Thalassas, 2005, 21 (2): 71-75 An International Journal of Marine Sciences THE INCREASING INCIDENCE OF IMPOSEX IN STRAMONITA HAEMASTOMA (MOLLUSCA: GASTROPODA: MURICIDAE) AFTER THE ESTABLISHMENT OF THE PECÉM HARBOR, CEARÁ STATE, NORTHEAST BRAZIL Ítalo Braga de Castro(1), Carlos Augusto Oliveira de Meirelles(1),(2), Jully Cristiny Lopes Pinheiro(2), Helena Matthews-Cascon(1),(2) and Cristina de Almeida Rocha-Barreira(1) Keywords: TBT, Imposex, Mollusks, Pollution, Harbor ABSTRACT INTRODUCTION Imposex is characterized by the development of Imposex is characterized by the development of masculine sexual organs in neogastropod females, masculine sexual organs, like penis and vas deferens, caused by organotin contamination of the seawater. in neogastropod females (Smith, 1971). This These compounds, as tributyltin (TBT), are used abnormality is a consequence of an endocrine world-wide as biocidal agents in antifouling paints, disruption induced by marine environment commonly used to covering boats and off shore contamination by tin organic compounds, as tributyltin metallic structures. Five sampling sites were (TBT) and triphenyltin (TPT), (Morcillo & Porte, determined along the coast of Ceará State, Northeast 1999; Depledge & Billinghurst, 1999). Brazil in March 2001. In each site were collected 50 These compounds are used world-wide as biocidal adults of neogastropod Stramonita haemastoma and agents in antifouling paints, commonly used to indexes that quantify imposex (% imposex, RPLI, covering boats and off shore metallic structures. The RPSI and VDSI) were observed. Other two collecting use of TBT and/or TPT-based antifouling paints started times were conducted (April 2003 and March 2004) at during the 60's, when it was known that these the same sites and imposex indexes were observed compounds had biocidal action and we can find the again. The indexes observed in the three collecting first report of harmful effects of these paints during the times showed an increase in the incidence of imposex 70's (Alzieu et al., 1982; Ruiz et al., 1996). Nowadays, at the sampling sites, probably caused by the more than 70% of world's fleet (Champ, 2000) is still establishment of Pecém Harbor and its increasing using organotin copolymers, releasing approximately shipping activities 4ug/cm2 of tin compounds per day to the seawater 1Laboratório de Zoobentos, Instituto de Ciências do Mar (Horiguchi et al., 1994). (LABOMAR)/Universidade Federal do Ceará- Av. da Abolição, Morphological changes induced by imposex are 3207 Bairro Meireles, 60165-081, Fortaleza, Ceará, Brazil. E- very visible, so, they became an important way to mail: [email protected] 2Laboratório de Invertebrados Marinhos - LIMCe - detect marine ecosystems contamination by organotin Universidade Federal do Ceará, Centro de Ciências, compounds. This kind of detection was done in Departamento de Biologia, Campus do Pici, 60455-970, England, where the study of imposex in Nucella Fortaleza, Ceará, Brazil lapillus allowed a large monitoring of TBT 71 Ítalo Braga de Castro, Carlos Augusto Oliveira de Meirelles, Jully Cristiny Lopes Pinheiro, Helena Matthews-Cascon, and Cristina de Almeida Rocha-Barreira contamination (Gibbs & Bryan, 1987). Monitoring MATERIALS AND METHODS works that used neogastropods to detect this kind of contamination were made in the coast area of many It was determined 5 points as sampling sites along countries: Portugal (Spence, 1990), Singapore (Ellis & the coast of Ceará State, Northeast Brazil. The Pattisina, 1990), New Zealand (Stewart et al.,1992; predominant maritime currents of the region as well as the proximity in relation to the Pecém Harbor were ATLANTIC OCEAN considered to the determination of these points: Cauípe's Beach (E1), Pecém Harbor (E2), Pecém's E5 Beach (E3) and Taíba's Beach (E4 e E5) (Fig. 1). E4 E3 E2 Three collecting times were conducted: March PECÉM E1 HARBOR 2001, April 2003 and March 2004. In each site were 5 Km collected, manually, 50 adults specimen of neogastropod Stramonita haemastoma. The animals were kept plastic boxes containing water from the respective site and they were led to the laboratory. The mollusks were preliminary narcotized with magnesium BRAZI chloride solution 3.5% (Huet et al., 1995). Subsequently length of each shell was measured from the apex to the distal end of the siphonal canal using Fig. 1. calipers and they were removed to reach the soft parts. Sampling sites in the coast of Ceará State, Northeast Brazil. The sexual determination was made based on the presence of the sperm-ingesting gland, observed only Smith,1996), Japan (Horiguchi et al., 1995; Horiguchi in females, the presence of prostate, exclusive to et al.,1997), Canada (Tester et al., 1996), Ireland males, and the coloration of the gonads. All males as (Minchin et al., 1997), Spain (Morcillo & Porte, 1998), well as the females that presented imposex had their Thailand (Bech, 1998) and Hong Kong (Blackmore, penises measured with a millimetric slide and the 2000). All these works showed clearly the influence of formation of the vas deferens in the females was also harbor areas on the results of indexes that quantify observed. imposex. For the quantification of imposex, three index The first report of imposex in the Atlantic coast of initially developed for the neogastropod Nucella South America was in Brazil, where this abnormality lapillus (Gibbs and Bryan, 1987; Gibbs et al., 1987) was observed in the neogastropod Stramonita and successfully used for others neogastropods haemastoma in the coast of São Paulo City (Magalhães (Minchin et al., 1996; Gooding et al., 1999) were et al., 1997), Fortaleza City (Castro et al., 2000) and used: the RPLI (relative penis length index), the RPSI Rio de Janeiro City (Fernandez et al., 2002). Further (relative penis size index) and the VDSI (vas deferens monitoring works were made in Maceió City (Camillo sequence index), scale that was originally developed et al., 2004) and Natal City (Castro et al., 2004), where by Gibbs et al. (1987) and later adapted for the use in it was studied imposex in Stramonita rustica. Stramonita haemastoma in the Brazilian coast To improve industry products transportation in (Fernandez et al., 2002; Fernandez et al., 2005). Ceará State, the state government started to construct, The RPLI is an index that quantifies the degree of in 1996, the Pecém Harbor. The location, before harbor imposex in the population and is obtained from the establishment, was not developed and there was not equation: (Mean length of female penis) / (Mean ship activity. The purpose of the present paper is to length of male penis) X 100. This index is better verify the organotin contamination of the area near applied in low contaminated areas. The RPSI Pecém Harbour, using imposex in Stramonita quantifies the degree of imposex in the population by haemastoma as bioindicator the equation: (Mean length of female penis) 3 / (Mean length of male penis) 3 X 100. This cubical index is better applied in highly contaminated areas, when the length of the female penis approaches the length of the 72 The increasing incidence of imposex in S. haemastoma Table 1. Imposex indexes observed in the three collecting times (March 2001, April 2003 and March 2004). 120 Collecting Sample n female % VDSI RPLI RPSI 100 Time size imposex 80 Site E1 mar/01 60 03/2001 50 30 68,42 0,20 0,10 0,01 abr/03 04/2003 50 26 69,2 0,85 1,33 0,01 40 03/2004 50 29 100,0 1,70 1,40 0,24 mar/04 % imposex Site E2 20 03/2001 50 24 66,6 0,50 1,20 0,01 04/2003 50 24 100,0 1,30 4,90 0,01 0 03/2004 50 27 100,0 2,00 28,37 2,33 Site E3 12345 03/2001 50 32 81,81 0,63 3,76 0,01 Sites 04/2003 50 30 100 1,45 11,27 0,14 03/2004 50 34 100 1,86 14,05 0,27 Fig. 2. Site E4 03/2001 50 28 7,4 0,01 0,53 0,01 % imposex observed in S. haemastoma from 2001 to 2004. 04/2003 50 30 13,0 0,13 2,55 0,01 03/2004 50 30 20,0 0,25 2,63 0,01 Site E5 03/2001 50 26 0,0 0,00 0,00 0,00 2,5 04/2003 50 24 6,6 0,08 1,41 0,01 03/2004 50 29 0,0 0,00 0,00 0,00 2 mar/01 male penis. 1,5 abr/03 The VDSI quantifies the degree of imposex in the VDSI 1 mar/04 animal through the sequence of the penis and vas deferens formation. This scale is composed of 7 levels: 0,5 stage 0, a healthy female without a sign of penis or vas 0 deferens; stage I, the beginning of the penis formation, 12345 characterized by a small wart; stage II, the penis Sites appears formed with approximately 1mm in length and Fig. 3. is observed the beginning of the vas deferens VDSI (vas deferent sequence index) values observed in S. formation; stage III, the penis is already developed, haemastoma from 2001 to 2004. with size superior to 1mm and it is also observed an VDSI showed a gradual decrease when the distance of incomplete vas deferens in the bottom of the palial the sites from the harbor was increased (Table 1). cavity; stage IV, the penis is developed, with size RPSI oscillations were low (almost all of sites superior to 1mm and a complete vas deferens can be oscillated between 0.00 and 0.27, except E2), however, observed; stage V, characterized by the blockage of the % imposex, RPLI and VDSI showed important vulva and stage VI where the presence of aborted variations, as we can see in figures 2, 3, 4 and 5.