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ᮊ 2000 Kluwer Academic Publishers. Manufactured in The Netherlands.
Effects of Endocrine Disruptors on Prosobranch Snails
( Mollusca:
Gastropoda in the Laboratory.
Part II: Triphenyltin as a Xeno-Androgen
ULRIKE SCHULTE-OEHLMANN, MICHAELA TILLMANN, BERND MARKERT Internationales Hochschulinstitut Zittau, Lehrstuhl Umwelt¨erfahrenstechnik, Fachgruppe Human- Okotoxikologie, Markt 23, D-02763 Zittau, Germany Abstract.
In laboratory experiments the effects of suspected endocrine disrupting chemicals on freshwa- ter and marine prosobranch species were analysed. In this second of three publications the responses ofthe freshwater ramshorn snail Marisa cornuarietis and of two marine prosobranchs Žthe dogwhelk Nucellalapillus and the netted whelk Hinia reticulata to the xeno-androgenic model compound triphenyltin TPT are presented. Marisa and Nucella were exposed via water Žnominal concentrations 5᎐500 ng L and Hinia via sediments Žnominal concentrations 50᎐500 ␮g TPT-Snrkg dry months. Female ramshorn snails but not the two marine species developed imposex in a time andconcentration dependent manner ŽEC described for tributyltin. TPT reduced furthermore the fecundity of Marisa at lower concentrations ŽEC104 months: 5.59 ng TPT-Snr.
L with a complete inhibition of spawning at nominal concentrations G250 ng TPT-SnrL Žmean measured "SD: G163"97.0 ng TPT-Snr.
organs Žpenis with accessory structures and prostate gland of male ramshorn snails and dogwhelks were reduced by up to 25% compared to the control but not in netted whelks. Histopathological analyses forM. cornuarietis and H. reticulata provide evidence for a marked impairment of spermatogenesis Žbothspecies. and oogenesis Žonly netted whelks. The test compound induced a highly significant andconcentration independent increase in the incidence of hyperplasia on gills, osphradia and other organsin the mantle cavity of N. lapillus indicating a carcinogenic potential of TPT. The results show thatprosobranchs are sensitive to endocrine disruption at environmentally relevant concentrations of TPT.
Also, M. cornuarietis is a promising candidate for a future organismic invertebrate system to identifyendocrine-mimetic test compounds.
Keywords: endocrine disruptors; xeno-androgen; triphenyltin; imposex; snails *To whom correspondence should be addressed: IHI Zittau,Markt 23, D-02763 Zittau, Germany; E-mail: oehlmann@ihi-zittau.de.
Introduction
September 1997 and May 2000. The objective was There is growing evidence from a number of to develop an organismic invertebrate test system studies that certain xenobiotics in the environ- for the simultaneous identification of either femi- mentᎏreferred to as hormone-mimetic sub- nisation or masculanisation responses. Recently, stances, endocrine disruptors or modulatorsᎏare gonochoristic prosobranchs were rated as the most capable of interfering with endocrine function in promising candidates for this purpose next to a variety of animals resulting in adverse effects on insects and crustaceans ŽdeFur et al., reproductive capability thereby affecting the sur- publication is focussed on the effects of the sus- vival of natural populations. For humans, the reported results are conflicting but some epidemi- was shown that this compound exhibits an impo- ological investigations show that decreased sperm sex-inducing effect in a prosobranch snail from counts, increased cases of breast, testicular and Asia, the rock shell Thais cla¨igera ŽHoriguchi et other forms of reproductive cancers, genital ab- cated to the xeno-estrogens bisphenol A and mature puberty in females, and increased cases of octylphenol; the last publication in this series will endometriosis might be induced by compounds deal with the xeno-antiandrogen vinclozolin.
which are suspected as endocrine disruptors ŽGist, 1998. Most ecotoxicological studies on the effects agricultural fungicides Žmainly as triphenyltinhy- of hormone-mimetic industrial chemicals are fo- cussed on findings in vertebrates although inver- tebrates represent more than 95% of the known early 1960s to combat a range of fungal diseases species in the animal kingdom ŽdeFur et al., in various crops, particularly potato blight Ž Phyto- The limited number of examples for endocrine phthora infestans., leaf spot ŽCercospora beticola, disruption in invertebrates is partially due to the beticola and powdery mildew Ž Erysiphe fact that their hormonal systems are rather poorly betae. on sugar beet, peanuts and celery, Pseu- understood in comparison with vertebrates. Dele- doperonospora humuli on hop, brown rust on terious endocrine changes following an exposure beans, grey moulds on onions, rice blast and to certain compounds may therefore easily be missed or simply not be measurable at present, more, TPT compounds are used in certain an- even though a number of field investigations and tifouling paints, mainly in combination with laboratory studies show that endocrine disruption or modulation has probably occurred Žfor review: production of TPT is unknown, but in Germany it is less than 1,000 t ŽLaughlin et al., The example of the masculinising effects of world usage of TPT in fungicides is estimated to be several thousand tons annually according to species of prosobranch molluscs shows that ap- parently alterations of a simple step in biochemi- Based on a number of studies, Federoff et al.
cal pathwaysᎏthe inhibition of aromatase activ- 1999 reviewed the environmental fate of tri- phenyltin compounds. According to their review, up to the population and community levels, and the aerobic and anaerobic soil half lives of TPT there is no reason to assume that such far- are 21 and 36 days, respectively. Other literature reaching changes are in any sense unique ŽMat- reports half lives range from 47 to 140 days ŽKan- This publication is the second in a series which Transport to surface water would most likely investigates effects of compounds suspected to act be in association with eroded soil particles during as endocrine modulators on freshwater and ma- rain or irrigation events or via spray drift during rine prosobranch species in the laboratory. Most application. Given that TPT is resistant to hydrol- of the results were obtained during a research ysis and aqueous photolysis, it is considered quite project for the German Federal Environmental persistent and partitioning will take place to the Endocrine Effects on Prosobranchs. II: Xeno-Androgens sediment in aquatic systems. The maximum solu- bility of TPT in water is between 1.2 and 8 mgrL yearly mean between 102 Žpotatoes or beets. and 1590 ng TPT-SnrL Žpecans. The predicted envi- 1999. Due to the low solubility in water and the ronmental concentration in ground water using the US-EPA SCIGROW worst case scenario was primarily adsorbed on the suspended particles and sediments in natural waters. TPT exhibits aconsiderable bioaccumulation Materials and methods
volatility is low Žvapour pressure at 20ЊC: 3.8= The experiments were performed with three dif- ferent gonochoristic prosobranch species, the In general, organotins with small alkyl chains freshwater ramshorn snail, Marisa cornuarietis degrade slowly in the environment. TPT is less stable and may, under certain conditions Že.g.
snails, the dogwhelk Nucella lapillus ŽNeogas- intense UV radiation. rapidly loose phenyl sub- tropoda: Muricidae. and the netted whelk Hinia stituents as long as it is in water; nevertheless the reticulata ŽNeogastropoda: Buccinidae. The organotin compound accumulates both in sedi- Marisa specimens for the experiments were im- ments and especially in organisms ŽSturgeon and ported from Florida. The two marine species were consider TPT as resistant to photodegradation and hydrolysis with half lives of 93 to 111 days in irradiated water samples and 155 days in dark Marisa and Nucella were exposed to the test compound triphenyltin chloride ŽTPT, Merck The occurrence of triphenyltin compounds in the environment was currently reviewed by ter. These experiments were conducted as 24 h Žweekends: 48 h. semi-static renewal systems in concentrations in freshwater ecosystems are 108 60 litre glass aquaria filled with tap water Žfor ng TPT-SnrL in lakes ŽBecker-van Slooten and Marisa or artificial seawater Žfor Nucella; salinity 35‰ and provided with an Eheim power filter.
The sediment dwelling netted whelks were ex- of 353 ng TPT-SnrL for the marine environment posed to TPT via artificial sediments consisting of 90% quartz sand and 10% peat Žuntreated. All concentrations in freshwater and marine sedi- tests were performed under constant conditions ments were 309 and 1860 ␮g TPT-Snrkg Ždry with a temperature of 22 "1ЊC for the freshwater wt. , respectively ŽBecker et al., 1992; Ceulemans and 14 "1ЊC for the two marine snails; the light 1998. Predicted environmental concentra- dark rhythm was adjusted to 12:12 h.
tions ŽPEC., derived from tissue residue measure- Three different series of exposure experiments ments of TPT in biota and the known bioconcen- tration factors, range from 46 to 216 ng TPT-SnrLfor freshwater and 9.2 to 56 ng TPT-SnrL for 1. Marisa test: Adult M. cornuarietis were ex-
coastalrmarine ecosystems. An alternative at- posed to nominal aqueous concentrations of tempt to estimate the predicted environmental TPT concentrations in inland waters was per- for 4 months, including a solvent control Žethanol; concentration: 12.5 ␮gr.
standard scenario PRIZMrEXAMS with a 20,000 mens from each group were analysed at the m3 pond adjacent to a 10 ha field. The fungicide beginning of the experiment and in monthly was applied to the field at the maximum label intervals. Additionally a complete life cycle rate and at the shortest allowable interval. Ac- test was planned with an exposure ex o¨o over cording to their scenario, the peak concentrations a period of 12 months until the hatched F1 were in the range between 475 Žpotato as specimens were one year old Žcomp. Oehlmann daily intervals for Marisa and twice a week for the test compound on reproductive perfor- mance of Marisa this was not possible.
All specimens were narcotised prior to analysis 2. Nucella test: Adult N. lapillus were exposed to
nominal aqueous concentrations of 5; 50 and 100 ng TPT-SnrL for 3 months, including a shell and aperture height were measured to the solvent control Žglacial acetic acid; concentra- nearest 0.1 mm before the shell was cracked. The presence, normal appearance and extension Žto identical nominal tributyltin concentrations was run in parallel. 30 specimens from each as well as the occurrence of oocytes and sperm in group were analysed at the beginning of the the genital system and of visible excrescences on experiment and in monthly intervals with an genital and other organs with a dissection micro- additional analysis of 30 dogwhelks in the 100 scope. Additionally, imposex parameters like the VDSI Žvas deferens sequence index smean value of imposex stages in a sample with values from 0 3. Hinia test: Adult H. reticulata were exposed
to 3 in M. cornuarietis, 0 to 6 in N. lapillus and 0 to nominal concentrations of 50; 125 and 500 reticulata were calculated Žfor details see Oehlmann et al., 1991; Stroben et al., 1992; for 3 months, including a solvent control Žgla- cial acetic acid; concentration: 5 mgr.
Furthermore, a histopathological analysis of comparison, a test with identical nominal tri- the gonads was performed for the Marisa Žonly butyltin concentrations was run in parallel.
30 specimens from each group were analysed at the beginning of the experiment and in female specimens from each sample were fixed in Carnoy's and Bouin's fluid, respectively, and thenpreserved in ethanol. After embedding in Para- The nominal TPT concentrations were checked plast, serial sections Ž5᎐7 ␮.
for the Marisa test analytically using the GC-MS stained with haemalum-chromotrope. The sec- method described by Kalbfus et al. Ž tions were analysed using an image analysis sys- shown in Table 1, the measured concentrations in the exposure groups ranged between 57.8 to 94.3% For Hinia sections Ž3᎐7 ␮.
stained with haemalum-chromotrope, haema- spawning masses with the number of eggs in each toxylin-eosin and periodic-acid-Schiff. All speci- of the aquaria and the mortality were recorded in mens found to be afflicted with parasites, mainly Comparison of nominal and analytically determined aqueous triphenyltin concentrations during the exposure experiments with Marisa cornuarietis Nominal concentrationsŽng TPT as Snr.
The water samples were taken during a 24 hour cycle, beginning 15 minutes before the change of the test water in theexposure tanks and ending 1 day later before the water was changed again Žn s.
Endocrine Effects on Prosobranchs. II: Xeno-Androgens trematode larvae, were excluded from the evalua- differences to the control were significant throughout the experiment for 500 ng TPT-SnrL, Standard statistical analyses of the results Že.g.
for 250 ng TPT-SnrL from the second, for 150 ng TPT-SnrL from the third and for 75 ng TPT-SnrL not before the last month of the test ŽANOVA of samples according to Tukey Žlow n. or with multiple comparison of samples according to calculations Žprobit analyses, maximum likelihood Originally, it was planned to expose Marisa for five months to TPT but due to an unexpected values. were performed according to Weber Ž high mortality in all groupsᎏexcept for the sol- vent controlᎏthe experiment had to be aborted after four months and after three months in the500 ng TPT-SnrL group.
TPT affected not only the female specimens by Results and discussion
the development of imposex but had furthermorean adverse impact on the extension of the pallial sex organs in males Žprostate gland, penis, penissheath, penis Fig. 1c. The reduction of the mean penis size by The normal morphological and histological struc- up to 25% compared to the control was only ture of the male and female genital system of the statistically significant for the two highest expo- changes during imposex development following a the experiment ŽANOVA with multiple compari- TBT exposure is documented by Schulte-Oehl- son of samples according to Student-Newman- exposure to TPT resulted in a time and concen- Another effect of the test compound was a tration dependent enhancement of the imposex negative impact on the fecundity, measured as intensities. Not only the vas deferens sequence ŽFig. 2a. and eggs ŽFig. 2b. For the two lowest nominal concentrations Ž75 and 150 ng TPT- thermore also the length of the female penis and the extension of the female penis pouch Ždata not was observed, and in the 250 and 500 ng TPT- SnrL exposure groups, a complete cessation of spawning occurred. An ANCOVA analysis with groups exposed to the three highest nominal TPT multiple comparison of samples according to Stu- concentrations Ž150, 250, 500 ng TPT-Snr.
dent-Newman-Keuls revealed that the spawning the control were statistically significant during the mass and egg production in all TPT treated groups entire experiment including the first month but was significantly lower than in the control group with the exception of month 2 for the 150 ng TPT-SnrL group ŽWeir test for classified values, The Marisa females in the control group pro- 0.05. At a concentration of 75 ng TPT-SnrL duced a total number of 53 spawning masses the increase of the VDSI compared to the control containing 2850 eggs while the corresponding was not significant before the fourth month of numbers were 47% and 53% lower in the 75 ng TPT-SnrL and 93% and 98% lower in the 150 ng values for the control group were not statistically TPT-SnrL group Žvalues for spawning masses significant different from each other ŽANOVA, and eggs, respectively. The reduced fertility or complete lack of any eggs in the experimental The statistical analysis of the development of groups was the reason why the originally planned the mean female penis sheath length revealed complete life cycle test with M. cornuarietis could comparable results as indicated for the VDSI: the Marisa cornuarietis. Cumulative numbers of spawn- ing masses Ža. and eggs Žb. produced by the females duringexposure to different nominal triphenyltin concentrations: Ž ؒ.
solvent control, Ž.
As the sex ratios were slightly different in the test groups, the fecundity parameters were alsoanalysed per female for the experiment ŽFig.
Those females which received TPT via ambientwater produced 14% Ž75 ng TPT-Snr.
L fewer spawning masses ŽFig. 3a. and 24% 3b. than the control females. Furthermore, themean number of eggs per spawning mass was Marisa cornuarietis. Development of the imposex statistically significant lowered by the test com- intensities, measured as the vas deferens sequence index pound ŽFig. 3b; ANOVA with multiple compari- VDSI Ža. and mean penis sheath length in females Žb., and of the mean penis length in males Žc. during exposure to different nominal triphenyltin concentrations: Ž ؒ. solvent con- Interestingly, the histopathological analyses of the gonads showed no evidence for any distur- q 500 ng TPT-SnrL. In Ža. the scale of the y bances of oogenesis in the TPT treated Marisa axis represents the species-specific minimum and maximumvalues for the VDSI. In specimens compared to the control but there were very marked differences for the males. As Endocrine Effects on Prosobranchs. II: Xeno-Androgens males and females from the control group andthe highest TPT concentration Ž500 ng TPT-Snr.
were analysed from each sample in monthly inter-vals. One month after the start of the experi-ments, no statistically significant differences inthe incidence or intensity of spermatogenesis dis-turbances could be detected. After 2 and 3 monthshowever, all analysed males from the TPT treatedgroup exhibited not only a severe disruption ofgerm cell formation with a 100% incidence of theeffect, which was statistically significant compared intensity of the disturbances. These males werefound to be sterile with an azoospermia Žsperma-togenesis index G2.0 according to Schulte-Oehl-mann et al., indicated that the TPT induced reduction of thefecundity during the experiment might be due toan impairment of spermatogenesis.
For M. cornuarietis no alterations of the exten- sion of the accessory female sex glands Žalbumenand capsule sic tissue alterations were found during the TPTexperiment.
In contrast to Marisa cornuarietis, no imposexdevelopment was observed when dogwhelks wereexposed to TPT in the tested nominal concentra-tion range Ž5 to 100 ng TPT-SnrL; ANOVA,p ) 0.05. In a parallel experimental series the effects of identical nominal tributyltin Ž centrations Ž5, 50, and 100 ng TBT-Snr.
Nucella lapillus were tested and this experimentresulted in the expected time and concentrationdependent increase of the imposex intensities ŽFig.
4a. The VDSI values in all TBT treated groupswere statistically significantly higher than in thecontrol during the entire experiment except for100 ng TBT-SnrL after 1 week ŽWeir test forclassified values, p - Marisa cornuarietis. Comparison of spawning mass As in ramshorn snails, TPT also produced an numbers per female Ža., egg numbers per spawning mass Žb.
and egg numbers per female adverse impact on the extension of the pallial sex nominal triphenyltin concentrations. In Žb. mean values and organs Žprostate gland and penis. in male N.
standard deviations are shown. Asterisks indicate statistical lapillus. The mean length of the prostate gland significant differences to control ŽANOVA with multiple com- was reduced by up to 18% and of the penis by up to 21% compared to the control but these differ- ences were only statistically significant for the end of the experiment the percentage of speci-mens with tissue excrescences ᎏmainly epithelialhyperplasiaeᎏon the gill, osphradium or pallialsex organs increased in all TPT exposed groups.
In the control only 8% of all snails exhibitedexcrescences while the corresponding values were52% Ž The results for the netted whelk Hinia reticulataare mostly in line with the findings for Nucellalapillus. While an exposure to TBT via the sedi-ments led to the expected statistically significantŽWeir test for classified values, p - concentration dependent increase of imposex in-tensities, TPT exhibited no comparable andro-genic activity in the applied nominal concentra-tion range Ž50 to 500 ␮g TPT-Snrkg dry VDSI values in all TPT test groups and the con-trol remained on the same level with no indica-tion for any differences between the groups orwithin a group during the three months experi-ment ŽANOVA, p ) Development of the imposex intensities, measured The reduced length of the penis, which was found for the two other prosobranch species, was Ža. and Hinia reticulata Žb. during the triphenyltin Žcontinuouslines. and tributyltin Žbroken lines. experiments. The scales of not confirmed for H. reticulata. The extension of the y axes represent the species-specific minimum and maxi- the copulatory organ was comparable in all groups mum values for the VDSI. Ža. aqueous exposure groups: Ž ؒ.
accessory female sex glands Žalbumen, ingestion 100 ng as SnrL. Žb. sediment exposure groups Ždry wt. basis.: there was also no indication of an increase of thepercentage of males or females with hyperplasictissue alterations on the gill, osphradium or pal- highest exposure group Ž100 ng TPT-Snr.
lial sex organs in the netted whelk following an end of the experiment ŽANOVA with multiple comparison of samples according to Student- The gonads of H. reticulata revealed in both sexes marked disturbances in differentiation and Possible effects of TPT on the fecundity of the maturation processes. In 20% of females exposed dogwhelks were not assessed during the experi- to 50 ␮g TPT-Snrkg for 1 and 2 months and in ments because N. lapillus does normally not 83% after 3 months, the follicles remained at a produce egg capsules when specimens are trans- low maturation level. They were predominantly ferred from the field into the laboratory. In con- filled with oogonia, incorporation of vitelline was trast to M. cornuarietis and Hinia reticulata no absent. At a concentration of 125 ␮g TPT-Snrkg, histopathological analyses were performed.
60% of females exhibited gonads without follicle In N. lapillus the extension of the accessory maturation after 1 month, 100% after 2, and 50% female sex glands Žalbumen, ingestion and cap- after 3 months, respectively. Females exposed to 500 ␮g TPT-Snrkg showed inhibition of matura- Endocrine Effects on Prosobranchs. II: Xeno-Androgens secondary spermatogonia and to a lower degreewith spermatids after 1 month. All stages fromprimary spermatogonia to ripe sperm could befound after 2 months. In the 50 ␮g TPT-Snrkgexposure group, 75% of the males showed anatrophy in the form of a ''Sertoli only'' changeafter 1 month, 50% after 2 and 3 months. Asimilar situation was present in animals exposedto 125 and 500 ␮g TPT-Snrkg whereas the high-est degree of testis atrophy occurred after 1 monthand dropped thereafter down to 80% and 50%ŽFig. 5b. Seminiferous tubules with ''sertoli only''changes and a maturation depletion containeddegenerated spermatogonia and spermatids in thelumen. In the 125 ␮g TPT-Snrkg exposure group,massive degeneration of all stages of germ cells,necrosis and shrinking of tubules were present.
A direct comparison of the adverse effects of TPTduring the experimental series with the threedifferent prosobranchs shows clear species-specific differences ŽTable.
oped imposex following an exposure to thefungicidal compound. It has been accepted that Hinia reticulata. Incidence of atrophy in the female imposex is induced almost exclusively by tri- Ža. and male gonad Žb. during the triphenyltin experiment.
Concentrations Ždry wt. basis.: Ž ؒ. solvent control, Ž.
on results of laboratory experiments using about Asterisks indicate statistical significant differences to control 15 mainly marine prosobranch species world-wide.
␹ test.: ଙ, p-0.05; ଙଙ, p-0.01.
The effects of TBT on gastropods have beenrated as the most complete example of the effectsof an endocrine disrupting chemical on inverte- tion of follicles in 40% after 1, 100% after 2, andin 83% after 3 months ŽFig. 5a. This phe-nomenon was accompanied by an increasing rate Comparison of adverse effects found during the of atresia but a low degree of resorption. The triphenyltin exposure experiments with Marisa cornuarietis, presence of ''nurse cells'' was lower than in speci- Nucella lapillus and Hinia reticulata mens of the control group. The follicle epithelium appeared after 2 months translucent with small Abundant yellow lipopigment, autochrome, in- soluble in organic solvents and PAS positive, was present in the follicle epithelium in the control group and filled the lumina of follicles and the oviduct to a high degree in all exposed specimens.
At concentrations of 125 and 500 ␮g TPT-Snrkg the oviduct was clogged with this lipopigment.
The gonads of male Hinia in the control group were predominantly composed of primary and q, effect observable; y, no effect; n.d., not determined.
brates. Although originally observed as morpho- list were subject to TBT laboratory experiments logical changes in individuals, there is now an understanding of the effects at all levels of organ- abilities of this compound. Therefore, it cannot isation from the molecular to the population and be excluded that in an unknown number of fur- possibly the community, all of which present op- ther prosobranch species, TPT and TBT are portunities for observation of effects in field sam- equally potent compounds with regard to the ples. At the molecular level, TBT interferes with induction of imposex. Even for TBT it is known hormone metabolism, possibly via aromatase inhi- that not all prosobranch species develop imposex bition ŽBettin et al., 1996; Matthiessen and Gibbs, following an exposure in the laboratory Žcomp.
1998 , increasing the androgen levels in the snails.
Gibbs et al., 1997; Schulte-Oehlmann et al., The first reports that not only TBT but also According to a recent study of the United States Environmental Protection Agency, TPT was prosobranch, the rock shell Thais cla¨igera, were tested positive for the induction of pituitary and published by Horiguchi and co-workers based on testicular tumours in rats ŽUS-EPA, 1999; study field analysis and laboratory experiments Žinclud- ing direct injection of different organotin com- results, the US-EPA stated that several indica- pounds ŽHoriguchi et al., 1995, 1997a,b, tions imply that TPT may cause endocrine disrup- The potency of TPT for promoting the develop- ment of imposex in the rock shell is estimated to The highest test concentrations of TPT led to a be approximately the same as that of TBT ŽHori- significant size reduction of the pallial sex organs in male Marisa cornuarietis and Nucella lapillus, Horiguchi and co-workers, T. cla¨igera develops but not in Hinia reticulata while the extension of imposex in the field when the TBT or TPT con- the corresponding female sex organs was affected centration in the tissue exceeds 10᎐20 ␮grkg, in none of the three species. This observation and equivalent to an aqueous concentration of F1 the adverse effects of TPT on spermatogenesis in ngrL for TPT-Sn with regard to the species- ramshorn snails and netted whelks Žcomp.
offer a probable explanation for the drop of re- The results from Horiguchi and co-workers productive parameters during the exposure exper- were contradictory to results of Bryan et al. Ž iments with Marisa cornuarietis. A successful cop- who tested 6 different organotin compounds Žin- ulation requires a minimum length of the penis and penis sheath of male ramshorn snails. Other- ting ability in the dogwhelk Nucella lapillus. In wise, the copulatory organ cannot be introduced agreement with our own findings for Nucella, into the female oviduct and sperm cannot be Bryan and co-workers were not able to find any transferred ŽSchulte-Oehlmann et al., increase of imposex intensities in the analysed reduction of the mean penis length of male Marisa laboratory groups either after an aqueous expo- by 25% compared to the control might be suffi- cient to already cause a reduced copulatory suc- To our present knowledge, no other research group has conducted TPT-exposure experiments Literature reports on reproductive effects of with prosobranch molluscs with an assessment of TPT on the gonad level are poor and of limited imposex as a toxicological endpoint. On the basis value. Male rats fed 400 mgrkg of fentin hydrox- of laboratory results with four different species from the list of all known 160 imposex-affected plete testicular atrophy 7᎐34 days after onset of the experiment. But they were apparently re- that in at least 2 species ŽThais cla¨igera and pelled by the compound and starved. Thus, the cornuarietis TPT is a potent promoter of testicular atrophy was supposed as caused by star- imposex, i.e. in 50% of the currently analysed prosobranchs. It has to be considered that only less, TPT is regularly listed in reviews as a testicu- about 15 of the mentioned 160 species from the lar atrophy inducing agent ŽNolte et al., Endocrine Effects on Prosobranchs. II: Xeno-Androgens Exposure to a related organotin compound in- genic effect of TPT in mammals are conflicting.
duced atrophy of the testes and accessory genital organs, as observed by Magee et al. Ž which had received 10᎐20 mgrkg of triethyl tin cally evident abnormalities in the testes and hydroxide for 3᎐5 months. In contrast to these ovaries of rats following an oral administration of investigations the two prosobranch species Marisa TPT with doses of 20 mgrkg body weight per day and Hinia showed marked signs of atrophy in the for less than 4 weeks. While Innes et al. Ž male testis and maturation inhibition in the report that TPT is non-carcinogenic, the US-EPA ovaries of netted whelks. The Marisa males in the rates TPT as positive for hepatocellular adeno- highest TPT concentration were already virtually mas and carcinomas in mice and positive for sterilised 8 weeks after the start of the experi- pituitary and testicular tumours in rats ŽUS-EPA, ment and exhibited a marked azoospermia. In Hinia the most prominent effect could be ob- served after 2 months with a lower intensity after for two different imposex and fecundity parame- 3 months. It can only be speculated whether this ters are compared based on nominal and analyti- was due to activation of detoxification pathways cally measured concentrations during the experi- or to a degradation of TPT in the sediments.
ments with M. cornuarietis. It is obvious that In all specimens histologically investigated, the mid-gut gland was checked for pathological alter- lower than the corresponding values for the vas ations and signs of starvation. In no case could deferens sequence index and the mean female penis sheath. If these data are compared with N. lapillus was the only test species which reports on acute and chronic effects in the litera- developed excrescences as a result of TPT expo- ture it is evident that the ramshorn snail is much sure, especially on the gill and osphradium of more sensitive to TPT than other test organisms.
both sexes and on the penis and vas deferens in The lowest reported acute effect concentrations males. This effect with an incidence of up to 52% at the end of the experiment was highly signifi- cant and interestingly not concentration depen- tebrates Žwater flea; Federoff et al., dent. The results concerning a possible carcino- ng TPT-SnrL for vertebrates ŽEuropean minnow Marisa cornuarietis: Comparison of calculated EC CI for imposex development Žincrease of vas deferens sequence index and mean penis sheath length of females. and female fecundity Žproduced spawning masses and nominal and analytically measured triphenyltin concentrations after 4 months Žmean values from Table.
The lowest values are underlined to highlight the most sensitive parameter. All concentrations are given inng TPT as SnrL.
Acknowledgements
centrations are generally lower than the acute The experiments with Marisa cornuarietis were values with the lowest reported concentrations in financially supported by the Federal Environmen- the literature of 200 ng TPT-SnrL for algae tal Agency, Berlin ŽR & D project 216 02 001r.
We would also like to thank Constanze Stark, Ulrike Schneider and Christina Schmidt for their ŽNOEC reproduction, water flea; Federoff et al., excellent technical assistance and furthermore Dr.
1999 , 34 ng TPT-SnrL for marine invertebrates Siegfried Korhammer, Anke Laufer and Dr. Olaf ŽLOEC inhibition of arm regeneration, Ophio- for vertebrates ŽLOEC growth reduction, fatheadminnow; Federoff et al., References
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