Imposex, a pseudo-hermaphroditic condition characterized by the development of male sexual characteristics in female prosobranch gastropods, is used as a bioindicator of pollution by tributyltin (TBT) -based antifoulants. The incidence and the intensity of imposex, evaluated from the vas deferens sequence index (VDSI) and the female penis length (FPL), were investigated in Hexaplex trunculus populations from seven locations in Kaštela Bay (Adriatic Sea, Croatia). Analysis of 131 females revealed the absolute prevalence of imposex at all investigated locations. Furthermore, most of the females showed signs of advanced imposex development (VDSI=4.4±0.8, range=2-5). Female penis homologues were found to be well developed (max=20 mm) with mean values ranging from 5.2-12.6 mm. Significant differences were detected regarding both the VDSI (H=36.24, p<0.001) and the FPL (F=7.08, p<0.001) between the locations. The results presented in the study confirm the widespread incidence of imposex and can provide reference values for future monitoring programs.Imposeks, pseudohermafroditizam karakteriziran pojavom muških spolnih obilježja u ženki prednjoškržnih puževa, upotrebljava se kao bionidikator zagađenja tributil-kositrom (TBT), sastojkom protuobraštajnih boja. Za procjenu pojave i intenziteta imposeksa korištene su vrijednosti indeksa stupnja vas deferensa (VDS I) i dužine penisa ženki (FPL ) dobivene analizom primjeraka vrste Hexaplex trunculus prikupljenih sa sedam postaja u Kaštelanskom zaljevu. Na istraživanim postajama analizom 131 ženke (visina kućice=66,3±7,5 mm) utvrđena je potpuna zahvaćenost imposeksom. Štoviše, većina zahvaćenih ženki bila je u uznapredovalom stadiju imposeksa (VDS I=4,4±0,8; raspon vrijednosti=2-5). Penisi su bili dobro razvijeni kod ženki (max=20 mm) sa srednjim vrijednostima od 5,2 do 12,6 mm. Utvrđene su statistički značajne razlike u VDS I (H=36,24, p<0,001) i FPL (F=7,08, p<0,001) između postaja. Rezultati istraživanja potvrđuju široku rasprostranjenost imposeksa i mogu se koristiti kao referentne vrijednosti za daljnje praćenje

Dajana BRAJČIĆ

Daniela BLAŽEVIĆ

Katarina MADIRAZA

Katja JELIĆ

Marijana ZOKO

Mark PRIME

Melita PEHARDA

Nika STAGLIČIĆ

Željka ERAK

English

STAGLIČIĆ, Nika

PRIME, Mark

ZOKO, Marijana

ERAK, Željka

BRAJČIĆ, Dajana

BLAŽEVIĆ, Daniela

MADIRAZA, Katarina

JELIĆ, Katja

PEHARDA, Melita

Hrčak - Portal of scientific journals of Croatia

ISSN: 0001-5113AADRAYACTA ADRIAT.,49(2): 159 - 164, 2008 UDC: 594.3: 591.167 (262.3.04 Kaštela)Imposex incidence in Hexaplex trunculusfrom Kaštela Bay, Adriatic Seanika staGlIČIĆ1*, Mark PRIME2,  Marijana zoko3, Željka erak3, dajana BrajČIĆ3, daniela BlaŽevIĆ3, katarina MadIrazza3, katja jelIĆ3and Melita PEhARDA11Institute of Oceanography and Fisheries, Šet. I. Meštrovića 63,P.O. Box 500, 21 000 Split, Croatia*Corresponing author, e-mail: nika@izor.hr2School of Ocean Science, Menai Bridge, Anglesey, Wales, LL59 5AB, UK3Centre for Marine Studies, University of Split, Livanjska 5/III, 21 000 Split, CroatiaImposex, a pseudo-hermaphroditic condition characterized by the development of male sexual characteristics in female prosobranch gastropods, is used as a bioindicator of pollution by tributyltin (TBT) -based antifoulants. The incidence and the intensity of imposex, evaluated from the vas deferens sequence index (VDSI) and the female penis length (FPL), were investigated in hexaplex trunculus populations from seven locations in Kaštela Bay (Adriatic Sea, Croatia). Analysis of 131 females revealed the absolute prevalence of imposex at all investigated locations. Furthermore, most of the females showed signs of advanced imposex development (VDSI=4.4±0.8, range=2-5). Female penis homologues were found to be well developed (max=20 mm) with mean values ranging from 5.2-12.6 mm. Significant differences were detected regarding both the VDSI (H=36.24, p<0.001) and the FPL (F=7.08, p<0.001) between the locations. The results presented in the study confirm the widespread incidence of imposex and can provide reference values for future monitoring programs.Key words: imposex, Hexaplex trunculus, kaštela Bay, adriatic sea, croatiaINTRODUCTIONMany worldwide studies relate imposex development, a pseudo-hermaphroditic condi-tion characterized by the development of male sexual characteristics in female prosobranch gastropods, to the use of organotin-based anti-fouling paints (aXIak et al., 1995; TERLIZZI et al., 1998; TERLIZZI et al., 1999; poWer & keeGan, 2001; aXIak et al., 2003; VASCONCELOS et al., 2006). Imposex is considered to be mediated by organotin interference with steroid metabo-lism (BETTIN et al., 1996; oBerdÖrster et al., 1998; oBerdÖrster & Mcclellan-Green, 2002; nIsHIkaWa, 2005). Since elevated organotin concentrations are most commonly measured near yacht marinas and  shipyards, this study aimed to investigate the incidence and the intensity of imposex in Hexaplex trunculus populations from kaštela Bay. The bay is a semi-enclosed, relatively small body of water, historically known as a ‘’Black Spot’’ due to intensive anthropogenic pollution (BarIĆ, 1989) exacerbated by poor 160  ACTA ADRIATICA, 49(2): 159 - 164, 2008flushing characteristics (ZORE-ARMANDA, 1980) and is currently characterized by intense shipping activity which encompasses a large shipyard and a growing number of nautical marinas.The target species, H. trunculus, is one of the most common muricids in the Mediterranean (TERLIZZI et al., 1998). It appears to be highly sus-ceptible to the development of imposex and thus an important indicator species for organotin con-tamination (aXIak et al., 1995). Since H. trunculus is also a direct developer species (PELLIZZATO et al., 2004) it could possibly be used to indicate point sources of organotin pollution. MATERIAL AND METHODSBetween 40 and 60 adult Hexaplex trunculus specimens (≥4 cm in length) were collected by snorkelling and SCUBA diving at seven loca-tions in kaštela Bay: Bene, Marina kaštela and kaštel stari in winter 2004, followed by lora, lučica, resnik and arbanija in summer 2006 (fig. 1). Sampling locations were chosen to cover the entire bay area. Collected H. trunculus specimens were brought to the laboratory where the shell length (SL) was measured to the near-est 0.1 mm using vernier callipers, upon which they were cracked in a bench vice and then the soft body was removed and examined.Males were recognized by the presence of only one cavity opening (anus), penis and vas deferens and by the absence of an egg capsule gland. females, both normal and affected, were recognized by the presence of a second opening (vagina) and the egg capsule gland (aXIak et al., 1995). The incidence and the intensity of imposex in females were evaluated from the mean female penis homologue length (fPL) and the vas def-erens sequence index (VDSI). Using graduated graph paper, fPL was measured, to the near-est 0.1 mm, from the tip to the base without straightening the penis. VDSI was calculated as the mean of the imposex stages present in the sample. Different imposex stages were deter-mined using aXIak et al. (1995) and TERLIZZI et al. (1999) classifications as shown in Table 1.One-way ANOVA was used to test for signifi-cant differences in the fPL between locations while a kruskal-Wallis analysis determined the differences in the VDSI between locations. All of these analyses were carried out on the statisti-cal package “Minitab 13”.RESULTS AND DISCUSSIONA total of 131 females (length=66.3±7.5 mm) were analyzed from seven locations in kaštela Bay: Bene (n=20), Marina kaštela (n=19), kaštel stari (n=18), lora (n=14), lučica (n=20), resnik (n=20) and arbanija (N=20). Imposex data is summarized in fig. 2 and Table 2. All collected females showed signs of imposex development (VDSI=4.4±0.8), with observed imposex stages ranging from 2 Fig. 1. (A) Position of Kaštela Bay relative to the Adriatic Sea, (B)  Map of Kaštela Bay showing the positions of the sampling locations (●) and shipyards (▼); SS - Shipyard Split, ST - Shipyard Trogir161stagličić et al.: Imposex incidence in Hexaplex trunculus from kaštela Bay, adriatic seato 5. The most advanced imposex stage 5 was observed at all sampling locations, showing a 20-90% frequency. female penis homologues were found to be well developed (max=20 mm) with mean values ranging from 5.2 mm to 12.6 mm. Some females also exhibited excrescences on the penis. Significant differences were detected regard-ing both the vdsI (kruskal-Wallis, H=36.24, p<0.001) and the fPL (ANOVA, f=7.08, p<0.001) between the locations. The most severely affected of the analyzed locations, yacht marina lučica and navy harbour lora, are the closest to the Split shipyard and are also by themselves a source of contamination (fig. 1). the results show that lučica and lora have the highest proportion of specimens exhibiting stage 5 development (fig. 2) and in turn also the high-est VDSI values of 4.9 and 4.7, respectively. Only in samples from Resnik and Arbanija was imposex not so pronounced, as indicated by the lowest proportion of advanced imposex stages with less than 30% of females in stages higher than 4.7, and minimal values of VDSI  and fPL being 3.8 and 6.7 mm for Resnik and 3.9 and 5.2 mm for Arbanija. These two locations are the furthest from the main point sources of orga-notin contamination in kaštela Bay,  the split shipyard situated at the opposite end of the bay and numerous dispersed marinas. Even though Resnik and Arbanija exhibited the lowest values in this study, when compared to a few detailed studies carried out in the eastern Adriatic (TERL-IZZI et al., 2004; GARAVENTA et al., 2006b; PRIME et al., 2006), the results correspond to polluted loca-tions. Since the impact of organotin toxcitiy may be seen over several kilometres from a known pollution source (aXIak et al., 2000), it indicates that the proximity of another smaller shipyard in the town of Trogir (fig. 1) may also impact upon these locations. Although the intensity of imposex is pri-marily related to organotin concentrations in the tissue, and in turn to the concentrations measured in the environment (aXIak et al., 2000; ChIAVARINI et al., 2003; PELLIZZATO et al., 2004; GARAVENTA et al., 2006b), the precise biochemical mechanism of imposex induction still remains unresolved. futhermore, the studies of aXIak et al. (2003) and GARAVENTA et al. (2006a) have sug-gested that there is incidence of early imposex development as a result of various stresses.VDSI stage DescriptionStage 0 female with no male sexual characteristicsStage 1 presence of incipient penis behind the right ocular tentacle, vas deferens is still not developedStage 2 penis with initiation of vas deferensStage 3 more prominent penis and vas deferens still not reaching the vaginal openingStage 4 vas deferens reaches the vaginal openingStage 4.3 vas deferens passes the vaginal openingStage 4.7 vas deferens extends beyond the point in stage 4.3 and now runs along the capsule gland by approximately 30% of its lengthStage 5 vas deferens runs along the entire length of the capsule gland. No vulva is visible and the capsule gland is swollen, frequently split with its lumen open into the mantle cavity. Egg capsule release is prevented and aborted capsules are often present. These females can be regarded as sterileTable 1. Classification of  vas deferens sequence index (VDSI) stages162  ACTA ADRIATICA, 49(2): 159 - 164, 2008All of the studies which determined the inci-dence of imposex along the eastern coast of the Adriatic Sea (TERLIZZI et al., 2004; GARAVENTA et al., 2006b; PRIME et al., 2006), including this one, confirm its widespread incidence and can provide reference values for future monitoring programs. Especially now, with Croatia’s recent adoption of the total ban on the use of organotin-based anti-foulants (NN, 17/2006) there is a need for further research aimed at understanding the imposex induction mechanism and assessing the effective-ness of  the regulations’ implementation. CONCLUSIONSAll analyzed female H. trunculus (N=131) were affected by imposex. The most advanced imposex stage 5 was observed in 46% of females. The low-est imposex stage found, stage 2, accounted for 2% of the total number of analyzed females.Location SL (mm) fPL (mm) VDSIBene 67.5±4.6 8.1±2.4 4.5±0.4Marina kaštela 65.0±4.0 12.6±5.0 4.4±0.7kaštel stari 66.3±9.2 6.8±3.0 4.6±0.5Lora 72.0±11.8 10.6±4.5 4.7±0.7lučica 70.7±5.7 11.8±3.3 4.9±0.2Resnik 64.8±6.3 6.7±2.6 3.8±1.0Arbanija 59.4±2.9 5.2±2.4 3.9±0.8Total 66.3±7.5 8.7±4.3 4.4±0.8Table 2. Summary of mean ± SD shell length (SL), female penis homologue length (FPL) and the vas deferens sequence index (VDSI) measurements of hexaplex trunculus from seven locations in Kaštela BayFig. 2. Percentage of imposex stages for hexaplex trunculus at each of the sampling locations in Kaštela Bay163stagličić et al.: Imposex incidence in Hexaplex trunculus from kaštela Bay, adriatic seaREFERENCESaXIak, v., a.j. vella, d. MIcallef, p. cHIrcopa & B. MINTOff. 1995. Imposex in Hexaplex trunculus (Gastropoda: Muricidae): first results from biomonitoring of tributyltin contamination in the Mediterranean. Mar. Biol., 121: 685-691. aXIak, v., a.j. vella, d. aGIus, p. BonnIcI, G. cas-SAR, R. CASSONE, P. ChIRCOP, D. MICALLEf, B. MINTOff & M. SAMMUT. 2000. Evaluation of environmental levels and biological impact of TBT in Malta (central Mediterranean). Sci. Total Environ., 258(1-2): 89-97.aXIak, v., d. MIcallef, j. Muscat, a. vella & B. MINTOff. 2003. Imposex as a biomonitoring tool for marine pollution by tributyltin: some further observations. Environ. Int., 28(8): 743-749. BarIĆ, a. 1989. stupanj eutrofikacije u kaštelan-skom zaljevu (Eutrophication level in the kaštela Bay). pogledi, 19(1): 127-141.BETTIN, C., J. OEhLMANN & E. STROBEN. 1996. TBT-induced imposex in marine neogastro-pods is mediated by an increasing androgen level. helgol. Mar. Res., 50: 299-317. ChIAVARINI, S., P. MASSANISSO, P. NICOLAI, C. NOBILI & R. MORABITO. 2003. Butyltins con-centration levels and imposex occurrence in snails from the Sicilian coasts (Italy). Chem-osphere, 50(3): 311-319.GARAVENTA, f., M. fAIMALI & A. TERLIZZI. 2006a. Imposex in pre-pollution times. Is TBT to blame? Mar. Pollut. Bull., 52: 696-718.GARAVENTA, f., f. PELLIZZATO, M. fAIMALI, A. terlIzzI, d. MedakovIĆ, s. GeracI & B. PAVONI. 2006b. Imposex in Hexaplex truncu-lus at some sites on the North Mediterranean Coast as a base-line for future evaluation of the effectiveness of the total ban on organo-tin based antifouling paints. hydrobiologia, 555: 281-287.nIsHIkaWa, j. 2005. Imposex in marine gastro-pods may be caused by binding of orga-notins to retinoid X receptor. Mar. Biol., 149(1): 117-124.NARODNE NOVINE - Croatian Official Gazette, No.17/2006. lista opasnih kemikalija čiji je promet zabranjen, odnosno ograničen (the list of dangerous chemicals whose use is forbidden or restricted).  NN 17/2006, pp. 1068-1078.oBerdÖrster, e., d. rIttscHof & p. Mcclel-LAN-GREEN. 1998. Testosterone metabolism in imposex and normal Ilyanassa obsoleta: Comparison of field and TBTA Cl-induced imposex. Mar. Pollut. Bull., 36(2): 144-151.oBerdÖrster, e. & p. Mcclellan-Green. 2002. Mechanisms of imposex induction in the mud snail, Ilyanassa obsoleta: TBT as a neurotoxin and aromatase inhibitor. Mar. Environ. Res., 54(3-5): 715-718.PELLIZZATO, f., E. CENTANNI, M.G. MARIN, V. MOSChINO & B. PAVONI. 2004. Concentra-tions of organotin compounds and imposex in the gastropod Hexaplex trunculus from the Lagoon of Venice. Sci. Total Environ., 332(1-3): 89-100.poWer, a.j. & B.f. keeGan. 2001. The Signifi-cance of Imposex Levels and TBT Contami-nation in the Red Whelk, Neptunea antiqua (L.) from the Offshore Irish Sea. Mar. Pollut. Bull., 42(9): 761-772.prIMe, M., M. peHarda, k. jelIĆ, I. MladIneo & C.A. RIChARDSON. 2006. The occurrence of imposex in Hexaplex trunculus from the Croatian Adriatic. Mar. Pollut. Bull., 52(7): 810-812.TERLIZZI, A., S. GERACI & V. MINGANTI. 1998. Tributyltin (TBT) pollution in the coastal waters of Italy as indicated by imposex in Hexaplex trunculus (Gastropoda, Murici-dae). Mar. Pollut. Bull., 36(9): 749-752.TERLIZZI, A., S. GERACI &  P.E. GIBBS. 1999. Trib-utyltin induced imposex in the Neogastropod Hexaplex trunculus in Italian coastal waters: morphological aspects and ecological impli-cations. Ital. J. Zool., 66: 141-146.TERLIZZI, A., A. L. DELOS, f. GARAVENTA, M. fAIM-ALI & S. GERACI. 2004. Limited effectiveness of marine protected areas: imposex in Hex-aplex trunculus (Gastropoda, Muricidae) populations from Italian marine reserves. Mar. Pollut. Bull., 48(1-2): 188-192.164  ACTA ADRIATICA, 49(2): 159 - 164, 2008VASCONCELOS, P., M. B. GASPAR & M. CASTRO. 2006. Imposex in Hexaplex (Trunculariopsis) trunculus (Gastropoda: Muricidae) from the Ria formosa lagoon (Algarve coast - southern Portugal). Mar. Pollut. Bull., 52(3): 337-341.ZORE-ARMANDA, M. 1980. Some dynamic and hydrographic properties of the kaštela Bay. Acta Adriat., 21(2): 55-74.Received: 17 March 2007Accepted: 27 October 2008Rasprostranjenost imposeksa kod vrste Hexaplex trunculusu Kaštelanskom zaljevu, Jadransko morenika staGlIČIĆ1*, Mark PRIME2,  Marijana zoko3, Željka erak3, dajana BrajČIĆ3, daniela BlaŽevIĆ3, katarina MadIrazza3, katja jelIĆ3 i Melita PEhARDA11Institut za oceanografiju i ribarstvo, P.P. 500, 21 000 Split, Hrvatska*Kontakt adresa, e-mail: nika@izor.hr2Fakultet za znanosti mora, Menai Bridge, Anglesey, Wales, LL59 5AB, UK3Centar za studije mora, Sveučilište u Splitu, Livanjska 5/III, 21 000 Split, HrvatskaSAŽETAKImposeks, pseudohermafroditizam karakteriziran pojavom muških spolnih obilježja u ženki prednjoškržnih puževa, upotrebljava se kao bionidikator zagađenja tributil-kositrom (tBt), sas-tojkom protuobraštajnih boja. Za procjenu pojave i intenziteta imposeksa korištene su vrijednosti indeksa stupnja vas deferensa (vdsI) i dužine penisa ženki (fpl) dobivene analizom primjeraka vrste Hexaplex trunculus prikupljenih sa sedam postaja u kaštelanskom zaljevu. na istraživanim postajama analizom 131 ženke (visina kućice=66,3±7,5 mm) utvrđena je potpuna zahvaćenost imposeksom. Štoviše, većina zahvaćenih ženki bila je u uznapredovalom stadiju imposeksa (vdsI=4,4±0,8; raspon vrijednosti=2-5). penisi su bili dobro razvijeni kod ženki (max=20 mm) sa srednjim vrijednostima od 5,2 do 12,6 mm. utvrđene su statistički značajne razlike u vdsI (H=36,24, p<0,001) i fpl (f=7,08, p<0,001) između postaja. rezultati istraživanja potvrđuju široku rasprostranjenost imposeksa i mogu se koristiti kao referentne vrijednosti za daljnje praćenje.Ključne riječi: imposex, Hexaplex trunculus, kaštelanski zaljev, jadran, Hrvatska

Rasprostranjenost imposeksa kod vrste Hexaplex trunculus u Kaštelanskom zaljevu, Jadransko more

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Rasprostranjenost imposeksa kod vrste Hexaplex trunculus u Kaštelanskom zaljevu, Jadransko more

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