Marine Pollution Bulletin Vol. 38, No. 12, pp. 1227±1231, 1999 Ó 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0025-326X(99)00167-8 0025-326X/99 $ - see front matter Imposex in Three Marine Gastropod Species in Chile and Potential Impact on Muriciculture

MEREDITH GOODING *, CARLOS GALLARDOà and GERALD LEBLANC Department of Toxicology, North Carolina State University, Raleigh, NC 27695-7633, USA àInstituto de Zoologõa, Universidad Austral de Chile, Valdivia, Chile

A survey of imposex was conducted between the Bay of Mediterranean (Gibbs et al., 1987; Tan 1997; Smith, San Vicente, Concepcion, Chile (36° 500) and the Bay of 1981; Wilson et al., 1993; Axiak et al., 1995) indicate the Renocavi, Puerto Montt, Chile (41° 350), to determine if global nature of this phenomena but to our knowledge imposex is occurring in an area of collection and cultiva- no surveys of imposex have been conducted on the Pa- tion of the gastropod, . Imposex was ci®c Coast of South America. Imposex is causally as- recorded in three chilean muricids C. giganteus, Xantho- sociated with tributyltin (TBT), a component of marine chorus cassidiformis and Nucella crassilabrum complex. anti-fouling paints, and can be induced in neogastopod Percentage of imposex in females sampled ranged from and mesogastropod species at concentrations as low as 0% to 100%. The degree of expression of imposex was 0.5 ng/l (Bryan et al., 1986). The e€ects of this com- not so severe as to elicit sterility in the individuals sur- pound on non-target organisms were ®rst noted in veyed. Incidence of imposex was as high as 47% in areas oysters in France in the 1970s and were linked to de- of harvest of C. giganteus for reproductive and cultivation velopment of a penis-like outgrowth in female gastro- studies for commercial production of this species. A six pods in the early 1980s (Alzieu, 1991; Smith, 1981). percent incidence of imposex was observed in Nucella Incidence of imposex is correlated to ambient water proximal to the facility for cultivation of C. giganteus. TBT concentration with higher induction linked to Gastropods of the Nucella are used in other coun- yacht docking facilities, ship repair docks and marine tries to monitor incidence of imposex and have demon- trac (Smith, 1981; Gibbs et al., 1987). The use of TBT strated sensitivity toward tributyltin (TBT), the associated is prohibited or severely restricted in some countries but causal agent. Since sex of individuals in muriciculture is there is no uniformity of regulations worldwide and determined by the presence or absence of a penis, imposex TBT use continues. may lead to erroneous classi®cation of males and females. Imposex is a graded response with greater penis and The incidence and severity of imposex in C. giganteus and vas deferens development associated with higher related species may have a negative impact on sustainable concentrations of TBT (Bettin et al., 1996). In some cultivation and harvest of gastropods in Chile. Ó 1999 species such as Nucella lapillus, found in Great Britian, Elsevier Science Ltd. All rights reserved. imposex has resulted in local population declines (Gibbs and Bryan, 1986). In this species the growth of the vas Keywords: imposex; gastropod; tributyltin; Chile; Chorus deferens blocks the ovipore of the female preventing giganteus; Nucella. release of egg capsules from the capsule gland. In Ocenebra erinacea, the morphological response to TBT is manifest as a split bursa copulatrix and capsule gland (Gibbs et al., 1990). These species and related gastro- Introduction pods are used to monitor TBT levels in countries where use of TBT anti-fouling paints are regulated. In other Imposex (Smith, 1971) or pseudohermaphrodatism, the stenoglossans such as Ilyanassa obsoleta, reproductive superimposition of male characteristics such as a penis ®tness of the population is seemingly una€ected and vas deferens on female dioecious gastropods is a although a penis, vas deferens, and convoluted oviduct widespread phenomena in the marine environment. are present (Smith, 1981). Imposex is currently documented in over 118 species in Gastropods of the genus Nucella have demonstrated 63 genera worldwide (Bettin et al., 1996). Data from sensitivity to imposex and have previously been used as Europe, Asia, North America, Australia, and the biomonitors for TBT levels in the marine environment (Gibbs and Bryan, 1986; Bright and Ellis, 1990). In *Corresponding author. southern Chile, populations of the Nucella crassilabrum

1227 Marine Pollution Bulletin complex are distributed throughout the intertidal rocky The purpose of this study was to determine if imposex shore. Chile has an extensive coastline over 4200 km is occurring in three marine gastropod species in Chile to long, with over 602 species of gastropods including some a degree that may adversely impact cultivation and 40 nominal muricean species (Ramirez, 1998). Muricids harvest of commercially important species. including Concholepas concholepas, chocolata, and C. giganteus are harvested for local consumption and Materials and Methods export. Excessive harvesting of C. concholepas has led to legal measures to control this threatened mollusc and The , N. crassilabrum, X. cassidiformis, and has increased pressure on other species with potential C. giganteus were collected between February and May, commercial value such as X. cassidiformis and Trophon 1998, from selected sites between the Bay of San Vi- geversianus. cente, Concepcion, (36° 500) (Region IX, Chile) and the E€orts are currently underway to develop sustainable Bay of Renocavi (Metri) Puerto Montt, (41° 360) (Re- cultivation methods for C. giganteus in Chile. One gion X, Chile) (Fig. 1). Each site was classi®ed as an component of the cultivation of muricid gastropods or enclosed bay or open coast and predominate land use muriciculture, is to develop extensive knowledge of the was noted to determine proximity to industry and har- reproductive cycles and life history of this species to bors (Table 1). Areas of high boating activity were optimize production. In muriciculture of C. giganteus, harbors with more than ten ships within visible range of sex determination is based on the presence or absence of the site. Medium boating activity was considered an a penis. If imposex is occuring in areas where gastropods area where less than ten ships were in proximity to the are being collected for cultivation, females may be mis- site or where there are harbor facilities for docking of identi®ed as males and prove detrimental to reproduc- large ships. In the medium boating activity areas, ar- tive studies and commercial production of C. giganteus. tesanal ®shing was frequent. Low shipping activity was a

Fig. 1 Location of sampling sites for the imposex survey. The muriciculture facility is indicated on the map as a star.

1228 Volume 38/Number 12/December 1999

TABLE 1 Site characteristics and sampling dates of imposex survey at eleven sites on the coast of Chile.

Site Coordinates Species sampled Date sampled Site characterization

Lenga 36° 40 S 71° 53 W X. cassidiformis 5/15/98 Open coast, high boating activity, industrial Concepcion 36° 48 S 73° 10 W N. crassilabrum 3/28, 5/11/98 Open coast, high boating activity, industrial Maule 36° 50 S 73° 10 W N. crassilabrum 5/30/98 Open coast, high boating activity, industrial Mancera 39° 55 S 73° 23 W C. giganteus 3/23/98 Bay, medium boating activity, rural Corral 39° 05 S 73° 25 W C. giganteus 4/15/98 Bay, medium boating activity, rural X. cassidiformis 4/15/98 Chaihuin 39° 56 S 73° 37 W C. giganteus 4/2/98 Bay, low boating activity, rural San Carlos 39° 15 S 73° 26 W N. crassilabrum 3/9/98 Bay, medium boating activity, rural Metri 41° 36 S 72° 43 W N. crassilabrum 2/10/98, 3/4/98 Bay, medium boating activity, rural Carelmapu 41° 44 S 73° 44 W N. crassilabrum 3/11/98 Open coast, medium boating activity, rural Guar 41° 40 S 73° 59 W N. crassilabrum 4/28/98 Bay, medium boating activity, rural Caicaen 41° 47 S 73° 11 W N. crassilabrum 3/15/98 Bay, medium boating activity, rural

rural area with no shipping observed and limited artes- were held no more than seven days before anal ®shing. processing. Shell length and shell height were measured The classi®cation of Nucella in Chile is controversial to the nearest 0.1 mm using vernier calipers. Snails were so for the purposes of this survey all Nucella were opened with a hammer and soft tissue mass was weighed grouped together as ``Nucella crassilabrum'' complex. to the nearest 0.01 g. Snails were sexed based on the Nucella were collected at low tide from the intertidal presence of the reproductive glands (albumen gland, zone. X. cassidiformis and C. giganteus were collected sperm-ingesting gland and capsule gland in females, from the subtidal zone by divers at depths from 5 to 20 prostate gland in males). Juveniles and parasitized in- m. When possible, the three species were collected from dividuals were excluded from analyses of imposex inci- the same site. If available, 40 or more adults were col- dence. lected from each site (Table 2). An e€ort was made to The presence or absence of a penis in adults was noted collect all species during their reproductive season. and penis length was measured to the nearest 0.1 mm Nucella were also collected from Metri, the location of using a micrometer graticle (0±10 mm). Penis length was the Center of Aquaculture and Marine Sciences (CEA- measured from the side of the right tentacle where the CIMA) of University of Los Lagos where cultivation of penis joins the body to the tip. Measurements were C. giganteus is currently underway (Fig. 1). Water from conducted on unnarcotized snails. The percent of fe- this bay is pumped into the muriciculture system and males with imposex, average female penis length, Rela- used in the holding tanks. No individuals of C. giganteus tive Penis Size Index and Vas Deferens Sequence Index were collected from the bay adjacent to the muricicul- was calculated for each species at each site. The Vas ture facility because the natural distribution of this Deferens Sequence Index was used to determine the species does not extend this far south. stage of imposex development (Stroben et al., 1992). All snails were transferred to the Universidad Austral This index ranked snails from 0 to 6. A value of zero is a de Chile in Valdivia and maintained under constant normal female with no imposex. Stages 1±4 represent aeration and temperature in a static renewal seawater increasing development of the penis and vas deferens system and a 12/12 h photoperiod until analysis. sequence. At stages 4+ and above the female is infertile

TABLE 2 Suvey results of imposex incidence at 11 sites on the Chilean coastline.

Site Species sampled N Males Male penis length N Females % Imposex Female penis length RPSa VDSb (mm) (mm)

Lenga X. cassidiformis 33 8.62 ‹ 2.11 44 100 3.66 ‹ 2.74 7.68 2.47 Concepcion N. crassilabrum 33 3.86 ‹ 0.84 45 100 0.61 + 0.23 0.39 1.89 Maule N. crassilabrum 11 4.37 ‹ 0.72 23 100 0.75 ‹ 0.27 0.5 1.57 Mancera C. giganteus 37 10.95 ‹ 2.02 42 48 0.72 ‹ 0.89 0.028 0.61 Corral C. giganteus 19 9.34 ‹ 2.09 32 47 0.97 ‹ 1.24 0.125 0.59 X. cassidiformis 36 8.65 ‹ 1.87 30 0 0 0 0 Chaihuin C. giganteus 26 10.94 ‹ 2.64 20 0 0 0 0 San Carlos N. crassilabrum 22 3.83 ‹ 1.02 18 56 0.36 ‹ 0.37 0.082 0.77 Metri N. crassilabrum 31 4.02 ‹ 0.87 33 6 0.03 ‹ 0.10 0 0.09 Carelmapu N. crassilabrum 24 3.92 ‹ 0.65 17 0 0 0 0 Guar N. crassilabrum 10 4.6 ‹ 0.72 34 21 0.13 ‹ 0.31 0.002 0.29 Caicaen N. crassilabrum 16 3.46 ‹ 0.85 26 0 0 0 0 a RPS: Relative Penis Size Index. b VDS: Vas Deferens Sequence Index.

1229 Marine Pollution Bulletin with stage 6 indicating a female with aborted egg cap- an average Vas Deferens Sequence Index of <1. How- sules (Stroben et al., 1992). In this study, Stage 1 was ever, this species was not found in the areas re¯ecting de®ned as a white thickening of skin or penis scar noted the highest percentage of imposex and Vas Deferens in the area behind the right tentacle and without a def- Sequence Index values with the resident species inite penis. The Relative Penis Size Index, de®ned as the N. crassilabrum and X. cassidiformis. The absence of cube of the length of the female penis divided by the Chorus from these areas may be due to overharvesting, cube of the length of the male penis ´ 100 was calculated competition with the resident species, or susceptibility to for each sample (Gibbs et al., 1987). TBT contamination which is likely responsible for the high imposex incidence. Results Potential impact of imposex on muriciculture opera- tions include errors in sex determination of breeding Geographic distribution of imposex in south-central adults of C. giganteus and development of imposex in Chile appears to be widespread with imposex observed snails under cultivation. Adults of C. giganteus are col- in open coastal areas and regions of limited boating lected at sites north of 39° 600 (the southernmost dis- activity as well as in harbors (Table 1). Imposex was tribution for this species) and transported to the holding identi®ed in C. giganteus, X. cassidiformis, and N. facilities at Metri. Snails are sexed based on presence or crassilabrum complex. (Table 2). The percentage of fe- absence of a penis and placed in suspended enclosures in males with imposex in the three species varied according the bay for reproduction and food preference studies. to locale and ranged from 0% to 48% in C. giganteus, 0± Eggs and juveniles are maintained in aquaria with cir- 93% in X. cassidiformis, and 0±100% in Nucella. The culating water from the bay. If adults are collected from Vas Deferens Sequence Index ranged from 0 to 0.6 in C. a site where imposex is present females may be mis- giganteus, 0±2.47 in X. cassidiformis and 0±1.89 in N. identi®ed as males complicating reproductive e€orts in crassilabrum. Individuals with a Vas Deferens Sequence this species. Index of 4+ or above were not found in this survey but Nucella may serve as a bioindicator for imposex levels this degree of imposex is known to occur in other mu- in the commercially valuable species X. cassidiformis ricid gastropods. The Relative Penis Size Index values and C. giganteus when these species are found proximal ranged from 0 to 7.68 but a large variation of this index to each other. Percentage of Nucella females with was found from individuals collected from the same site imposex was higher than the other two species surveyed on di€erent dates indicating that this index may be af- in all study sites. This species is also widely distributed fected by seasonal variations in penis size. A six percent along the Chilean coast and has direct development incidence of imposex in individuals of the genus Nucella (Gallardo, 1979) in contrast to X. cassidiformis and was found in the bay adjacent to muriciculture opera- C. giganteus with indirect planktotropic development tions for C. giganteus indicating a potential impact on and a short lecitotrophic veliger stage respectively commercial production of this species. Average size of (Gallardo, 1981; Gallardo and Gonzalez, 1994; adult C. giganteus was larger in Chaihuin (south of Gonzalez and Gallardo, in press). Nucella populations Corral) where limited extraction is occurring compared may be more susceptible to localized TBT contamina- to the Bay of Corral where this gastropod is intensively tion since they do not have a larval stage for dispersion. harvested by the local ®shing community. In contrast, imposex populations that exhibit a larval Morphological response in the three species was stage may be recolonized by larvae originating at other similar with a vas deferens originating from the area of sites. Normally the sex ratios of these muricid gastro- the penis and developing posteriorly. In Nucella, a pods approaches 1:1. Studies conducted in the 1970s in variation on this pattern occurred with a middle section Mehuin (north of Valdivia) indicated that one of the of the vas deferens present in some individuals that did populations of Nucella had a clear predominance of not originate at the base of the penis. It was not possible males (Gallardo, 1983). In this area, anti-fouling paints to determine the e€ect of imposex on reproduction on were used in ecological studies to study the e€ects the the Muricids sampled. No individuals in any species absence of vegetation on intertidal rocky shore com- surveyed were found with split capsule glands or an munities. It is possible that the use of these paints pro- ovipore blocked by the growth of the vas deferens al- duced a gradient of imposex that would explain the though in some areas where Nucella were previously apparent predominance of males. At the time of this abundant no individuals were found (Gallardo, unpub- observation the phenomena of imposex was not con- lished data). No aborted egg capsules were present and sidered and sex was not veri®ed by examination of the no incidence of sterilization due to overgrowth of the gonads. vulva by the vas deferens was evident in this survey. Imposex was recorded in six percent of Nucella sur- veyed in the bay adjacent to the muriciculture opera- Discussion tions for C. giganteus. At this facility, water from the bay is pumped into a series of aquaria where eggs, lar- The greatest incidence of imposex noted for the eco- vae, and juveniles of C. giganteus are monitored for nomically important species C. giganteus was 48% with growth and viability. Adults of C. giganteus are main-

1230 Volume 38/Number 12/December 1999 tained in suspended enclosures on long lines in the bay. Gallardo, C. S. (1981) Posturas y estadio de eclosion del gastropodo Long-term exposure to the waters of the bay where low Muricidae Chorus giganteus (Lesson: 1829). Studies on Neotropical Fauna and Environment 16, 35±44. levels of imposex were recorded in Nucella may cause Gallardo, C. S. (1983) Bionomische Untersuchungen uber den pseudohermaphrodatism in female and juvenile C. gig- Zusammenhang zwischen Habitat und Reproduktionsbiologie bei anteus. It will be necessary to monitor areas along the einigen marinen Prosobranchia. Inaugural-Dissertation, Fachbe- reich Biologie, Justus-Liebig-Universitat Giessen, Germany. south coast of Chile where C. giganteus is collected to Gallardo, C. S. and Gonzalez, K. (1994) Ovipostura y desarollo determine possible levels of contamination and predict intracapsular de Xanthochorus cassidiformis (Blainville, 1832) the e€ects on cultivation. Reproductive e€ects of ( Muricidae) de la costa sur de Chile. Gayana (Zool.) 58, 79±90. imposex in Chilean gastropods are not known and ad- Gibbs, P. E., Bryan, G. W., Pascoe, P. L. and Burt, G. R. (1990) ditional study is needed to evaluate the impact of Reproductive abnormalities in female Ocenebra erinacea (Gastro- imposex on muriciculture. poda) resulting from tributyltin-induced imposex. Journal of the Marine Biological Association UK 70, 639±656. Gibbs, P. E., Bryan, G. W., Pascoe, P. L. and Burt, G. R. (1987) The Funding for this project was provided by a Graduate Enhancement use of the dog-whelk, Nucella lapillus, as an indicator of tributyltin Fellowship from the Academy for Educational Development and ad- (TBT) contamination. Journal of the Marine Biological Association ditional support from Proyecto Fondecyt 1960488 and Proyecto UK 67, 507±523. Fundacion Volkswagen I 96-01 (to C. S. Gallardo). The assistance of Gibbs, P. E. and Bryan G. W. (1986) Reproductive failure in Carmen Gloria Narvaez, Eduardo Cardenas, Rodrigo Gonzalez, populations of the dog-whelk Nucella lapillus, caused by imposex Bruno Vital, and students and faculty of The Institute of Zoology, induced tributyltin from anti-fouling paints. Journal of the Marine Universidad Austral de Chile, Valdivia, Chile, and Centro EULA, Biological Association UK 66, 767±777. Universidad de Concepcion, Concepcion, Chile is much appreciated. Gonzalez, K. and Gallardo, C. S. Embryonic and larval development of the muricid snail Chorus giganteus (Lesson, 1879) with an Alzieu, C. L., Sanjuan, J., Deltreil, J. P. and Borel, M. (1991) assessment of the developmental nutrition source. Ophelia (in Environmental problems caused by TBT in France: assessment, press). regulations, prospects. Marine Environmental Research 32, 7±18. Ramirez, J. ed. (1998) Moluscos de Chile, vol. 3, . Axiak, V., Vella, A. J., Micallef, D. and Chircop, P. (1995) Imposex in Santiago de Chile. Hexaplex trunculus (Gastropoda:Muricidae): ®rst results from Smith, B. S. (1981) Tributyl compounds induce male characteristics on biomonitoring of tributyltin contamination in the Mediterranean. female mud snails. Nassarius obsoletus ˆ Ilyanassa obsoleta. Journal Marine Biology 121, 685±691. of Applied Toxicology 3, 141±144. Bettin, C., Oehlmann, J. and Stroben, E. (1996) TBT-induced imposex Smith, B. S. (1971) Sexuality in the American mud snail, Nassarius in marine neogastropods is mediated by an increasing androgen obsoletus: abnormalities in the reproductive system. Journal of level. Helgolander Meeresunters 50, 299±317. Molluscan Studies 46, 247±256. Bright, D. A. and Ellis D. V. (1990) A comparative survey of imposex Stroben, E., Oehlmann, J. and Fioroni, P. (1992) The morphological in northeast Paci®c neogastropods (Prosobranchia) related to expression of imposex in Hinia reticulata (Gastropoda: Buccinidae): tributyltin contamination, and choice of a suitable bioindicator. a potential indicator of tributyltin pollution. Marine Biology 113, Canadian Journal of Zoology 68, 1915±1924. 625±636. Bryan, G. W., Gibbs, P. E., Hummerstone, L. G. and Burt, G. R. Tan, K. S. (1997) Imposex in three species of Thais from Singapore, (1986) The decline of the gastropod Nucella lapillus around South- with additional observations on T. clavigera (Kuster) from Japan. West England: evidence for the e€ect of Tributyltin from anti- Marine Pollution Bulletin 34, 577±581. fouling paints. Journal of the Marine Biological Association UK 66, Wilson, S. P., Ahsanullah, M. and Thompson, G. B. (1993) Imposex in 611±640. neogastropods: an indicator of tributyltin contamination in Eastern Gallardo, C. S. (1979) Developmental pattern and adaptations for Australia. Marine Pollution Bulletin 26, 44±48. reproduction in N. crassilabrum and other muricacean gastropods. Biological Bulletin 157, 453±463.

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