L. & Butt, G. R., 1988. Sex change in the female dog-whelk, P4ucefla lapillus,
induced by tributyltin from antifouling paints. - J. mar. biol. Ass. U.K. 68, 715-731.
Hall, L. W. & Pinkney, A. E., 1985. Acute and sublethal effects of organotin compounds in aquatic
biota: an interpretative literature evaluation. - CRC crit. Rev. Toxicot. 14, 159-209.
Henderson, D., 1987. Aromatase inhibitors: their biochemistry and clinical potential. - J. Steroid
Biochem. 27, 905-914.
Ibrahim, A.-R. & Abul-Hajj, Y. J., 1990. Aromatase inhibition by fiavonoids. - J. Steroid Biochem.
molec. Biol. 37, 257-260.
Jenner, M. G., 1979. Pseudohermaphroditism: a newly recognized sexual p h e n o m e n o n in I1yanassa
obsoleta and other Neogastropoda. - Diss. Abstr. int. 40B, 117.
Joosse, J., 1984. Recent progress in endocrinology of molluscs. In: Biosynthesis, metabolism and
mode of action in invertebrate hormones. Ed: by J. Hoffmann & M. Porchet. Springer, Berlin,
19-35.
Joosse, J. & Geraerts, W. P. M., 1983. Endocrinology. In: The Mollusca. Ed. by A. S. NI. Saleuddin &
K. M. WilbuL Acad. Press, London, 4, 317-406.
Kirchin, M. A., Wiseman, A. & Livingstone, D. R., 1988. Studies on the mixed-function oxygenase
system of the marine bivalve M y t i l u s edulis. - Mar. environ. Res. 24, 117-118.
Langston, W. J., Butt, G. R. & Mingjiang, Z., 1987. Tin and organotin in water, sediments, and
benthic organisms of Poole Harbour. - Mar. Pollut. Bull. 18, 634-639.
Laughlin, R. B., French, W. & Guard, H. E., 1986. Accumulation of bis(tributyltin) oxide by the marine
mussel M y t i l u s edufis. - Environ. Sci. Technol. 20, 884-890.
TBT-induced
i m p o s e x in s n a i l s
317
Lee, R. F., 1985. M e t a b o l i s m of tributyltin oxide by crabs, oysters a n d fish. - Mar. environ. Res. 17,
145-148.
Lee, R. F., 1986. M e t a b o l i s m of bis(tributyltin)oxide by estuarine animals. In: Oceans '86 C o n f e r e n c e
Record. VoL 4: O r g a n o t i n Symposium, Washington, D.C. IEEE Service Center, Piscataway, N.J.,
1182-1188.
Le Guellec, D., Thiard, M.-C., Remy-Martin, J. P., Deray, A., Gomot, L. & Adessi, G. L., 1987. In vitro
m e t a b o l i s m of a n d r o s t e n e d i o n e a n d identification of e n d o g e n o u s steroids in Helix aspersa. Gen. comp. Endocrinol. 66, 425-433.
Livingstone, D. R., Arnold, R., C h i p m a n , K., Kirchin, M. A. & Marsh, J., 1990. The m i x e d - f u n c t i o n
o x y g e n a s e s y s t e m in molluscs: metabolism, r e s p o n s e s to xenobiotics and toxicity. - Oc~anis 16,
331-347.
Livingstone, D. R., Kirchin, M. A. & Wiseman, A., 1989. C y t o c h r o m e P-450 and oxidative m e t a b o l i s m
in molluscs. - Xenobiotica 19, 1041-1062.
Lupo di Prisco, C. & Dessi'Fulgheri, F., 1975. Alternative p a t h w a y s of steroid biosynthesis in g o n a d s
a n d h e p a t o p a n c r e a s of Aplysia depilans. - Comp. Biochem. Physiol. 50B, 191-195.
Martin, R. C., Dixon, D. G , Maguire, R. J., Hodson, P. V. & Tkacz R. J., 1989. Acute toxicity, uptake,
d e p u r a t i o n a n d tissue distribution of tri-n-butyltin in r a i n b o w trout, Salmo gairdneri. - Aquat.
Toxicot. I5, 37-52.
O e h l m a n n , J., Stroben, E. & Fioroni, P., 1991. The morphological expression of i m p o s e x in Nucella
lapillus (Linnaeus) (Gastropoda: Muricidae). - J. moll. Stud. 57, 375-390.
O e h l m a n n , J., Stroben, E. & Fioroni, P., 1992. The r o u g h tingle Ocenebra erinacea (Gastropoda:
Muricidae): an exhibitor of i m p o s e x in c o m p a r i s o n to Nucella lapillus. - H e l g o l a n d e r M e e r e s unters. 46, 311-328.
O e h l m a n n , J., Stroben, E. & Fioroni, P., 1996. Tributyltin (TBT) effects on Ocinebrina aciculata
(Gastropoda: Muricidae): i m p o s e x d e v e l o p m e n t , sterilization, sex c h a n g e a n d population
decline. - Zool. Anz. (In press).
Payne, E., Smith, J. F., Cope, B. C. & M c G o w a n , L. T., 1991. Studies on the role of hver c y t o c h r o m e
P-450 a n d oestradiol m e t a b o l i s m in the effects of nutrition a n d p h e n o b a r b i t a l on ovulation rate in
the ewe. - Reprod. Fertil. Dev. 3, 725-736.
Smith, B. S., 1971. Sexuality in the A m e r i c a n m u d snail, Nassarius obsoletus Say. - Proc. malac. Soc.
Lond. 39, 377.
Smith, B. S., 1980. T h e estuarine m u d snail, Nassarius obsoletus: abnormalities in the r e p r o d u c t i v e
system. - J . moll. Stud. 46, 247-256.
Smith, B. S., 1981a. Male characteristics in the female Nassarius obsoletus: variations r e l a t e d to
locality, s e a s o n a n d year. - Veliger 23, 212-216.
Smith, B. S., 1981b. Reproductive anomalies in s t e n o g l o s s a n snails related to pollution from marinas.
- J . appl. Toxicol. 1, 15-21.
Smith, B. S., 1981c. Male characteristics on female m u d snails c a u s e d by antifouling b o t t o m paints. J. appl. Toxicol. 1, 22-25.
Smith, B. S., 1981d. Tributyltin c o m p o u n d s i n d u c e m a l e characteristics on female m u d snails
Nassarius obsoletus = Ilyanassa obsoleta. - J . appl. Toxicol. I, 141-144.
Spooner, N., Gibbs, P. E., Bryan, G. W. & Goad, L. J., 1991. The effect of tributyltin u p o n steroid titres
in the female d o g w h e l k , Nucella lapillus, a n d t h e d e v e l o p m e n t of imposex. - Mar. environ. Res.
32, 37-49.
Stroben, E., O e h l m a n n , J. & Bettin, C., 1991. T B T - i n d u c e d i m p o s e x a n d the role of steroids in m a r i n e
snails. In: P r o c e e d i n g s of the T e n t h World M e e t i n g ORganoTin Environmental P r o g r a m m e
(ORTEP) Association, Berlin, S e p t e m b e r 26/27, 199I. Vlissingen, ORTEP Ass., 68-73.
Stroben, E., O e h l m a n n , J. & Fioroni, P., 1992a. The morphological expression of i m p o s e x in Hinia
reticulata (Gastropoda: Buccinidae): a potential biological indicator of tributyltin pollution. Mar. Biol. 113, 625-636.
Stroben, E., O e h l m a n n , J. & Fioroni, P., 1992b. Hinia reticulata a n d Nucella l a p i l l u s - c o m p a r i s o n of
two TBT bioindicators. - Mar. Biol. 114, 289-296.
Thain, J. E., 1986. Toxicity of TBT to bivalves: effects on reproduction, growth a n d survival. In:
O c e a n s '86 C o n f e r e n c e Record. Vol. 4: O r g a n o t i n Symposium, Washington, D.C. IEEE Service
Center, Piscataway, N.J., 1306--1313.
Thain, J. E. & Waldock, M. J., 1986. The impact of tributyltin (TBT) antifouling paints on molluscan
f i s h e r i e s . - Wat. Sci. Technol. 18, 193-202.
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