THAIS ( STRAMONITA ) RUSTICA ( LAMARCK , 1822 ) ( MOLLUSCA : GASTROPODA : THAIDIDAE ) , A POTENTIAL BIOINDICATOR OF CONTAMINATION BY ORGANOTIN NORTHEAST BRAZIL

The use of antifouling paints containing the biocide compound tributyltin (TBT) has been shown as an inductor of imposex in neogastropods mollusks. Imposex is characterized by the development of male features in females, mainly the appearance of a no functional vas deferens and penis. Samples of Thais rustica were collected in eight sites in the metropolitan area of Natal city, Rio Grande do Norte state, northeast Brazil, and examined for occurrence of imposex, which was found in many females. The higher imposex levels were presented by samples from sites near city harbor.


INTRODUCTION
Antifouling paints are used world-wide as covering of boats and other metallic structures in intention to prevent incrustations and corrosion which are processes that drastically reduce the income of boats and the durability of these structures.
To solve these problems, during the decades of 60 and 70, highly efficient paints were developed using tin organic compounds as tributyltin (TBT) and triphenyltin (TPT) as biocide agent.Since the 70's, it was known that these compounds were harmful to the marine environment, been detected an increase in the mortality rate and serious deformities in shells of Crassostrea gigas cultivated, in the Arcachon bay in France (Alzieu et al., 1982;Alzieu et al., 1986;Alzieu et al., 1989).
Imposex was verified in gastropods (Smith, 1971) and is characterized by the development of non functional masculine sexual organs, like penis and vas deferens, in females.This phenomenon is mainly caused by organotin contamination of the seawater and/or food cases.The problem was initially observed in Nucella lapilus from the southwest coast of England, where some populations of this prosobranch mollusk had severely declined (Bryan et al., 1987b).
Many countries around the world had already evidenced imposex in gastropods.As a result some have implemented severe legislations that banned the use of TBT based paints in boats smaller than 25 meters long (Minchin et al., 1995).
Imposex in Thais rustica was reported for the first time in the coast of the state of Alagoas, where monitoring works were made using this species as a possible indicator of organotin contamination (Quadros, personal communication)*.
The purpose of the present paper is to provide a preliminary biomonitoring of imposex in Thais rustica from Natal City coast, Northeast Brazil.

MATERIALS AND METHODS
It was determined 8 points as sampling sites along the coast of the city of Natal-RN, Brazil.The predominant maritime currents of the region as well as the proximity in relation to the Natal Harbor, situated in the estuary of the Potengi River were considered to the determination of these points (Fig. 1).

Field Procedures
In each site were collected, manually, 25 adults of Thais rustica, except in the pier of Redinha's Beach where, despite the effort, only 8 individuals were collected.These animals were kept plastic boxes containing water from the respective sites and they were led to the laboratory.

Laboratory Procedures
The mollusks were preliminary narcotized with magnesium chloride solution 3.5% (Huet et al., 1995).Subsequently length of each snail was measured from the apex to the distal end of the siphonal canal using callipers.The shell of the animals was crushed with a hammer, soft parts were placed in a petri dish, and examined using a binocular microscope.
The sexual determination was made based on the presence of the sperm-ingesting gland, observed only in females, the presence of prostate, exclusive to males, and the coloration of the gonads.
All males as well as the females that presented imposex, had their penises measured with a millimetric slide and the formation of the vas deferens in the females was also observed.The percentage of females presenting imposex was calculated.__________ (*) Quadros, J. 2002.Universidade Estadual do Rio de Janeiro.
For the quantification of imposex, three index initially developed for the neogastropod Nucella lapillus (Gibbs & Bryan, 1987;Gibbs et al., 1987) and successfully used for others neogastropods (Minchin et al., 1996;Gooding et al., 1999) were used: the RPLI (relative penis length index), the RPSI (relative penis size index) and the VDSI (vas deferens sequence index), scale that was originally developed by Gibbs et al (1987) and later adapted for the use in Thais haemastoma in the Brazilian coast (Fernandez et al., 2002).
The RPLI is an index that quantifies the degree of imposex in the population and is obtained from the equation: (Mean length of female penis) / (Mean length of male penis) X 100.This index is better applied in low contaminated areas.The RPSI quantifies the degree of imposex in the population by 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 male penis.
The VDSI quantifies the degree of imposex in the animal through the sequence of the penis and vas deferens formation.This scale is composed of 7 levels: stage 0, a healthy female without a sign of penis or vas deferens; stage I, the beginning of the penis formation, characterized by a small wart; stage II, the penis appears formed with approximately 1mm in length and is observed the beginning of the vas deferens formation; stage III, the penis is already developed, with size superior to 1mm and it is also observed an incomplete vas deferens in the bottom of the palial cavity; stage IV, the penis is developed, with size superior to 1mm and a complete vas deferens can be observed; stage V, characterized by the blockage of the vulva and stage VI where the presence of aborted capsules in the capsule gland is observed.

RESULTS
Four of the eight sampling sites presented animals with imposex.The most elevated indexes observed were in the area of the port of Natal (site 6) where it was found RPLI = 42.1,RPSI = 7.7 and the animals were all in stage IV of VDSI.The indexes in the estuary of the Potengi River (site 5) were: RPLI = 27.3RPSI = 2.03 and VDSI oscillating between I and III.In the Forte's beach (site 4) observed RPLI corresponded to 7.2, RPSI to 0.03 and VDSI oscillated between 0 and III.The site that presented the lowest indexes was the beach of Redinha (site 7) where they were RPLI < 0.1, RPSI < 0.01 and VDSI = I.The others sites (1,2,3 and 8) didn't present imposex (Table 1).

DISCUSSION
The occurrence of imposex is a consequence of contamination by anti-fouling paints and, in particular, by their main biotoxic ingredient, the tributyltin that acts on the endocrine control of the reproductive system of some gastropods (Gibbs et al., 1997;Mathiessen & Gibbs, 1998).For this reason, the occurrence of imposex in Gastropoda has been used to monitor the contamination by tributyltin in several countries, as England (Bryan et al., 1987a, b, c), Japan (Horigushi et al., 1994, 1995, 1998;Tan, 1997) and Thailand (Evans et al., 1995;Bech, 1998Bech, , 1999)).
The occurrence of imposex in Thais rustica populations was observed in this study in the area around Natal, Northeast Brazil.Neverthless, the indices were very low even in the sites close to the city harbor.The same fact was observed for Thais rustica populations from Maceió city (Quadros, personal communication)*.The low indices of imposex in these populations could be due to low levels of contamination in these areas or to a low sensibility for the organotin compounds by this species.

Table 1 .
Results of the monitoring of Natal -RN