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 envi- ronment, 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 wide- spread 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 ap- erture 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 se- quence = 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 scan- ning 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.