Imposex and Reproductive Failure in Hydrobia Ulvae (Gastropoda: Prosobranchia)

Home , Hydrobia, Hydrobiidae, Peringia ulvae

Marine Biology (1997) 128: 257–266  Springer-Verlag 1997

U. Schulte-Oehlmann · J. Oehlmann · P. Fioroni · B. Bauer Imposex and reproductive failure in Hydrobia ulvae (Gastropoda: Prosobranchia)

Received: 28 September 1996 / Accepted: 7 November 1996

Abstract Hydrobia ulvae specimens collected from 16 sification schemes (Gibbs et al. 1987; Oehlmann et al. stations along the German North Sea and Baltic coasts 1991; Bauer et al. 1995; Schulte-Oehlmann et al. 1995) exhibited imposex (occurrence of male parts in addition based on different stages of virilisation culminating in to the female genital system). For the purposes of the functional sterilisation and ultimate death of fe- comparison, a description of both the male and unaf- males. Virilisation is induced by TBT, a substance used fected female genital systems is presented. Four different in antifouling paints on ships, boats and off-shore in- imposex stages with two types of development were stallations, as a fungicide in agriculture and in the identified and documented with scanning electron mi- preservation of wood, and is added to a variety of ma- crographs for the first time. The percentage of imposex- terials as a catalyst (e.g., polyurethane foams) and pro- affected females (an average over all the localities sam- tectant against microbial decomposition (e.g., textiles, pled) was about 44.3%, and 12.9% were definitively dispersion paints, PVC and other plastics). During the sterilized. The phenomenon of sex change was not ob- last decade, increasing environmental TBT concentra- served. The vas deferens sequence index, imposex inci- tions have led to an increase of the number and intensity dence, percentage of sterilized females and the average of imposex occurrences in prosobranch populations. To female penis length are recommended as biological effect date, more than 120 prosobranch species are known to monitoring parameters in response to tributyltin pollu- be affected by these pathological alterations, which tion. demonstrates the widespread occurrence of this very common problem. The degree of virilisation, because of its dependence on the TBT contamination of the environment, may be fruitfully used for TBT effect moni- Introduction toring purposes. Many investigations have already used prosobranchs, especially the dogwhelk Nucella lapillus, Imposex (Smith 1971), also known as pseudohermaph- as sentinel organisms to assess the actual TBT contam- roditism (Jenner 1979), in prosobranch species is the ination of the marine environment by means of pseu- most sensitive biological effect in response to TBT dohermaphroditism intensities (Bryan et al. 1986; Gibbs (tributyltin) pollution of the environment. Under the et al. 1987; Oehlmann et al. 1991, 1994; Minchin et al. influence of this biocide with androgenic activity, female 1995, 1996, 1997). Unfortunately, the distribution of snails develop male sex characteristics, e.g. a vas defer- N. lapillus is restricted. Dogwhelks are absent in the ens and/or a penis in addition to the female system. entire Baltic and, with few exceptions, also in the Masculinisation effects can be described by using clas- Skagerrak, Kattegat and the southern part of the North Sea. Therefore it is desirable to find a broader base of indicator organisms which should be indigenous within areas under investigation. The fact that OSPARCOM Communicated by O. Kinne, Oldendorf/Luhe (Oslo and Paris Commissions) included the intersex U. Schulte-Oehlmann (&) · P. Fioroni · B. Bauer phenomenon of Littorina littorea (Bauer et al. 1993) in Institut fu¨r Spezielle Zoologie und Vergleichende Embryologie, the JMP (Joint Monitoring Programme) underlines the Universita¨tMu¨nster, Hu¨fferstr. 1, D-48149 Mu¨nster, Germany importance of such studies (Oslo and Paris Commissions 1996). As far as biomonitoring purposes within the J. Oehlmann Internationales Hochschulinstitut, OSPARCOM convention area are concerned, the se- Lehrstuhl fu¨r Umweltverfahrenstechnik, Markt 23, lection of the periwinkle and dogwhelk seems to be D-02763 Zittau, Germany sufficient. Problems would certainly arise if TBT effect 258 monitoring surveys are extended to the Baltic Sea or to brackish estuaries. In this case the mud snail Hydrobia SD ulvae would offer a very suitable alternative. The widespread distribution area of the mud snail includes the % sexually mature North Sea, Baltic Sea and large parts of the Atlantic. Furthermore, mud snails are known to be an important part of the marine food web and possible effects of para- sited xenobiotics on population size and/or reproductive performance may well result in serious ecological prob- ±SD % lems. Generally this paper will give an idea of path- omorphoses which probably have a negative effect on the fecundity of female mud snails. A preliminary de- oviduct (mm) scription of the imposex development in Hydrobia ulvae

will be presented. The documentation of the variability ± SD Pallial of imposex expression and the degree of TBT pollution at diﬀerent localities will be done in another publication

(Schulte-Oehlmann et al. in preparation). gland (mm) ± SD Capsule Materials and methods

In 1994 and 1995 Hydrobia ulvae samples from 16 diﬀerent stations along the German North Sea and Baltic coasts were collected. A gland (mm) total number of 1783 mud snails was examined. The specimens were analysed (sample size ≥ 30 individuals) and narcotised in 2 to ± SD Albumen 7% MgCl2 (according to salinity at sampling stations) dissolved in distilled water. Before cracking the shell with a vice, shell and aperture height were measured, and after cracking, the external properties were analysed with a stereo dissecting microscope and all organs measured to an accuracy of 0.05 mm by eyepiece (Table 1). length (mm) For imposex measurements the snails were sexed, the penis length measured and the imposex stage (according to Oehlmann et al.

1991 and Stroben et al. 1992) determined. As imposex indices the ± SD Prostate VDS (Gibbs et al. 1987; Fioroni et al. 1991) (vas deferens sequence = average imposex stage of a population with values of 0 to 4inHydrobia ulvae) and the average female penis length (FPL) of a sample were calculated and imposex incidences determined. length (mm) For histological studies, individuals were fixed with Bouin’s fluid for 24 h and preserved afterwards in 70% ethanol. Serial ± SD Penis sections (5 lm), embedded in paraplast, were made and stained with haemalun-chromotrop (Romeis 1989). Complete series of histological sections exist for 15 males and 54 females. For scanning electron microscopy (SEM), individuals were also fixed in Bouin’s fluid, dehydrated via graded ethanol series, critical point height (mm) dried, coated with gold and examined with a Hitachi scanning electron microscope S-530. ± SD Aperture

Results Shell height (mm)

Male genital system %of females From proximal to distal the male genital system

(Fig. 1a) consists of testis, gonadial vas deferens, ve- not determined) . Morphometrical data of males, females and different imposex stages. For definition of imposex stages see ‘‘Results – Imposex measurement and indices’’ ( sicula seminalis, renal vas deferens, prostate gland %of total (pallial vas deferens) and penis. ND The yellow-coloured testis is clearly delimited from the digestive gland and extends along the columellar side of the visceral hump. The blind-ending seminiferous Number of specimens tubules are separated from each other by delicate layers Hydrobia ulvae of connective tissue. The coiled seminal vesicle runs standard deviation; Sex or imposex stage Males0 837123 46.94 5274 204 –5 1476 – 137 – 4.208 1 – 179 55.71 0.71 12 – 21.56Females 16 4.17 1.77 946 – 15.54 4.27 – 8 0.73 4.61 – 1 1.37 0.26 0.66 – 1.77 53.06 0.11 4.61 0.79 1.79 1.81 1.80 – – 4.70 1.89 1.27 – 0.91 0.48 4.31 0.28 1.69 4.56 0.26 0.99 0.00 – 1.94 0.47 4.28 0.84 4.08 0.29 0.11 0.77 0.11 0.00 0.43 1.80 4.61 0.30 0.74 0.55 1.70 0.26 0.06 – 1.81 6.30 – 0.65 0.33 0.53 – 1.80 1.71 0.20 – – – 1.84 0.30 0.58 0.00 0.28 0.19 – 0.07 – – 2.00 2.50 – 0.00 0.11 0.41 0.26 – 0.08 – 0.52 0.53 – – 0.25 0.56 – 0.54 – – – – 0.59 0.41 – 0.17 – 0.60 – – 0.16 0.46 0.98 – 0.17 1.03 – – – 0.25 0.52 1.13 – – 0.12 0.44 0.46 1.36 – – 1.09 0.26 0.53 0.55 1.45 0.60 0.13 0.41 – 1.60 0.35 0.09 0.23 1.00 0.40 11.20 0.17 0.41 0.84 1.54 0.47 – – 17.56 0.28 1.02 1.08 8.33 72.30 0.00 14.29 0.19 0.30 1.22 75.87 0.75 82.27 ND 0.90 0.31 0.25 1.05 77.57 30.77 0.37 1.40 1.24 0.00 – 0.38 66.67 0.94 47.06 – 0.44 0.62 33.33 1.50 43.75 0.28 13.32 43.75 ND 50.00 – 73.38 62.50 50.00 – 0.00 0.00 0.60 100.00 – 100.00 0.00 distally along the columellar side towards the prostate Table 1 259 gland. In sexually mature individuals this organ is cycle were not observed within the investigation period, characteristically white-coloured and sperm-filled from but the extension of the male copulatory organ can vary spring until late summer. In the distal direction the ve- considerably within the very same sample. sicula merges into a short renal vas deferens section which leads into the pallial glandular part of the prostate gland. The latter is a kidney-shaped, closed structure, Female genital system with the exception of a small valve in the organ’s distal region leading into the mantle cavity. This construction The proximal female genital tract (Fig. 1b) is, according represents a kind of safety precaution, guaranteeing a to its ontogenetic descent, composed of a gonadial and decrease of overpressure resulting from interruption renal section. Following the latter is a pallial region during copulation. The gland’s slit-like lumen is lined which is divided into a proximal albumen and a distal with a ciliated prismatic epithelium and surrounded by capsule gland by subdivided lumina. The tubular ovary voluminous subepithelial glandular tissue. The flask-like occupies a position similar to the testis in the male and is cells are arranged in clusters; their ducts penetrate the clearly separated from the midgut gland. The branched, lumen’s epithelium to discharge their secretions. More blind-ending ovary diverticles are surrounded by a thin distally the prostate continues in the pallial vas deferens layer of connective tissue and lined by a flat germinal section (Figs. 1a, 2a), which runs over the bottom of the epithelium. During the spawning season the gonad has a mantle cavity towards the massive copulatory organ. vivid white colour and is filled with oocytes. These are The muscular penis (Figs. 1a, 2a) is situated in the neck visible through a stereo microscope after removal of the region. The coiled vas deferens, which serves as a penis shell, with no further dissection necessary. The tubules duct, extends throughout its whole length. Penis size fuse to form the gonadial oviduct which runs into the variations during the different phases of the reproductive proximal coiled renal portion. This section enlarges

Fig. 1 Hydrobia ulvae. Recon- struction of the male (a) and female (b) genital system without testis and ovary (ag albumen gland; bc bursa copulatrix; c connective tissue; cg capsule gland; god gonadial oviduct; gt glandular tissue; oc ovipar channel; ool oriﬁce of the ovipar channel; ovl oriﬁce of the vaginal channel; p penis; pd penis duct; pg prostate gland; pl lumen of the prostate gland; pv prostate valve; rs receptaculum seminis; vc vaginal channel; vd vas deferens; vs vesicula seminalis) 260

Fig. 2 Hydrobia ulvae. a SEM photograph of a male mud snail. b Transverse section of the male penis. c SEM photograph of a normal female (Stage 0). d Transverse section of the capsule gland. e, f SEM photographs of sterilized females without imposex; in f with split capsule gland and abortive egg capsules (ac abortive capsules; cgo closed genital openings; ct ctenidium; r rectum; t tentacle; other abbreviations as in Fig. 1)

distally to form the loop-like receptaculum seminis. The muscular tissue. The pallial part of the female repro- blind-ending translucent bursa copulatrix is embedded ductive organ is represented by a glandular complex. in the proximal region of the albumen gland (Fig. 1b). This can be divided into a proximal albumen gland The duct of the organ is not visible through the glan- embedded in the tissue of the visceral hump and the dular tissue, but in serial sections it becomes obvious more distally situated capsule gland positioned in the that it leads into the distal region of the coiled re- central region of the mantle roof (Fig. 1b). Both glands ceptaculum. The black-pigmented renal part of the fuse to form a mass with a common lumen. The division oviduct is connected to the glandular tissue of the al- of the lumen of the capsule, or nidamental gland, into a bumen gland and joins the most proximal section of the dorsal ovipar and a ventral vaginal channel is a dis- capsule gland’s ventral channel (vaginal part of the or- tinctive mark dividing the glands (Fig. 1b). The central gan’s lumen). The distal region of the renal oviduct is channel of the albumen gland does not resemble this lined by a cylindrical, ciliated epithelium. In this section structure and has a nondivided lumen. Thus the ovipar sperm are stored and orientated with their heads to- channel extends through the whole length of the gland wards the epithelium and their tails towards the lumen. complex. Oocytes are discharged into the mantle cavity The whole renal part is enclosed by a thin wrap of from an inconspicuous opening hidden by the vaginal 261 lips (orifice of the vaginal part) (Figs. 1b, 2d). The (average over all localities), i.e. almost half of all females vaginal part runs from the proximal end, where it re- studied exposed clear signs of virilisation (Table 1) in ceives the receptaculum, in the distal direction as a varying degrees. It should be mentioned that this value clearly separated ventral channel. The structure finally includes considerable differences between single sam- ends in a second genital aperture which forms the clearly pling stations (maximum values: 100%; minimum values: visible lips of the vulva situated directly above the 6.3%). About 12.9% of all pseudohermaphrodites were opening of the ovipar channel (Figs. 1b, 2c, d). The definitively sterilized (Table 1, Stages 5 to 9; compare the tissue of the albumen gland is composed of numerous following explanation). Table 1 also shows that the oc- subepithelial gland cell clusters. Each of the cluster cells currence of Stages 1 and 2 in Hydrobia ulvae was sig- is provided with its own duct which penetrates the epi- nificantly higher than of the more advanced stages (i.e., thelium to discharge its secretion into the central lumen 3 to 9) of imposex development. of the gland. The capsule gland exhibits a corresponding In Hydrobia ulvae there were no signs of a sex change histological composition. Different staining reactions of of females as known for muricid species (Oehlmann et al. the tissues of both organs demonstrate that qualitatively 1991). different secretions are produced.

Imposex measurement and indices Imposex development In Hydrobia ulvae the morphological appearance of the As imposex describes a morphological phenomenon female genital apertures is very variable. This means based on the masculinisation of dioecious prosobranch that although most virilised mud snails exhibit open species, females exhibit a superimposition of male sex orifices (Figs. 2d, 4a, e), some also possess closed ori- characteristics. In extreme cases this leads to sterilisation fices (Fig. 2e) irrespective of their imposex stage. The and death of the affected specimens (Fig. 2e, f ). In VDS index represents a reliable parameter for imposex Hydrobia ulvae imposex development can be described intensities and its values exhibit a significant depen- by a classification scheme (Stages 0 to 4 and in addition dence on biocide concentrations (Gibbs et al. 1987; Stage 5 which represents a sterile female without male Fioroni et al. 1991). This index represents the arith- sex characteristics) (Fig. 3), including two different paths metic mean value of all imposex stages in a sample. The of evolution and a gradual increase of masculinisation in number of stages may differ according to species spe- females. The a-types within this scheme are females cificity. In H. ulvae four stages can be distinguished. which first produce a penis and then gradually develop a Although a Stage 5 (Figs. 2e, 3) is not explicitly in- vas deferens by Stage 3a. In the b-path the occurrence of cluded in the scheme, the value 5 should be added to male organs is vice versa; the formation of a vas deferens the imposex stage of all females which exhibit closed portion initiates the process which is completed with the genital apertures (Stages 6 to 9), similar to the original appearance of a penis in Stage 3b. schemes of Gibbs et al. (1987), Fioroni et al. (1991) and Within the a-line, Stage 1 (Figs. 3, 4a) represents a Oehlmann et al. (1991), where sterile females have been female with a penis primordium but without a penis duct ranked as stages ≥5. According to the scheme here (Fig. 4b). In Stage 2a (Figs. 3, 4c,d) a penis duct is then suggested for H. ulvae a virilised female of Stage 2 with formed and the extension of the copulatory organ in- closed female genital opening will be given the value creases (Table 1). A distal vas deferens section appears 7. As already stated the VDS index should provide a at the base of the penis in Stage 3a (Figs. 3, 4e) and realistic assessment of the reproductive capability in the grows toward the genital apertures. Stage 4 sees the populations. It should be mentioned at this point that completion of imposex development as a continuous vas morphologically different pathological conditions can deferens connects the female orifices with the penis base lead to an occlusion of the female openings. Although (Figs. 3, 4f ). masculinisation will not automatically lead to sterility Directly in front of the vulva a gutter-like depression in H. ulvae, proliferating vas deferens tissue can cause can be found in Stage 1b (Figs. 3, 5a). This is modified vulva closure. In most cases this is detected in speci- towards Stage 2b to become a completely closed vas mens which follow the b-path of imposex development. deferens running above the bottom of the mantle cavity, In these cases sterility manifests the beginning of mas- ending at the corresponding male position at the mud culinisation. Figure 6 illustrates the correlation between snail’s neck (Figs. 3, 5b). In contrast to the male vas the percentage of sterile females and the degree of deferens, the corresponding female structure is not nec- imposex intensity measured as the VDS index. In essarily of an elevated (Fig. 5b) but can also be of a H. ulvae populations with VDS values <0.5, no sterile lowered (Fig. 5c, d) appearance, making it more difficult females are usually found. In populations with a VDS to identify the structure. In Stage 3b a penis primordium range between 0.5 and 2.0 the first infertile females may without a penis duct, which increases in size and length occur. Sterile females are not found in every population is developed; a penis duct then appears in Stage 4 with VDS values in this range. In samples with VDS (Figs. 3, 5d). In general the percentage of imposex-af- values > 2.0, at least some females are sterilized by fected females in our investigation was about 44.3% vaginal blockade. 262

Fig. 3 Hydrobia ulvae. Imposex development scheme with four diﬀerent stages. Stage 5 represents a sterilized female without male characteristics (e eye; pp penis primordium; other abbreviations as in Figs. 1 and 2)

Parasitism found that the occurrence of small nonfunctioning penes in this species is closely related to the infestation with The investigations of Krull (1935) and Rothschild (1938) parasites. Therefore in our investigation we considered are important references for the ability of Hydrobia ulvae whether or not all cases of masculinisation in H. ulvae to exhibit signs of imposex irrespective of TBT exposure. are causally and exclusively correlated with parasitism. These papers give evidence that the masculinisation of Table 2 summarizes the incidence of parasites in males, H. ulvae females was already known well before envi- females and imposex-aﬀected females. It is apparent that ronmental organotin pollution existed. Both authors there is only a slight increase of parasitism in imposex- 263

Fig. 4 Hydrobia ulvae. Diﬀer- ent imposex stages of the a- path. a SEM photograph of Stage 1. b Transverse section of the female penis primordium of Stage 1. c SEM photograph of Stage 2. d Transverse section of the female penis of Stage 2. e SEM photograph of Stage 3. f SEM photograph of Stage 4 ( pp penis primordium; r rectum; t tentacle; other abbreviations as in Fig. 1)

aﬀected specimens in comparison with unaﬀected fe- thus not much higher than in normal forms without

2 X males (Chi-square test: v 8 24 , p < 0.01); in the imposex, the reason for masculinisation of the other comparison between males and unaﬀected females 84.1% cannot be explained by parasitism. On the other

2 X v 14 72 ( p < 1). The analysed infested H. ulvae hand, a high percentage of parasite infestation within an specimens harboured a great variety of larval trema- analysed sample will lead to a slight increase of VDS todes; sporocysts, redia and cercaria were observable but index values. Therefore for imposex analyses within a determination of the diﬀerent species was not per- biomonitoring programmes parasitized specimens formed. should be excluded. These results do not negate the fact that virilisation of female mud snails can also be caused by parasitism, but on no account can this be the decisive and only Discussion and conclusions reason for imposex development. If this were so, 100% of the imposex-aﬀected females should have parasites. Detailed descriptions of the genital system of Hydrobia As the percentage of parasitism in all analysed pseu- ulvae have already been presented by Krull (1935), dohermaphrodites was only approximately 15.9% and Johansson (1948), Falniowski (1988) and Fretter and 264

Fig. 5 Hydrobia ulvae. SEM photographs of the diﬀerent imposex stages of the b-path. a Stage 1; b Stage 2; c Stage 3; and d Stage 4 (cgo closed genital openings; oga open genital apertures; pp penis primordium; other abbreviations as in Fig. 1)

Graham (1994). Although the present results are basi- found in the vaginal channel of the nidamental gland cally in agreement with those of the authors above, there which continues in the coiled sperm-storing re- are minor differences in respect to the female reproduc- ceptaculum. Hershler and Davis (1980) reported that in tive tract. While Krull (1935), Falniowski (1988) and Hydrobia truncata sperm are also stored within these Fretter and Graham (1994) described the bursa copu- coils although a separated receptaculum was identified. latrix and the receptaculum seminis as two pouch-like They furthermore pointed out that in various species of bulges of the proximal oviduct section, we were unable the European hydrobiid genus Pyrgula, the seminal re- to discover a receptaculum in this form. Sperm are ceptacle is reduced to a simple bulging of the coiled stored in the distal region of the coiled part of the renal oviduct. Johansson (1948) and Fretter and Graham oviduct. In transverse serial sections sperm could also be (1994) concluded that the closure of the pallial duct is delayed in juvenile H. ulvae, and a ciliated groove runs posteriorly to form the vaginal channel. In contrast to this no individual analysed in the present study dis- played a complete closure of the pallial tract; we found two orifices in juveniles and adults alike (Figs. 2d, 4e). As this phenomenon was detectable in all analysed samples it does not seem to be a pathological condition comparable with intersex development in Littorina littorea (Bauer et al. 1995), where masculinisation of females is introduced by an inhibition of the ontogenetic

Table 2 Hydrobia ulvae. Occurrence of parasites in males, females with and without imposex characteristics

Males Females without Females with imposex imposex

Fig. 6 Hydrobia ulvae. Relationship between the percentage of sterile Number of specimens 837 527 419 females and the VDS index. The results of population analysis (data, Number parasitized 147 59 66

points) and the calculated exponential regression (line) are docu- Relative amount (%) 17.6 11.2 15.9 X

0 984 x 2 X ÿ X X ` X mented: y 2 79 e 2 78; n 64; r 0 444; p 0 0005 265 closure of the pallial oviduct. It is also worth noting that imposex development. In H. ulvae, on the other hand, Krull (1935) and Rothschild (1938) described parasitized infertility does not necessarily correlate with pseudo- but typical male mud snails with a reduced copulatory hermaphroditism; the mathematical parameters for as- organ or missing vas deferens. If a correct gonadial sex sessing imposex intensities within mud snail populations determination can be assumed, these results emphasize should therefore be adjusted. Former studies do not deal that males also face the possibility of feminisation when with sterile females without male characteristics. The they are affected by parasites. Therefore it might be VDS index was developed to measure imposex intensi- possible that under the influence of TBT as a xenobiotic ties in prosobranch populations and to give an estima- with androgenic activity, the phenomenon of femini- tion of the reproductive capability of females (Gibbs sation of males caused by gonadial castration can no et al. 1987); through this data TBT levels in the envi- longer be observed. Within our investigation such indi- ronment may be determined. For the analysis of mud viduals could not clearly be identified. However, an snail data, however, this system is unsuitable without analysis of preserved museum species sampled before adaption in order to account for sterile females without 1960 when organotin compounds in antifouling paints any male characteristics (Stage 5 at the beginning of the became available, would elucidate this problem. imposex development). For Hydrobia ulvae the imposex classification scheme Hydrobia ulvae’s life span of approximately 2 years is in accordance to the findings of Oehlmann et al. (1991). (Fish and Fish 1974; Lassen and Clark 1979) assures The stages of the a-path of H. ulvae are comparable with that the biological and chemical data thus gathered those of muricids and buccinids, while the b-path in the represent the current situation if the species is used in a mud snail represents a mixture of the b- and c-lines re- TBT effect monitoring programme. In this context the ported for muricids and is less complicated. Oehlmann problem of floating mud snails (Armonies and Hartke (1994) reported phenotypical imposex variabilities for 1995) has to be considered. H. ulvae juveniles, for the the dogwhelk Nucella lapillus according to a Robert- most part, exhibit high mobility (Armonies personal sonian polymorphism previously described by Staiger communication); adult mud snails are more or less sta- (1957). Chromosome dimorphism of dogwhelks from tionary, so that this species meets the requirements for a different geographical areas in Brittany is correlated with bioindicator. the determination of imposex development paths a and High abundance of mud snails with population den- b. Corresponding to the results for dogwhelks, all sities up to 70 000–90 000 individuals m)2 (Walters and H. ulvae exhibiting the b-path of masculinisation came Wharfe 1980) may lead to the opinion that imposex and from sampling stations on the Kiel Bight. For biomon- female sterility will have no serious effect on population itoring studies the VDS index (Gibbs et al. 1987; Fioroni levels. As already mentioned, an average over all the et al. 1991) is best suited for giving a realistic measure of localities sampled showed that >44% of mud snails ex- the virilisation and reproductive capability of females as hibit signs of masculinisation. Although imposex inci- well as for the corresponding TBT concentrations. For dences at different sampling stations varied H. ulvae imposex incidences, percentage of sterilized fe- considerably, the biological indices at single stations males and average female penis length may be added to remained quite constant (Schulte-Oehlmann et al. in the list of biological effect monitoring parameters. A preparation). In various laboratory experiments with combination of all parameters will guarantee the most marine and limnic prosobranch species it has been precise results. As male penis size in H. ulvae does not proven that above all the occurrence of female sterility is vary during phases of the reproductive cycle, the RPS a question of the degree of TBT pollution (Schulte- index [(average female penis length3 ÷ average male pe- Oehlmann et al. 1996). Considering the fact that Hy- nis length3) · 100] introduced by Gibbs et al. (1987), as drobia ulvae is a typical soft-sediment inhabitant and the well as the RPL index [(average female penis TBT half-life of aerobic and anaerobic harbour sedi- length) ÷ (average male penis length) · 100] (Stroben et al. ments is up to 15 years (de Mora et al. 1989), the eco- 1992) are further biological pollution parameters. logical consequences should not be underestimated. However, both parameters are of restricted practica- Reise (1985) has already reported the important role of bility in Hydrobia ulvae especially in only slightly pol- hydrobiid species for secondary production, and the luted areas. At these stations the average female penis occurrence of mud snails in the digestive tract of sea length values are very small compared to the extensions birds and fish has been documented (Clay 1960). Fur- of the copulatory organ of males. As a result, RPS and thermore, Drake and Arias (1995) described a significant RPL indices are near 0%, whereas it is possible to dif- positive correlation between population abundance of ferentiate VDS values at lower pollution levels as well. It Hydrobia species and the biomass of benthic macroal- is important to notice that the majority of the estab- gae. Although Wilhelmsen and Reise (1994) have lished imposex classifications refer to neogastropods pointed out that it still remains uncertain whether (Gibbs et al. 1987; Fioroni et al. 1991; Oehlmann et al. Hydrobia species feed on macroalgae themselves or 1991; Stroben et al. 1992). These descriptions indicate whether they consume microflora covering its surface, that sterility in female prosobranchs is likely to be seen the possible effects on coastal ecosystems if population as closely related to the masculinisation phenomenon. densities of the mud snail H. ulvae decrease should be Sterility in these species is always the end result of considered. 266

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