You are on page 1of 13

Marine Biology (2006) 149: 65–77

DOI 10.1007/s00227-005-0205-0

R ES E AR C H A RT I C L E

Alice Barbaglio Æ Daniela Mozzi Æ Michela Sugni


Paolo Tremolada Æ Francesco Bonasoro
Ramon Lavado Æ Cinta Porte
M. Daniela Candia Carnevali

Effects of exposure to ED contaminants (TPT-Cl and Fenarimol)


on crinoid echinoderms: comparative analysis of regenerative
development and correlated steroid levels

Received: 10 February 2005 / Accepted: 2 June 2005 / Published online: 6 January 2006
 Springer-Verlag 2006

Abstract Regenerative phenomena reproduce develop- the regenerative process from the macroscopic to the
mental processes in adult organisms and are regulated molecular level. The present investigation employs an
by neuro-endocrine mechanisms. They can therefore integrated approach which combines exposure experi-
provide sensitive tests for monitoring the effects of ments and biological analysis utilizing microscopy,
exposure to endocrine disrupter contaminants (EDs) immunocytochemistry and biochemistry. The experi-
which can be bioaccumulated by the organisms causing ments were carried out on experimentally induced arm
dysfunctions in steroid hormone metabolism and activ- regenerations in semistatic controlled conditions with
ities and affecting reproduction and development. Ech- exposure concentrations comparable to those of mod-
inoderms are prime candidates for this new erately polluted coastal zones. The bulk of results ob-
ecotoxicological approach, since (1) they offer unique tained so far provide indications of significant sublethal
models to study physiological regenerative processes and effects from exposure to TPT-Cl and Fenarimol and
(2) in echinoderms vertebrate-type steroids can be syn- mechanisms of toxicity related to developmental physi-
thesized and used as terminal hormones along the neuro- ology, which are associated with variations in steroid
endocrine cascades regulating reproductive, growth and levels in the animal tissues. The results indicate that
developmental processes. We are currently exploring the these two substances (1) affect growth and development
effects on the regenerative potential of echinoderms of by interfering with the same basic cellular mechanisms
different classes of compounds that are well known to of regeneration, such as cell proliferation, migration and
have ED activity. The present paper focuses on the differentiation/dedifferentiation, which are possibly
possible effects of well-known compounds with sus- controlled by steroid hormones; and (2) can induce a
pected androgenic activity such as TPT-Cl (Triphenyl- number of significant modifications in the timing,
tin-chloride) and Fenarimol [(±)-2,4-dichloro-a- modalities and pattern of arm regeneration, which may
(pyrimidin-5-yl) benzhydryl alcohol]. The selected test- involve the activation of cell mechanisms related to
species is the crinoid Antedon mediterranea, a tractable steroid synthesis/metabolism.
and sensitive benthic filter-feeding species which repre-
sents a valuable experimental model for investigation on

Communicated by R. Cattaneo-Vietti, Genova Introduction


Physical and Chemical Impacts on Marine Organisms, a Bilateral ‘‘Endocrine disrupters’’ (EDs) are xenobiotic com-
Seminar Italy-Japan held in November 2004
pounds that are persistent and widespread in the envi-
A. Barbaglio Æ D. Mozzi Æ M. Sugni Æ P. Tremolada ronment, and can be bioaccumulated by exposed
F. Bonasoro Æ M. D. C. Carnevali (&) organisms, affecting significantly their physiology, par-
Dipartimento di Biologia, Università degli Studi di Milano, ticularly in terms of reproduction, development and
Via Celoria 26, 20133 Milano, Italy
E-mail: daniela.candia@unimi.it growth. These contaminants exert their effects by mim-
Tel.: +39-02-50314788 icking the action of natural hormones, particularly ste-
Fax: +39-02-50314781 roids, interfering synergistically or antagonistically with
their synthesis, metabolism or activity and interacting
R. Lavado Æ C. Porte with their nuclear receptors (Cadbury 1998; Colborn
Environmental Chemistry Department, IIQAB-CSIC,
C/ Jordi Girona, 18, 08034 Barcelona, Spain et al. 1993; Cooper and Kavlock 1997; Fairley et al.
66

1996; Gray et al. 1996; Soto et al. 1995). There is a long they possess physiological mechanisms rather similar to
list of compounds (including pesticides, fungicides, those of vertebrates, in terms of molecules and actions.
insecticides, industrial and commercial chemicals and In some echinoderm classes there is limited but signifi-
drugs) known or suspected to act as hormonal modula- cant published evidence for the disruptive effects of
tors in human and wildlife populations. There are many contaminants on steroid metabolism and steroid levels
categories of pollutants displaying potential estrogenic/ and on the cytochrome P450 monooxygenase (MO)
anti-estrogenic activity, as well as androgenic/anti- system (den Besten 1998; den Besten et al. 1989, 1990,
androgenic activity. However, a complete understanding 1991a, b).
of their mechanisms of action on different organisms is A final important point is that echinoderms have
far from being achieved. This gap in knowledge affects spectacular capacity for regeneration, in addition to the
particularly invertebrates which represent more than normal processes of sexual reproduction. Regenerative
95% of the extant animal species in natural ecosystems. phenomena, which represent developmental processes in
In recent years aquatic invertebrates have been em- adult organisms, are characterized by enhanced and
ployed extensively for monitoring environmental haz- active cell proliferation, morphogenesis, differentiation
ards and as sensitive test models for EDs (Candia and tissue renewal, which are modulated by endocrine
Carnevali 2005; Mu and LeBlanc 2002). and neurohumoral mechanisms comparable, if not
The presence of ED contaminants in the sea has an identical, to those involved in reproductive and devel-
obvious impact on the physiology of marine inverte- opmental processes. Vertebrate-type regulatory factors,
brates (Depledge and Billinghurst 1999), with particular including peptides and steroids, are also likely to be in-
reference to benthic species living in close contact with volved in the regeneration processes (Candia Carnevali
contaminated sediments. The phenomena of imposex et al. 2001b; Thorndyke and Candia Carnevali 2001).
and intersex described in gastropod molluscs are the For this reason regenerating echinoderms can be used as
best-documented examples of adverse effects of ED experimental models to test the effects of exposure to
contaminants in marine animals (Bryan et al. 1986; different types of EDs (Candia Carnevali 2005). Expo-
Gibbs et al. 1988; Horiguchi 1995). On the other hand, sure to pseudohormonal contaminants has already been
with regard to important marine macro-invertebrates shown to induce variations, in the timing, mechanisms
such as echinoderms, the available data are still rather and actions of regenerative development, which is
limited. Although they offer a wide range of sensitive amongst the most sensitive phenomena with respect to
and amenable models, they have been only occasionally environmental stress. Previous data obtained from the
employed in ecotoxicological tests using adult organisms ophiuroids Ophioderma brevispina (Walsh et al. 1986)
(Anderson et al. 1994; Békri and Pelletier 2004; Coteur and Microphiopholis gracillima (D’Andrea et al. 1996)
et al. 2001; den Besten 1998; den Besten et al. 1989, 1990, showed clearly that exposure to organotin compounds
1991a, b; Kobayashi 1984) or developmental stages and metals significantly affects arm regeneration pro-
(Novelli et al. 2002). There are many factors that make cesses, and demonstrates the usefulness of studying
echinoderms the prime candidates for studying the ef- regenerative development in adult organisms. Regener-
fects of exposure to ED contaminants. Firstly, echino- ating echinoderms thus appear to be ideal bioindicators
derms are benthic animals and are particularly for ED-induced stress at the whole organism, cellular
susceptible to the presence of micropollutants stored in and molecular levels.
marine sediments. Primary uptake across external epi- An important goal in studying EDs is establishing the
thelia (respiratory surfaces, epidermis, etc.) or secondary most sensitive test-species and the most specific forms of
uptake from food, represent important routes of entry response (endpoints) at which hormonal dysfunction is
for many dissolved aquatic pollutants which can be expressed unequivocally. Unique endocrine-regulated
rapidly bioaccumulated by these organisms (Smith et al. processes, such as echinoderm regeneration, can provide
1981; Tremolada et al. 2004). Secondly, regulatory fac- an important target of toxic action and original and
tors and hormones similar to those of vertebrates have quantifiable endpoints which can provide indications for
been detected recently in echinoderms (Dieleman and the specific effects of these persistent pollutants.
Schoenmakers 1979; Hines et al. 1994; Janer et al. 2004). The present work is focused on the effects of two
In particular, vertebrate-type steroids, both androgens potential endocrine disrupter compounds, TPT-Cl (tri-
and estrogens, can be synthesized (Aminin et al. 1995; phenyltin-chloride) and Fenarimol ((±)-2,4-dichloro-a-
den Besten et al. 1989; LeBlanc et al. 1999; Voogt et al. (pyrimidin-5-yl) benzhydryl alcohol), on the regenera-
1984, 1990, 1991; Shirai and Walker 1988; Schoenmak- tive potential of crinoid echinoderms, with particular
ers 1979, 1980; Schoenmakers and Voogt 1980; Shubina reference to the well-known process of arm regeneration.
et al. 1998). Current research is focused on echinoderm TPT-Cl is an organotin compound which is used
endocrinology and knowledge of the specific mecha- extensively in agriculture and in antifouling paints. TPT
nisms involved, including steroid metabolism, is concentrations have been detected in terrestrial, fresh-
expanding rapidly (Janer et al. 2004; Lutz et al. 2004). water and coastal/marine environments. This compound
Echinoderms are deuterostome invertebrates and are is well known for its androgenic activity (Fait et al. 1994;
phylogenetically closer to chordates than to other Matthiessen and Gibbs 1998), although no reliable data
invertebrate groups. It is not surprising, therefore, that are presently available on its specific mechanisms of
67

action. There is some published information on the ef- different concentrations of the selected compounds
fects of organotin compounds on marine invertebrates, (TPT-Cl: 50, 100, 225, 500, 1,000 ng/l; Fenarimol: 24,
including a few echinoderms (Depledge and Billinghurst 240, 2,400 ng/l) for prefixed periods (72 h, 1 and
1999; Walsh et al. 1986). 2 weeks): in this way the exposure period corresponded
Fenarimol is a halogenated pesticide, particulary a to well defined and established regenerative stage (Can-
fungicide, employed against several plant diseases (IPCS dia Carnevali and Bonasoro 2001). As far as the selected
2004). There are no available data about environmental TPT-Cl exposure concentrations are concerned, the
levels of this pesticide. The presence of Fenarimol in the maximum concentration was close to LC50 experimental
sediments is suspected to be dangerous for aquatic spe- values quoted in the literature for molluscs (Rippen
cies, particularly for benthic organisms. 1990), the minimum to NOEC experimental values
Our specific aim is to explore the impact of these known for echinoderms (O. brevispina, Walsh et al.
pollutans on crucial developmental processes such as 1986). With regard to Fenarimol, the maximum and the
repair, growth and differentiation at the whole organ- minimum concentrations were chosen on the basis of the
ism, tissue and cellular level. The selected experimental minimum EC50 available data (0.18 mg/l for Daphnia
animal is the crinoid Antedon mediterranea, an echino- magna—Bell 1994) reduced by factors of 10 and 10,000.
derm species representative of marine benthic fauna and The TPT-contaminated medium was obtained by
a typical microfilter-feeding animal on which persistent adding to the aquaria (50 l artificial seawater) 1.25 ml
sediment-bound micropollutants have an immediate ethanol–TPT-Cl solution (TPT-Cl concentrations: 2, 4,
impact. This experimental model was employed suc- 9, 20, 40 lg/ml, respectively) at the start of the experi-
cessfully in our laboratory for pilot ecotoxicological ment and 0.25 ml ethanol–TPT-Cl solution (TPT-Cl
studies. Definitive or preliminary results obtained so far concentrations: 2, 4, 9, 20, 40 lg/ml, respectively) day by
on the effects on crinoid regeneration of a wide range of day. The Fenarimol-contaminated medium was ob-
different ED compounds, such as PCBs (Polychlorinated tained by adding to the aquaria (50 l artificial seawater)
biphenyls), nonylphenols, organotins (Barbaglio et al. 1 ml ethanol–Fenarimol solution (Fenarimol concen-
2004; Candia Carnevali 2005; Candia Carnevali et al. trations: 1.2, 2, 12, 120 lg/ml, respectively) at the start
2001a, b, c), showed that the regenerative response of of the experiment and 0.2 ml ethanol–Fenarimol solu-
echinoderms is a valuable test for EDs and strongly tion (Fenarimol concentrations: 1.2, 2, 12, 120 lg/ml,
encouraged us to develop this applied research field. respectively) day by day. The final ethanol concentration
This paper focuses on the effects of TPT-Cl and in exposure and in solvent control aquaria was 0.025 and
Fenarimol on arm regeneration of the echinoderm 0.020 ml/l for TPT-Cl and Fenarimol, respectively: these
A. mediterranea. It provides a comparative account of concentrations are much lower than that (0.1 ml/l) offi-
(1) the possible damage and alterations to the regener- cially allowed in long-term ecotoxicity tests with aquatic
ative development detectable at whole organism, tissue invertebrates (see Annex V, Dir67/548/EEC, EEC 1967).
and cellular levels due to exposure to these suspected At each selected regenerative stage, chemical analyses
endocrine disrupter compounds, and (2) the correlated of water and echinoderm tissues were performed in or-
fluctuations of testosterone and estradiol in the animal der to check the variability of exposure concentration
tissues. and bioaccumulation (Tremolada et al. 2005). TPT-Cl
Preliminary results were presented at the 11th Inter- analyses were performed by gas-chromatographic sepa-
national Echinoderm Conference, Munich 2003 (Bar- ration and mass-spectrometry detection after derivati-
baglio et al. 2004). zation of the original compound (TPT-Cl) in the
extraction medium. Fenarimol analyses were performed
by gas-chromatographic separation. In terms of chemi-
Materials and methods cal parameters, a detailed chemical analysis of water and
tissue samples from our exposure experiments is still in
Exposure experiments progress (Dagnac et al., unpublished; Sakkas et al.,
unpublished).
Specimens of A. mediterranea, collected from the Tyr-
rhenian coast of Italy (Giglio Island), were maintained in
aquaria of artificial seawater at 14C, and fed with In- Biological analyses
verteMin (Tetra Marin). Exposure tests for both TPT-Cl
(Merck) and Fenarimol (Riedel) were performed in Histological analysis
semistatic conditions (20% water renewal in 24 h).
Groups of 30 specimens (TPT tests) and 35 specimens Standard methods for morphological analysis by both
(Fenarimol tests) were employed in each aquarium stereomicroscope and light microscope, as described in
(exposure, control and solvent control aquaria). In each previous papers (Candia Carnevali et al. 1993), and
exposed or control specimen, experimental regeneration specific immunocytochemical protocols for monitoring
was induced by amputating three arms at the autotomy cell proliferation (BrdU methods) were employed. A
plane. Immediately after amputation, the experimental statistical analysis of the quantitative results was per-
animals were put in the test-aquaria and exposed to formed whenever appropriate.
68

Exposed and control regenerating arms (three arms using commercial RIA kits. Standard curves with the
from each individual) were prefixed with 2% glutaral- steroids dissolved in the same phosphate buffer were
dehyde in 0.1 M cacodylate buffer for 4–5 h, then, after performed in every run. The detection limits were of
overnight washing in the same buffer, postfixed with 1% 30 pg/g for E2 and 130 pg/g for T. Intra-assay coeffi-
osmium tetroxide in the same buffer. After standard cients of variation were of 6.1 (T) and 3.3% (E2). In-
dehydration in an ethanol series, the samples were terassay coefficients of variation were 9.3 (T) and 3.5%
embedded in Epon-Araldite 812. In particular, four ex- (E2).
posed samples per each exposure concentration and four
control samples at all the regenerative stages were
analysed microscopically. The semithin sections, cut Statistical analysis
with a Reichert Ultracut E, were stained by conventional
methods (crystal violet-basic fuchsin) and then observed Growth
in a Jenaval light microscope.
Cell proliferation was monitored using in vivo Lengths of regenerating arms were measured using the
incorporation of the substituted nucleotide, 5-bromo- software ArcView GIS 3.2 and, with regard to TPT-Cl
deoxyuridine (BrdU), then revealed by a monoclonal tests, comparison of significant difference (P<0.05) was
antibody against BrdU (Cell proliferation kit: Amer- made with multifactorial ANOVA.
sham). This same DNA synthesis labelling technique has
been previously used successfully to monitor early and
advanced stages of arm regeneration in Antedon (Candia Steroid levels
Carnevali et al. 1995, 1997). For use with semithin
Epon-Araldite sections, the standard BrdU-immunocy- Results are presented as mean values ± SEM. Statistics
tochemistry protocol for paraffin sections was modified were applied on log-transformed data, because depen-
as described in detail by Candia Carnevali et al. (1995). dent variables did not satisfy the assumption of para-
In particular, two exposed samples per each exposure metric tests. Comparison of significant differences
concentration and two control samples at all the (P<0.05) was made with one-way ANOVA followed
regenerative stages were analysed microscopically. with Tukey’s test multiple range test and Student’s t test.
The results of the exposure tests were compared with All statistics and graphics were analysed using the
those obtained by a parallel analysis of normal regen- software SPSS v10.0 and Excel 2000.
erating samples in standard conditions.
Results
Biochemical analysis
Mortality
Steroid analysis was performed on whole animal sam-
ples, as described in Janer et al. (2004). Briefly, indi- The concentration of 1,000 ng/l TPT-Cl appeared to be
viduals (1.0–1.5 g wet weight) were homogenized in close to the acute toxicity threshold for A. mediterranea.
ethanol, and frozen overnight at 80C. Homogenates In specimens exposed to this concentration 50% mor-
were then extracted with ethyl acetate twice. The organic tality was evident within the first 72 h (LC50), whereas in
extract was evaporated under nitrogen, and redissolved the surviving samples growth did not progress further
in 80% methanol. This solution was then washed with and regenerative development appeared to stop at early
petroleum ether to remove the lipid fraction and evap- stages of 24–72 h postamputation (p.a.).
orated to dryness. The dry residue was redissolved in In all the other TPT-Cl experiments, as well as in all
4 ml Milli-Q water and passed through a C18 cartridge Fenarimol exposure tests, the overall mortality in both
(Isolute, 1 g, 6 ml) that had been sequentially precon- exposed and control samples was less than 20%, a value
ditioned with methanol (4 ml) and milli-Q water (8 ml). which is considered acceptable for the validity of eco-
After finishing the concentration step, cartridges were toxicological results (see Annex V, Dir67/548/EEC, EEC
washed with milli-Q water (8 ml), dried, and connected 1967).
to a NH2 cartridge (Waters, Sep-Pack Plus). The C18-
NH2 system was then washed with 8 ml hexane, and the
steroids eluted with 9 ml dichloromethane:methanol Growth and malformations
(7:3). This fraction was collected and evaporated to
dryness. The efficiency of the extraction and delipidation Morphological and morphometric analyses using the
procedure was 74±3% for T, and 80±3% for E2 (Janer stereomicroscope allowed us to quantify the overall re-
et al. 2004) and 95 (E2) to 97% (T) for the purification growth of the regenerates, particularly at the advanced
step (SPE cartridges) (Janer et al. 2004). stages, and to identify possible malformations in the
Dry extracts were resuspended in 50 mM potassium exposed samples which could be attributed to individual
phosphate buffer pH 7.6 containing 0.1% gelatin, and compounds in concordance with what had been seen
assayed for estradiol and testosterone concentration previously (Barbaglio et al. 2004; Candia Carnevali
69

2005; Candia Carnevali et al. 2001a, c, 2003). The results malformations need to be confirmed by histopathologi-
obtained from the different series of tests performed with cal analysis (see below).
TPT-Cl and Fenarimol, respectively, were neither uni-
form nor easily interpretable from a qualitative or
quantitative point of view. As far as overall growth is Regeneration
concerned at the advanced stage of 1 and 2 weeks that
corresponded to long-term exposure periods, both TPT- As far as TPT-Cl is concerned, for the reasons stated
Cl and Fenarimol samples displayed great variability in above, the highest concentration of 1,000 ng/l could not
terms of overall growth in comparison with the uniform be considered further in the present analysis. On the
growth of the controls. With regard to TPT-Cl-exposed other hand, the 50 ng TPT-Cl/l-exposed samples did not
samples alone, a clear effect in terms of enhanced growth show at any developmental stage any significant differ-
could be detected at the advanced stage of 2 weeks and ence from the controls. The following results therefore
with the intermediate-high concentration employed refer to all the other TPT-Cl and Fenarimol exposure
(225 ng/l), as shown by a quantitative analysis carried experiments. They indicate clearly that exposure to these
out on the measured lengths of all the experimental compounds significantly affects regenerative develop-
regenerates in both exposed and control samples. Fig- ment, all the exposed samples showing obvious anom-
ure 1 shows that, according to the statistical analysis alies in microscopic anatomy (Figs. 2, 4). Preliminary
carried out for 1 and 2 weeks p.a. stages, 225 ng/l ap- results were also obtained regarding cell proliferation,
pears to induce a significant (P<0.05) increase in the by employing the BrdU method (Candia Carnevali et al.
growth of the regenerate. 1995, 1997).
The presence of possible malformations in terms of
general morphology and external anatomy was also ex-
plored preliminarily in specimens exposed to TPT-Cl Early regenerative phase (72 h p.a.)
and Fenarimol in comparison with controls. During the
advanced regenerative phase (1–2 weeks), abnormalities In terms of general morphology, at the early regenera-
in overall morphology and posture were observed fre- tive stage of 72 h p.a., which corresponded to a short-
quently in the exposed regenerating arms (about 77% in term exposure period, although the exposed samples did
samples exposed to the highest concentration for Fe- not show any appreciable external alterations when
narimol) and this encouraged us to take into account compared to the controls, some significant differences in
this parameter as an easily quantifiable endpoint. terms of histological pattern were detectable.
However, these effects could overlap partly to, and be Sagittal histological sections of TPT-Cl-exposed
masked easily by, extrinsic effects due to other factors, samples showed the development of what could be de-
i.e. tissue shrinkage caused by fixation, contraction fined a pseudoblastema. The regenerate, externally rec-
reactions to traumatic amputation, etc. For these rea- ognizable as a regrowing bud, did not consist of the
sons the morphological results related to the external usual blastema of undifferentiated cells (Fig. 2a), but

Fig. 1 Overall regenerative


growth (expressed as length of
regenerating arms) at 1 and
2 weeks postamputation (p.a.)
in samples exposed to TPT-Cl.
Data are presented as
mean ± SEM. At
concentration of 225 ng/l the
regenerate growth increases in
comparison with the control
(CTL) and the solvent control
(CTLs). The 50 ng/l-exposed
samples do not show any
difference in comparison with
the controls
70

Fig. 2 Histological sagittal sections of control and exposed namely skeletal spicules (arrow) usually not present at this early
regenerating arms at 72 h p.a.: a Control. The blastema consists stage. d Fenarimol-exposed sample. 240 ng Fenarimol/l. Tissue
of morphologically undifferentiated cells. b, c TPT-Cl-exposed repair is still incomplete: in spite of the presence of a thin,
samples. b 100 ng TPT-Cl/l. The blastemal bud appears to be continuous external cicatricial layer, a wide empty area (asterisk) is
formed mostly from ectopic myocytes (arrow head) supplied by the clearly evident at the level of the brachial nerve b blastema; cc
apparent rearrangement of the muscle bundle of the stump. coelomic canals; m muscle bundles; n brachial nerve
c 225 ng TPT-Cl/l. The blastemal bud consists of ectopic elements,

was characterized by the presence of many differentiated term exposure periods, significant anomalies could be
nonblastemal elements, such as myocytes, apparently detected particularly in the samples exposed to inter-
migrating from the distal muscles of the stump (Figs. 2b, mediate or higher concentrations of both the com-
5b) and scattered skeletal spicules (Fig. 2c). pounds (Fig. 4).
Another abnormal feature concerned the presence In sagittal sections the aberrant anatomy of the
and the functional state of granulocytes, which are regenerate could be appreciated even at low magnifica-
normally employed in crinoid repair processes (Heinz- tion. It was generally characterized by an unusual
eller and Welsch 1994). In TPT-Cl-exposed samples a developmental pattern of tissues, with particular refer-
large number of granulocytes were detected close to both ence to a pronounced abnormal development of the
the coelomic canals and brachial nerve, where they ap- components of the skeletal tissue (Fig. 4b, c, e, f). All
peared to release their cytoplasmic granules (Fig. 3). TPT-Cl- and Fenarimol-exposed samples exhibited
Similar degranulation phenomena were found also in the scattered skeletal spicules (Fig. 4b, c). The phenomenon
advanced regenerative stages. was particularly evident in Fenarimol samples: in the
As far as Fenarimol-exposed samples are concerned, regenerating arm the ossicles were smaller on the whole
the atypical features were less conspicuous. At the than in the controls (Fig. 4a) and looked much less
highest concentrations employed, the sagittal sections of developed than other anatomical elements (ambulacral
the regenerating arms displayed an unusual empty area grooves, coelomic cavities, brachial nerve, etc.) (Fig. 4c).
delimited by a thin cicatricial layer, in correspondence The only exception was the first ossicle of the series
with the brachial nerve end (Fig. 2d). (proximal with respect to the amputation surface) which
As far as cell proliferation is concerned, our BrdU appeared always to have developed normally. In addi-
incorporation experiments (not shown) indicated that in tion, in Fenarimol samples (Fig. 4f), the ossicles were
both TPT-Cl and Fenarimol samples, at this early stage, characterized by a less compact and irregular stereom
the cell proliferation pattern was only slightly different macro- and microstructure than in the controls
from that of the controls, the possible differences (Fig. 4d).
becoming evident only at more advanced stages (see Besides these abnormalities related to the skeleton,
below). TPT-Cl- and Fenarimol-exposed samples showed an
extensive rearrangement and/or dedifferentiation of
Advanced regenerative phase (1 and 2 weeks p.a.) other differentiated tissues, specifically involving the
muscle bundles of the stump (Fig. 5a–c). In particular,
During the advanced regenerative phases of 1 and the 1-week samples exposed to the highest concentra-
2 weeks p.a., which corresponded to medium and long- tions of Fenarimol exhibited also a conspicuous
71

nerve and the coelomic epithelium. However, in all the


exposed samples, the labelling at these sites appeared to
be less strong and widespread than in the controls
(Fig. 6b).
A similar situation was detected in 1-week Fenarimol
samples (Fig. 6c), whereas in 2 weeks samples the pro-
liferation pattern appeared to be more similar to the
standard level for this stage.

Tissue steroid levels

Steroid levels were determined in whole animal samples


after 2 weeks exposure. A significant increase in testos-
terone levels was detected in those organisms exposed to
225 ng/l TPT. The amount of testosterone detected was
440±140 pg/g wet weight, double the levels detected in
the control group. Interestingly, this effect was associ-
ated with a corresponding significant decrease in estra-
diol levels at the same exposure level (Fig. 7a). This
apparent androgenization effect was more clearly ob-
served when the ratio E2/T was calculated. This ratio
ranged from 1.22±0.44 (control) and 0.83±0.34 (sol-
vent control) to 0.19±0.04 in individuals exposed to
225 ng/l TPT (P<0.05).
In contrast to TPT, exposure to fenarimol resulted in
no significant differences in testosterone levels between
control and exposed groups; only estradiol levels in-
creased significantly (230±128 pg/g wet weight in
organisms exposed to the medium dose (225 ng/l) when
compared to controls (28–44 pg/g wet weight) (Fig. 7b).

Discussion and conclusions

In order to elucidate the possible pseudohormonal ac-


tion of ED compounds, the tendency in the past was to
develop in vitro assays. However, a complete under-
standing of the different mechanisms of action of diverse
substances requires parallel in vivo studies, which are
necessary, because in vitro and in vivo effects can be
different (Degen and Bolt 2000). It is therefore necessary
to identify clear and reproducible effects of exposure to
Fig. 3 Light microscopy details of control and exposed regerating
ED compounds in vivo by means of sensitive and rele-
arms at 72 h p.a. showing the coelomic canals of the stump. a vant experimental models. For this reason our ecotoxi-
Control. The granulocytes located close to coelomic canals (arrow) cological study of the effects of two well-known
appear to be intact. b 225 ng TPT-Cl/l. Remarkable degranulation endocrine disrupters on the regenerative development of
of granulocytes is evident. Many cytoplasmatic granules (arrow) a common marine species, the echinoderm A. mediter-
are free and clearly detectable close to the coelomic canals. Only a
few granulocytes are still intact (arrowhead). cc coelomic canal ranea (tested recently in pilot-experiments: Barbaglio
2004; Candia Carnevali 2005; Candia Carnevali et al.
2001a, c, 2003), is particularly timeous.
bi-directional migration of myocytes between adjacent As seen above, TPT-Cl is an organotin widely
muscle bundles (Fig. 5c). employed in agriculture and in antifouling paints. TPT
As far as cell proliferation is concerned, our BrdU concentrations has been detected in all the environ-
incorporation experiments showed that in TPT-Cl mental compartments. As far as the Mediterranean
samples, at both the stages of 1 and 2 weeks, cell Sea is concerned, Caricchia et al. (1992) reported a
proliferation phenomena were always localized in the concentration of 0.011 lg TPT/l in La Spezia gulf,
same proliferation sites as in the controls (Fig. 6a), whereas Tolosa et al. (1996) measured a concentration
namely at the level of the apical blastema, the brachial range from 1 to 29 lg TPT/l in the Côte d’Azur
72

Fig. 4 Histological sagittal sections of control and exposed and appear to be poorly developed if compared with other
regenerating arm at 1-week p.a. a Control. Normal tissue histological elements (ambulacral grooves, coelomic cavities,
differentiation is in progress in the regenerate. The proximal–distal brachial nerve). d Detail of a control sample to show the compact
development of the brachial ossicles is evident: the proximal and structure of a differentiating ossicle. e Detail of 225 ng TPT-Cl/l-
the distal ossicles are indicated by arrow and arrowheads, exposed sample. The ossicle structure appears to be poorly
respectively. b 225 ng TPT-Cl/l. The regenerating arm shows developed and rather disorganized. f Detail of 240 ng Fenarimol/
anomalous histological features: the developing distal ossicles l-exposed sample. The development of the skeletal tissue is clearly
(arrowheads) are similar in size to the proximal one (arrow). c delayed. Several skeletal spicules are scattered and disarranged. cc
240 ng Fenarimol/l. With the only exception of the proximal ossicle coelomic canals; ga ambulacral grove; m muscle bundle; n rachial
of the series (arrow), all the ossicles (arrowheads) are generally small nerve

coastline. This contaminant tends to be accumulated available data about environmental levels of this pesti-
in soil and sediments (Federoff et al. 1999), but due to cide. In spite of this, its Henry’s law constant
its strong affinity for tissues (Kow=3.1; Federoff et al. (6.93 s·10 4 Pa·m3/mol) suggests a moderately low
1999), it can be found at quite a high level in volatility (Mackay et al. 1997), implying that it may
organisms. This compound is well known for its reach surface waters quickly by direct overspray or by
androgenic activity (Fait et al. 1994; Matthiessen and run off from treated fields (IPCS 2004). It is rapidly
Gibbs 1998), although no reliable data are presently transferred to the sediments (Jackson and Lewis 1994)
available on its specific mechanisms of action. There is where it tends to persist because of its stability (Fitox
some published information on the effects of organo- 1999; Kline and Knox 1981; IPCS 2004). For these
tin compounds on marine invertebrates, including a reasons the presence of Fenarimol in the sediments is
few echinoderms (Depledge and Billinghurst 1999). suspected to be dangerous for benthic organisms. Fe-
Particularly relevant are their effects on reproductive narimol shows moderate values of toxicity in aquatic
biology (see imposex in gastropod molluscs, Horiguchi organisms: EC50=0.18 mg/l in D. magna (Bell 1994),
et al. 1995), and development (see the benthic filter- NOEC=0.113 mg/l again in D. magna (Hoffman et al.
feeding ascidian Styela plicata, Cima et al. 1996; Ya- 1987). Fenarimol lowers ecdysone levels in this organ-
llapragada et al. 1990). With regard to echinoderms, ism, which results in embryo abnormalities (Mu and
toxicity tests on the sea urchin Paracentrotus lividus LeBlanc 2002).
emphasized that TPT can cause critical damage to On the whole, our present results confirm that se-
sperm motility and development (Moschino and Marin lected androgenic compounds (TPT-Cl and Fenarimol)
2002; Novelli et al. 2002). As previously mentioned, impact strongly on regenerative development and induce
data on the ophiuroid O. brevispina (Walsh et al. obvious malformations in the internal anatomy and
1986) showed that exposure to TPT affects signifi- histopathological anomalies involving cell migration,
cantly arm regeneration processes. proliferation, histogenesis and differentiation.
Fenarimol is a halogenated pesticide extensively Our results indicate that both TPT and Fenarimol
employed in agriculture (IPCS 2004). There are no strongly affect skeletal development, as shown by both
73

appeared also to be less developed and compact in their


internal stereom structure (Smith 1990) when compared
to the controls. This is in agreement with previous re-
sults for other triphenyltin compounds, which showed
that they could affect significantly skeletogenesis in the
larvae of the echinoid P. lividus (Moschino and Marin
2002). Moreover, taking into account what happens in
vertebrates, where skeletogenesis mechanisms involving
osteoblasts and osteoclasts are regulated by sex hor-
mones (in particular estrogens and androgens, Lorenzo
2003), the teratogenic effects of suspected androgenic
compounds on skeletogenesis of A. mediterranea are
particularly intriguing, since they suggest that there is a
similar hormonal control of skeletal tissue development
in echinoderms. This was supported by our preliminary
biochemical results (Fig. 7) related to estradiol and tes-
tosterone levels in A. mediterranea. Furthermore, it has
been established that crinoids possess cellular elements
analogous to osteocytes/osteoblasts and osteoclasts in
vertebrates (Heinzeller and Welsch 1994). In conclusion
these data strongly support the hypothesis that chronic
exposure to low concentrations of both TPT and Fe-
narimol affects skeletogenesis in A. mediterranea by
interfering with sex steroid activity and metabolism.
ED compounds appear also to cause massive dedif-
ferentiation processes involving the old stump tissues of
the exposed regenerating animals. This phenomenon
involves specifically the muscle bundles, which in both
TPT- and Fenarimol-exposed samples undergo a sub-
stantial tissue rearrangement and cell recycling, often
resulting in extensive cell migration fluxes (towards the
apical blastema and the coelomic canals or, less fre-
quently, between the adjacent muscles bundles)
(Figs. 2b, 5). These results are in agreement with those
obtained from previous experiments with other EDs
(PCBs and 4-nonylphenol exposure tests, Candia Car-
nevali et al. 2001a, c, 2003) which also showed that
muscles of the exposed samples are employed as
recruitment sites for dediferrentiated cellular elements,
such as myocytes, that are used subsequently for
regenerative processes. Therefore, as well as PCBs and 4-
nonylphenol, TPT and Fenarimol appear to increase the
relative contribution of morphallactic mechanisms to
regeneration. It is important to stress that normal
regeneration depends mainly on epimorphic phenomena
employing undifferentiated progenitor elements (pre-
sumably neoblast-like cells) derived from specific
Fig. 5 Histological sagittal sections of control and exposed recruitment sites (coelomic epithelium and brachial
amputated arms at different regenerative stages. The details show nerve, Candia Carnevali and Bonasoro 2001).
the muscle bundles of the stump. a Control (1 week). The muscle
bundle shows a tightly packed and compact structure. b 100 ng Cell proliferation also appears to be affected by
TPT-Cl/l (72 h). An extensive muscle rearrangement is evident in exposure to ED compounds, as shown by our BrdU
the bundle. Many myocytes appear to migrate towards the experiments, in which both TPT and Fenarimol samples
blastema region (arrow). c 240 ng Fenarimol/l (1 week). A massive tended to show less extensive proliferation phenomena
rearrangement of the muscle bundle is evident. An unusual bi-
directional migration of myocytes between adjacent muscle bundles
as revealed by reduced cell labelling in comparison with
is detectable controls (Fig. 6). This effect, which is particularly
obvious in TPT samples, suggests an appreciable de-
external and internal anatomical malformations (Fig. 4). crease in proliferation of the ‘‘stem’’ elements involved
In particular, exposed samples are characterized by the in the standard epimorphic regenerative phenomena
anomalous differentiation of skeletal elements, which (Candia Carnevali and Bonasoro 2001) and explains the
74

Fig. 6 Histological sagittal sections of control and exposed labelled. b 225 ng TPT-Cl/l. The immunostaining appears to be less
regenerating arms at 1-week p.a. Immunocytochemistry for BrdU. evident in comparison with the control. c 240 ng Fenarimol/l. Only
a Control. Many blastemal and coelothelial cells are strongly a few labelled cells are detectable. cc coelomic canals

consequent increase in the relative contribution to the amputation area. However, in TPT-exposed sam-
regeneration of morphallactic mechanisms in these par- ples, they appear to be involved in widespread degran-
ticular stress conditions. However, the effects of the two ulation and cell lysis detectable in the connective tissues
compounds are not identical, since at the stage of of the stump, especially close to the coelomic canals
2 weeks Fenarimol samples tend to show a condition (Fig. 3). This phenomenon could be partly explicable in
analogous to the normal, in terms of cell proliferation terms of the well-known interference of organotin with
pattern, which is associated with a parallel reduction in Ca2+ metabolism and with proteins involved in cell
the rearrangement phenomena at the level of the stump homeostasis (for instance F1F0 ATPase, Na+K+AT-
tissues. These features suggest that after 2 weeks regen- Pase, Ca2+ATPase as shown in mammals, Powers and
eration in the Fenarimol-exposed animals was returned Beavis 1991).
to the normal condition. Although the histological results of the exposure tests
Granulocites are normally involved in repair with TPT are similar to those obtained with Fenarimol,
processes and discharge their granules in the tissues of these substances have a different chemical composition

Fig. 7 Whole tissue levels of testosterone and estradiol (expressed in estradiol levels is evident in samples exposed to 240 ng
as pg/g wet weight) in (a) TPT- and (b) Fenarimol-exposed Fenarimol/l. a Significant differences respect to control, b signif-
regenerating animals. Data are presented as mean ± SEM (n=5). icant differences respect to solvent control. C control, SC solvent
Samples exposed to 225 ng TPT-Cl/l show an increase in control
testosterone levels and a decrease in estradiol levels. An increase
75

and therefore could have different mechanisms of action found for other marine invertebrates (Depledge and
at different levels. It is not surprising, therefore, that the Billinghurst 1999).
effects on steroid levels are quite different for the two In conclusion, the present work further confirms that
compounds: TPT appears to produce a significant in- crinoid regeneration is a useful model for evaluating
crease in testosterone level accompanied by a relevant possible effects of ED compounds at whole organism,
decrease in the estrogen levels in samples exposed to tissue and cellular levels. It demonstrates that the
225 ng/l; in contrast, Fenarimol induces an increase in physiology of sensitive marine animals can be affected
estrogen levels in samples exposed to comparable con- significantly by these compounds, even at very low
centrations (240 ng/l). In agreement with the known concentrations, through their interaction with mecha-
activity of TBT (Tributyltin), TPT appears to interfere nisms regulating development and growth. Finally it
with steroid metabolism, possibly acting on synthesis by provides valuable indications of the actual threat to
cytochrome P450-dependent aromatase or excretion ra- marine ecosystems that is posed by EDs.
ther than on steroid receptors (Matthiessen and Gibbs
1998). While our results seem to confirm that TPT has Acknowledgements The present work has received financial support
an androgenic effect in echinoderms, Fenarimol seems to from the EU (COMPRENDO Project n EVK1-CT-2002-00129).
The authors are particularly grateful to Dr Ulrike Shulte-Oehl-
have an estrogenic action. The biochemical data show mann for her valuable coordinating activity and to all the partners
clearly that there are enhanced estradiol levels in Fe- of the COMPRENDO project for their direct or indirect support
narimol-exposed samples. This is in agreement with the and advice. Special thanks are addressed to Drs Simona Ceriani
results of recent in vitro experiments showing that Fe- and Angelita Doria for their valuable help and technical assistance.
All the experiments carried out for the research work are in accord
narimol can interact with both estrogen and androgen with the current laws of our country. The authors are grateful to
receptors, acting as an estrogen agonist or as an the anonymous reviewers for their invaluable suggestions and
androgen antagonist (Andersen et al. 2002). careful revision of the manuscript.
However, since the two compounds cause the same
skeletal anomalies and cell/tissue alterations (decrease in
cell proliferation and rearrangement/dedifferentiation of References
muscles) it appears that they have a range of different
mechanisms of action at the tissue and cellular level. On Aminin DL, Agafonova IG, Federov SN (1995) Biological activity
the one hand, they act differently on hormone metabo- of disulfated polyhydroxysterids from the Pacific brittle star
lism and have contrasting effects on the observed hor- Ophiopholis aculeata. Comp Biochem Physiol 112(C):201–204
Anderson S, Hose JE, Knezovic JP (1994) Genotoxic and devel-
mone levels; on the other hand, they appear to affect opmental effects in sea urchin are sensitive indicators of effects
other physiological mechanisms in a similar way and to genotoxic chemicals. Environ Toxicol Chem 13:1033–1041
induce a pattern of developmental dysfunctions resulting Andersen HR, Vinggard AM, Rasmussen TH, Gjermandsen IM,
finally in anomalous regeneration. They probably affect Bonefeld-Jørgensen EC (2002) Effects of currently used pesti-
cides in assays for estrogenicity, androgenicity and aromatase
growth and development by interfering with the same activity in vitro. Toxicol Appl Pharmacol 179:1–12
basic cellular mechanisms of regeneration, such as cell Barbaglio A, Sugni M, Mozzi D, Invernizzi A, Doria A, Pacchetti
proliferation, migration and differentiation/dedifferenti- G, Tremolada P, Bonasoro F, Candia Carnevali MD (2004)
ation, which may be controlled, directly or indirectly, by Exposure effects of organotin compounds (TPT-Cl) on regen-
erative potential of crinoids. In: Heinzeller, Nebelsick (eds)
steroid hormones or their quantitative ratios (Marsh and Echinoderms. Munchen Taylor & Francis Group, London, pp
Walker 1995). 91–95
Although definitive results from chemical analysis of Békri K, Pelletier E (2004) Trophic transfer and in vivo immuni-
water and tissue samples from Fenarimol exposure toxicological effects of tributyltin (TBT) in polar seastar Lep-
experiments are not available at the moment, pre- tasterias polaris. Aquat Toxicol 66:39–53
Bell G (1994) Rubigan 12 EC (EAF 457) acute toxicity to Daphnia
liminary data (Sakkas et al. unpublished) showed that magna. DowElanco Company report
actual concentration measured in exposure medium at Bryan GW, Gibbs PE, Hummerstone LG, Burt GR (1986) The
2 weeks were lower than the nominal ones (i.e. 4.5, 128 decline of the gastropod Nucella lapillus around south-west
and 1,285 ng/l, respectively, for the nominal concentra- England: evidence for the effect of tributyltin from antifouling
paints. J Mar Biol Ass UK 66:611–640
tions of 24, 240, 2,400 ng/l). On the other hand, as far as Cadbury D (1998) The feminisation of nature. Our future at risk.
TPT is concerned, analytical results for TPT exposure Penguin Books, London
experiments (Tremolada et al. 2004) clearly indicate that Candia Carnevali MD (2005) Regenerative response and Endocrine
actual concentrations measured in exposure medium are Disrupters in Crinoid Echinoderms: an old experimental model,
a new ecotoxicological test. In: Matranga V (ed) Echinoder-
much lower than the nominal ones (i.e. 5.5±3.1, 14±6.3 mata. Progress in molecular and subcellular biology. vol 39.
and 33±11 ng/l, respectively, for the nominal concen- Subseries marine molecular biotechnology. Springer, Berlin
tration of 100, 225 and 500 ng/l). Nevertheless, actual Heidelberg New York, pp 187–198
TPT exposure concentrations are rather similar to those Candia Carnevali MD, Bonasoro F (2001) Microscopic overview
recorded in polluted Mediterranean coastal areas (Tol- of crinoid regeneration. Microsc Res Tech 55:403–426
Candia Carnevali MD, Lucca E, Bonasoro F (1993) Mechanism of
osa et al. 1996). This means that TPT concentrations arm regeneration in the feather star Antedon mediterranea:
found commonly in coastal zones can affect significantly healing of wound and early stages of development. J Exp Zool
the physiology of echinoderms, as was already been 267:299–317
76

Candia Carnevali MD, Bonasoro F, Lucca E, Thorndyke MC Dieleman SJ, Schoenmakers HNJ (1979) Radioimmunoassay to
(1995) Pattern of cell proliferation in the feather star Antedon determine the presence of progesterone and estrone in starfish
mediterranea. J Exp Zool 272:464–474 Asterias rubens. Gen Comp Endocrinol 39:534–542
Candia Carnevali MD, Bonasoro F, Biale A (1997) Pattern of EEC (1967) Council Directive 67/548/EEC on the approximation
bromodeoxyuridine incorporation in the advanced stages of of laws, regulations and administrative provisions relating to
arm regeneration in the feather star Antedon mediterranea. Cell the classification, packaging and labelling of dangerous sub-
Tissue Res 289:363–374 stances. Official Journal of the European Communities, Lux-
Candia Carnevali MD, Bonasoro F, Patruno M, Thorndyke MC, emborg, 196, p.1
Galassi S (2001a) PCB exposure and regeneration in crinoids Fairley P, Roberts M, Stringer J (1996). Endocrine disruptor.
(Echinodermata). Mar Ecol Prog Ser 215:155–167 Chem Week May 8:29–36
Candia Carnevali MD, Bonasoro F, Patruno M, Thorndyke MC Fait A, Ferioli A, Barbieri F (1994) Organotin compounds. Toxi-
(2001b). Role of the nervous system in echinoderm regenera- cology 91:77–82
tion. In: Barker M (ed), Echinoderm 2000: Proceedings of 10th Federoff NE, Young D, Cowles J, Spatz D, Shamin M (1999)
international echinoderm conference, Dunedin 2000, Balkema, TPTH. Environmental fate and ecological risk assessment.
Rotterdam, pp 5–20 United States Environmental Protection Agency, Washington
Candia Carnevali MD, Galassi S, Bonasoro F, Patruno M, DC
Thorndyke MC (2001c) Regenerative response and endocrine Fitox (1999) Database on physical, chemical and ecotoxicological
disrupters in crinoid echinoderms: arm regeneration in Antedon properties of pesticides. In: Finizio A (ed) Environmental im-
mediterranea after experimental exposure to polychlorinated pact of pesticides: risk evaluation for non-target organism.
biphenyls. J Exp Biol 204:835–842 Ampa n 1 Roma
Candia Carnevali MD, Bonasoro F, Ferreri P, Galassi S (2003) Gibbs PE, Pascoe PL, Burt GR (1988) Sex changes in the female
Regenerative potential and effects of exposure to pseudo- dog-whelk, Nucella lapillus, induced by tributyltin from anti-
estrogenic contaminants (4-nonylphenol) in the crinoid Antedon fouling paints. J Mar Biol Ass UK 68:715–731
mediterranea. In: Feral JP (ed) Echinoderm research 2001. Gray LE Jr, Monosson E, Kelce WR (1996) Emerging issues: the
Balkema, Rotterdam, pp 201–207 effects of endocrine disrupters on reproductive development. In:
Caricchia AM, Chiavarini S, Cremisini C, Fantini M, Morabito R Di Giulio RT, Monosson E (eds) Interconnection between
(1992) Monitoring of organotins in the La Spezia Gulf – II. human and ecosystem health. Chapman & Hall, London, pp
Results of the 1990 sampling campaigns and concluding re- 47–81
marks. Sci Total Environ 121:133–144 Heinzeller T, Welsch U (1994) Crinoidea. In: Harrison F (ed)
Cima F, Ballarin L, Bressa G, Martinucci G, Burighel P (1996) Microscopic anatomy of invertebrates: Echinodermata, vol 14.
Toxicity of organotin compounds on embryos of a marine Wiley-Liss, New York, pp 9–148
invertebrate (Styela plicata; Tunicata). Ecotoxicol Environ Saf Hines GA, Watts SA, McClintock JB (1994) Biosynthesis of
35:174–182 estrogen derivatives in the echinoid Lytechinus variegates La-
Colborn T, von Saal FS, Soto AM (1993) Developmental effects of mark. In: David, Guille, Féral, Roux (eds) Echinoderms
endocrine disrupting chemicals in wildlife and humans. Environ through time. Balkema, Rotterdam, pp 711–716
Health Perspect 101:378–384 Hoffman DG, Grothe DW, Francis PC (1987) Chronic toxicity of
Cooper RL, Kavlock RJ (1997) Endocrine disruptors and repro- fenarimol to Daphnia magna in a static renewal life cycle test.
ductive development: a weight-of-evidence overview. J Endo- DowElanco Company report
crinol 152:159–166 Horiguchi T, Shiraishi H, Shimizu M, Morita M (1995) Imposex in
Coteur G, Danis B, Fowler SW, Teyssié J-L, Dubois Ph, Warnau Japanese gastropods (Neogastropoda and Mesogastropoda):
M (2001) Effects of PCBs on reactive oxygen species (ROS) effects of tributyltin and triphenyltin from antifouling paints.
production by the immune cells of Paracentrotus lividus (Echi- Mar Pollut Bull 31:402–405
nodermata). Mar Pollut Bull 42:667–672 IPCS (2004) (International programme on chemical safety), by
D’Andrea AF, Stancyk S, Chandler GT (1996) Sublethal effects of World Health Organization, United Nations Environmental
cadmium on arm regeneration in the burrowing brittlerstars, Programme, International Labour Organization, http://
Microphiopholis gracillima (Stimpson) (Echinodermata: www.inchem.org
Ophiuroidea). Ecotoxicology 5:115–133 Jackson R, Lewis C (1994) The degradation and retention of 14C
Degen GH, Botl HM (2000) Endocrine disruptors: update on xe- Fenarimol in water-sediment systems. DowElanco Company
noestrogens. Int Arch Occup Environ Health 73:433–441 report
den Besten PJ (1998) Cytochrome P450 monooxygenase system in Janer G, LeBlanc GA, Porte C (2004) A comparative study on the
echinoderms. Comp Biochem Physiol 121(C):139–146 metabolism of androgens in invertebrates and its modulation by
den Besten PJ, Herwig HJ, Zandee DI, Voogt PA (1989) Effects of xenoandrogens. In: Proceedings of CREDO cluster workshop
cadmium and PCBs on reproduction of the sea star Asterias on ecological relevance of chemically induced endocrine dis-
rubens: aberrations in the early development. Ecotoxicol Envi- ruption in wildlife, p 23
ron Saf 18:173–180 Kline RM, Knox JW (1981) Fenarimol: Interactions with sewage
den Besten PJ, Herwig HJ, Smaal AC, Zandee DI, Voogt PA microorganism. DowElanco Company report
(1990) Interference of polychlorinated biphenyls (Clophen A50) Kabayashi N (1984) Marine ecotoxicological testing with Echino-
with gametogenesis in the sea star Asterias rubens L. Aquat derms. In: Persoone G, Jaspers F, Claus C (eds) Ecotoxico-
Toxicol 18:231–246 logical testing for the marine environment. Vol.I, State
den Besten PJ, Elenbaas JM, Maas EJR, Dielman SJ, Herwig HJ, University of Ghent and Institute of Marine Scientific Re-
Voogt PA (1991a) Effects of cadmium and polychlorinated bi- search, Bredene, Belgium, pp 341–405
phenyls (Clophen A50) on steroid metabolism and cytochrome LeBlanc GA, Campbell PM, den Besten P, Brown RP, Chang ES,
P-450 monooxygenase system in the sea star Asterias rubens L. Coats JR, De Fur PL, Dhadialla T, Edwards J, Riddiford Lm,
Aquat Toxicol 20:95–110 Simpson MG, Snell TW, Thorndyke MC, Matsumura F (1999)
den Besten PJ, Mass JR, Livingstone DR, Zandee DI, Voogt PA The endocrinology of invertebrates. In: deFur P, Crane M,
(1991b) Interference of benzo[a]pyrene with cytochrome P450 Ingersoll C, Tattersfield L (eds) Endocrine dirsruption in
mediated steroid metabolism in pyloric caeca microsomes of the invertebrates: endocrinology, testing and assessment. Work-
sea star Asterias rubens L. Comp Biochem Physiol 100(C): shop on endocrine disruption in invertebrates: endocrinology,
165–168 testing and assessment: 1998 Dec 12–15. SETAC Noordwij-
Depledge MH, Billinghurst Z (1999) Ecological significance of kerhout, The Netherlands, pp 23–106
endocrine disruption in marine invertebrates. Mar Pollut Bull Lorenzo J (2003) A new hypothesis for how sex steroid hormones
39:32–38 regulate bone mass. J Clin Invest 111:1641–1643
77

Lutz I, Sugni M, Candia Carnevali MD, Lavado R, Porte C, Shubina LK, Fedorov SN, Levina EV, Andriyaschenko PV, Ka-
Schulte-Oehlmann U, Kloas W (2004) First evidence for specific linovsky AI, Stonik VA, Smirnov IS (1998) Comparative study
[3H]-testosterone and [3H]-estradiol binding sites in echino- on polyhydroxylated steroids from echinoderms. Comp Bio-
derms. In: Proceedings of CREDO cluster workshop on eco- chem Physiol 119(B):505–511
logical relevance of chemically induced endocrine disruption in Smith AB (1990) Biomineralization in Echinoderms. In: Carler JG
wildlife, p 43 (ed) Skeletal biomineralization: patterns, processes and evolu-
Mackay D, Shiu WY, Ma KC (1997) Illustrated handbook of tionary trends. vol I, pp 413–435
physical-chemical properties and environmental fate for organic Smith DF, Meyer DL, Horner SMJ (1981) Amino acid uptake by
chemicals. vol 5, Lewis Publishers, New York, p 812 the comatulid crinoid Cenometra bella (Echinodermata) fol-
Marsh A, Walker C (1995) Effect of estradiol and progesterone on lowing evisceration. Mar Biol 61:207–213
c-myc expression in the sea star testis and the seasonal regula- Soto AM, Sonnenschein C, Chung KL (1995) The E-Screen as a
tion of spermatogenesis. Mol Reprod Dev 40:62–68 tool to identify estrogens: an update on estrogenic environ-
Matthiessen P, Gibbs PE (1998) Critical appraisal of the evidence mental pollutants. Environ Health Perspect 103(Suppl):113–122
for tributyltin-mediated endocrine disruption in mollusks. Thorndyke MC, Candia Carnevali MD (2001) Regeneration and
Environ Toxicol Chem 17(1):37–43 neurohormones and growth factors in echinoderms. Can J Zool
Moschino V, Marin M (2002) Spermiotoxicity and embriotoxicity 79:1171–1208
of triphenyltin in the sea urchin Paracentrotus lividus Lmk. Tolosa I, Readman JW, Blaevoet A, Ghilini S, Bartocci J, Horvat
Appl Organomet Chem 16:175–181 M (1996) Contamination of Mediterranean (Côte d’Azur)
Mu XY, LeBlanc GA (2002) Environmental antiecdysteroids alter coastal waters by organotins and Irgarol 1051 used in anti-
embryo development in the crustacean Daphnia magna. J Exp fouling paints. Mar Pollut Bull 32:335–341
Zool 292(3):287–292 Tremolada P, Bristeau S, Mozzi D, Sugni M, Barbaglio A, Dagnac
Novelli AA, Argese E, Tagliapietra D, Bettiol C, Volpi Ghirardini T, Candia Carnevali MD (2005) A simple model to predict
A (2002) Toxicity of tributyltin and triphenyltin to early life- compound loss processes in aquatic ecotoxicological tests: cal-
stages of Paracentrotus lividus (Echinodermata: Echinoidea). culated and measured TPT-Cl levels in water and biota. Int J
Environ Toxicol Chem 21:859–864 Env Anal Chem 86:171–184
Powers MF, Beavis AD (1991) Triorganotins inhibit the mito- Voogt PA, Oudejans RCHM, Broertjes JJS (1984) Steroids and
chondrial inner membrane anion channel. J Biol Chem reproduction in starfish. In: Engels W, Clark WH, Fischer A,
266(26):17250–17256 Olive PJM, Went DF (eds) Advances in invertebrate repro-
Rippen G (ed) (1990) Handbuch Umweltechemikalien. Stoffdaten, duction. Elsevier North-Holland Inc, New York, pp 151–161
Pfüfverfahren, Vorschirften. 3. Auflage, 5. Ergänzungslieferung Voogt PA, den Besten PJ, Jansen M (1990) The D5 –pathway in
2/90. Ecomed, Landsberg am Lech steroid metabolism in the sea star Asterias rubens L. Comp
Schoenmakers HJN (1979) In vitro biosynthesis of steroids from Biochem Physiol 97(B):555–562
cholesterol by the ovaries and pyloric caeca of the starfish Voogt PA, den Besten PJ, Jansen M (1991) Steroid metabolism in
Asterias rubens. Comp Biochem Physiol (B) 63:179–184 relation to the reproductive cycle in Asterias rubens L. Comp
Schoenmakers HJN (1980) The variation of 3ß-hydroxysteroid Biochem Physiol 99(B):77–82
dehydrogenase activity of the ovaries and pyloric caeca of the Walsh GE, McLaughlin LL, Louie MK, Deans CH, Lores EM
starfish Asterias rubens during the annual reproductive cycle. (1986) Inhibition of arm regeneration by Ophioderma brevispina
J Comp Physiol 138:27–30 (Echinodermata, Ophiuroidea) by tributyltin oxide and triphe-
Schoenmakers HJN, Voogt PA (1980) In vitro biosynthesis of nyltin oxide. Ecotoxicol Environ Saf 12:95–100
steroids from progesterone by the ovaries and pyloric caeca of Yallapragada PR, Vig PJS, Desaiah D (1990) Differential effects of
the starfish Asterias rubens. Gen Comp Endocrinol 41:408–416 triorganotins on calmodulin activity. J Toxicol Environ Health
Shirai H, Walker CW (1988) Chemical control of asexual and 29:317–327
sexual reproduction in echinoderms. Alan R Liss Inc,
New York