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[ 28 ] J.A. Zirrolli, R.C. Murphy, J. Am. Soc. Mass Spectrom. 4 ( 1993 ) 223^229. [ 29 ] K.W. Nickerson, L.A. Bulla Jr., T.L. Mounts, J. Bacteriol. 124 ( 1975 ) 1256^1262. [ 30 ] W.C. Kossa, J. MacGee, S. Ramachandran, A.J. Webber, J. Chromatogr. Sci. 17 ( 1979 ) 177^187. [ 31 ] C. Abbas-Hawks, T.L. Had¢eld, K.J. Voorhees, Rapid Commun. Mass Spectrom. 10 ( 1996 ) 1802^1806. [ 32 ] A.D. Hendricker, K.J. Voorhees, in: Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, CA, June 1^5, 1997. [ 33 ] B.D. Nourse, R.G. Cooks, Anal. Chim. Acta 228 ( 1990 ) 1^21. [ 34 ] S.A. McLuckey, G.L. Glish, K.G. Asano, G.J. Van Berkel, Anal. Chem. 60 ( 1988 ) 2314^2317. [ 35 ] D.J. Harvey, Org. Mass Spectrom. 28 ( 1993 ) 287^288. Franco Basile, Ph.D. is an Assistant Research Professor at the Department of Chemistry, Colorado School of Mines. His research interests have involved the detection and identi¢cation of bacteria with mass spectrometry and laser-based £uorescence spectroscopy.

Michael B.Beverly is a graduate student working towards his Ph.D. at the Colorado School of Mines. His graduate research focuses on the use of pyrolysis mass spectrometry to study bacterial biomarkers useful in the identi¢cation of bacteria. Ted L. Had¢eld, Ph.D. is a Lt. Col. in the US Air Force. He is currently Chief of the Division of Microbiology and Associate Chairman for Administration of Infectious and Parasitic Diseases Pathology at the Armed Forces Institute of Pathology in Washington, DC. Kent Voorhees, Ph.D. is a professor of chemistry and geochemistry at the Colorado School of Mines. His major research interests have been pyrolysis, mass spectrometry, and chemometrics. His laboratory has been funded by the U.S. Army since 1989 to study the use of biomarkers for bacterial identi¢cation. During this period, ¢ve post doctoral associates, and six students have worked on the project. The biomarker investigation has utilized pyrolysis-mass spectrometry, tandem and high resolution mass spectrometry, and recently, ¢eld-portable ion trap mass spectrometerbased detectors.

Monitoring of organotin compounds and their effects in marine molluscs Yolanda Morcillo, Cinta Porte*

Department of Environmental Chemistry, CID, CSIC, Jordi Girona 18^26, 08034 Barcelona, Spain Organotin compounds are ubiquitous contaminants in the environment. The high biological activity of some compounds toward aquatic organisms leads to deleterious impacts in aquatic ecosystems. A comprehensive view is given of the occurrence, biochemical effects and impact of organotin contamination on molluscs along the Catalan coast (Northwestern Mediterranean ). Chemical analysis of biota samples revealed that several years after legislation to reduce tributyltin (TBT ) inputs, the concentration of this compound in mussels inhabiting Catalan harbours is still elevated. Likewise, levels of organotins outside the harbours are high enough to cause imposex in the commercial muricid Bolinus brandaris. The inter*Corresponding author. Tel.: +34 (3) 4006175; Fax: +34 (3) 2045904. E-mail: [email protected]

action of TBT with the molluscan cytochrome P450 and the consequences for the organism in terms of hormonal disruptions are discussed. z1998 Elsevier Science B.V. Keywords: Organotin compounds; Molluscs; Tributyltin; Cytochrome P450

1. Introduction Triorganotin compounds and particularly tributyltin (TBT ) are used in a variety of consumer and industrial products including antifouling paints, agricultural pesticides and material preservants. It is widely accepted that antifouling paints are the most important contributors of organotin compounds to the marine environment. A number of studies have demonstrated the deleterious effects of TBT on non-target marine

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organisms, viz. production decrease in mollusc farming [ 1 ], decline in dogwhelk populations [ 2 ] and potential reproductive impairment [ 3 ]. Accordingly, environmental concern led to the introduction of legislative controls in a number of countries, viz. the European Union in 1989 and the Mediterranean region in 1991 restricted the use of TBT-based paints on vessels under 25 m. However, even after regulation, monitoring programmes revealed the presence of organotin compounds in coastal areas [ 4^6 ]. Thus, marine organisms are still subjected to exposure from sediment, water-column and through their diet. Despite the fact that molluscs are known to be very sensitive to low concentrations of TBT, limited information concerning the mechanism of action of organotin compounds is available. Increases in testosterone titres or imbalance in the androgens / estrogens ratio have been described for several gastropod species after exposure to TBT and these ¢ndings have been associated with the phenomenon of imposex, the imposition of male characteristics in females of some gastropod species [ 7^9 ]. The inhibition of the P450-aromatase responsible for the conversion of androgens to estrogens has been anticipated as the causal mechanism [ 10 ]. Nevertheless, virtually nothing is known about potential interaction of organotin compounds with the molluscan cytochrome P450 system; this system plays a key role in the metabolism of xenobiotic compounds but also in the conversion of cholesterol into a variety of hormones. Thus, inhibition or stimulation of cytochrome P450 isozymes by TBT can result in changes in hormone production or clearance. Within this framework, several strategies have been used in order to assess the impact of organotin compounds in marine molluscs, namely ( a ) analysis of TBT body burdens, ( b ) interaction of TBT with the P450 monooxygenase system and sex hormone metabolism, and ( c ) induction of imposex in gastropods. This study will focus on the Catalan Coast (Northwestern Mediterranean ), a region that has become an important tourist destination over the past decade, and where an increasing number of yacht marinas have appeared.

2. Analysis of organotin body burdens in marine molluscs Due to the detrimental effects of organotin compounds in aquatic organisms, a variety of methods has been developed to measure their occurrence in

several matrices including water, sediments and biota. Many techniques were originally developed for aqueous matrices, and afterwards adapted to biota, which often present more problems in terms of interferences and stronger binding of the organotin to the matrix. A well established analysis procedure consisted of the extraction of the ionic metabolites by an organic solvent modi¢ed with a quelating agent such as tropolone, followed by alkylation with the Grignard reagent [ 11,12 ]. This method, however, is time consuming and requires a lot of manipulation of the sample, increasing the probability of errors in the ¢nal determination. Therefore, alternative methods based on aqueous ethylation of organotins and simultaneous extraction of ethylated derivatives have been developed recently [ 13,14 ]. Brie£y, the method used to assess organotin contamination in the present study consisted of the hydrolysis of the sample ( 1^2 g of tissue ) with tetramethylammonium hydroxide followed by aqueous ethylation with sodium tetraethylborate and extraction with n-hexane [ 15 ]. Afterwards, extracts were cleaned up with a short alumina column and analysed by gas chromatography ( GC ) coupled with £ame photometric detection ( FPD ). Tripentyltin and tetrabutyltin were added as internal standards and the accuracy of the method checked by analysing a certi¢ed reference material (NIES-11 ). Mussels Mytilus galloprovincialis were sampled in May 1996 and used to monitor pollution by organotin compounds in three harbours, namely ( a ) El Masnou, a recreational marina, ( b ) Barcelona, a commercial harbour, and ( c ) St. Carles, a ¢shing harbour located in the area of the Ebro Delta ( Fig. 1 ). The pattern of occurrence of TBT and its metabolites ( DBT, MBT ) as well as triphenyltin (TPhT ) in mussels is reported in Table 1. In general, TBT was present at higher levels than DBT or MBT in all the samples, with the exception of those organisms taken outside St. Carles harbour. This fact indicates that fresh inputs of the antifouling agent dominate the degradation products, probably because the sampling coincided with the beginning of the sailing / painting season. The highest concentration of TBT was found in mussels from Barcelona harbour; 1151 þ 1 ng / g wet weight ( w.w. ) of TBT as Sn in mussels sampled near the dry dock ^ where boats are painted / repaired ^ and 401 ng / g w.w. of TBT in mussels sampled elsewhere inside the harbour (Table 1 ). Similarly, high levels of TBT were seen in mussels from El Masnou ( 355 ng / g w.w. as Sn ), an area impacted predominantly by leisure vessels. Mussels taken outside St. Carles harbour showed the lowest concentration of TBT, presumably

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Fig. 1. Map showing sampling areas for organotin analysis and imposex measurements.

due to the low traf¢c of boats, and the continuous fresh-water £ushing from land to the sea, which may prevent TBT to accumulate. TPhT was detected in all the samples, although it represents only a small proportion of organotin body burdens ( 6 10%). In general, organotin body burdens are in the range of those reported previously in the area [ 15 ], with the exception of the increasing occurrence of TPhT, which is probably due to its recent use as a co-toxicant in some long-performance antifouling paints.

In summary, these results indicate that several years after legislation to reduce TBT inputs, the concentration of this compound in mussels inhabiting Catalan harbours is still elevated. The consequences of these levels of exposure for the organism are unknown, although at TBT concentrations above 400 ng / g w.w., signi¢cant adverse biological effects were measured in adult mussels [ 16,17 ]. It is also worth mentioning that organotin does not remain exclusively inside the harbours; contamination offshore is lower

Table 1 Organotin residues ( ng / g wet weight as Sn ) in mussels Mytilus galloprovincialis collected from three harbours along the Catalan coast (Western Mediterranean ) Harbour Masnou Inside ( I ) Inside ( II ) Barcelona Dry dock Inside St. Carles Inside ( I ) Inside ( II ) Outside

MBT

DBT

TBT

TPhT

TBT / DBT

78 þ 5 12 þ 6

287 þ 26 78 þ 15

356 þ 17 240 þ 36

29 þ 7 11 þ 1

1.2 þ 0.1 3.1 þ 0.1

204 þ 45 17 þ 1

1094 þ 63 171 þ 5

1151 þ 1 401 þ 14

92 þ 1 4þ1

1.1 þ 1 3.7 þ 0.1

13 þ 1 21 þ 6 10 þ 10

149 þ 3 109 þ 17 4þ4

201 þ 31 283 þ 69 1þ1

26 þ 7 3þ1 n.d.

1.5 þ 0.4 3.0 þ 0.1 0.2 þ 0.2

n.d. = below detection limit ( 3 ng / g w / w ).

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Fig. 2. Inhibition of ( A ) NADH- and ( B ) NADPH-dependent cytochrome c reductase after incubation of the microsomal fraction of Mytilus galloprovincialis, Ruditapes decussata, and Thais haemastoma with different concentrations of TBT. Values are mean þ S.E.M. ( n = 6 ). Signi¢cant differences with respect to controls indicated by *P 9 0.05. Speci¢c activities of NADHcytochrome c reductase were 77 þ 25 (M.g. ), 49 þ 2 (R.d. ), and 138 þ 4 (T.h. ); and NADPH-cytochrome c reductase, 16 þ 1 (M.g. ), 6 þ 1 (R.d. ), and 59 þ 9 (T.h. ) in nmol / min / mg protein.

but detectable, both TBT and TPhT have been determined in different ¢sh species from the Western Mediterraean [ 5,15 ].

3. Interaction of organotin compounds with the molluscan cytochrome P450 system and sex hormone metabolism The cytochrome P450 monooxygenase system is a multi-enzymatic system that plays a key role in the metabolism of both xenobiotic and endogenous compounds, such as hormones and fatty acids. It catalyses the hydroxylation of hundreds of structurally diverse compounds, whose only common feature appears to be the degree of lipophilicity. Several P450 isozymes are involved in the metabolism of xenobiotics, such as benzo[ a ]pyrene hydroxylation or 7-ethoxyresoru¢nO-deethylation; whereas others are involved in the synthesis of sex hormones, including the aromatization step that converts androgens ( testosterone / androstenedione ) to estrogens ( estrone / estradiol ) [ 18,19 ]. Hence, alterations of the cytochrome P450 system could have consequences for both the protection of the organism against pollutants and the metabolism of endogenous steroids, among other compounds. The interaction of organotin compounds with the cytochrome P450 system was ¢rst described for vertebrates, viz. the loss of cytochrome P450 after exposure to TBT [ 20,21 ]. More recently, some studies have provided evidence that both TBT

and TPhT have signi¢cant and selective effects upon different components of the cytochrome P450 system of marine and fresh water ¢sh [ 12 ]. In vitro exposure of the microsomal fraction to organotins resulted in a decrease of the total spectrally determined cytochrome P450, inhibition of the catalytic activity of CYP1A ( a cytochrome P450 isozyme ) and to some extent inhibition of the £avoproteins NAD(P )H cytochrome c reductases [ 22,23 ]. Species-related differences were observed with regard to sensitivity to organotins [ 24 ], but essentially in vivo exposure experiments con¢rmed previous in vitro data. Here we focus on the interaction of organotin compounds with the molluscan cytochrome P450 system, both in terms of main components of the system and associated activities. Due to the low or undetectable ability of molluscs to metabolize environmental pollutants [ 25 ], and the potential reproductive impairment caused by TBT in gastropod species [ 3 ], special emphasis will be placed on evaluating the effect of TBT on sex hormone metabolism. 3.1. In vitro studies

Similar effects of organotin compounds on the cytochrome P450 system of molluscs could be anticipated. Evidence of spectral changes of mussel cytochrome P450 after incubation with 1 mM TBT or 1 mM TPhT have been reported elsewhere [ 26 ]. However, the low levels of cytochrome P450 present in molluscs ( 14^45 pmol / mg protein ) made the accurate determi-

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Table 2 In vitro effect of TBT on Phase I metabolism of [14 C ]testosterone by digestive gland microsomal fractions of the clam Ruditapes decussata Metabolitea TLC metabolites Androstenedione DHA+DHT OH-testosterone Other metabolites Total metabolitesb Aromatase activity Estronec Estradiolc

Control

0.1 mM TBT

1 mM TBT

405.1 þ 45.0 6.4 þ 1.6 33.3 þ 3.1 3.0 þ 0.8 447.8 þ 45.9

200.1 þ 31.8* 4.1 þ 1.2 59.8 þ 4.8* 6.8 þ 2.0 270.9 þ 31.4*

185.2 þ 21.3* 21.1 þ 4.4* 80.0 þ 11.7* 34.8 þ 7.0* 321.1 þ 15.1*

100 þ 62 100 þ 16

32 þ 3 113 þ 42

91 þ 5 108 þ 30

Values expressed in pmol / h / mg protein as mean þ S.E.M. ( n = 4 ). Signi¢cant differences vs. control indicated by *P 9 0.05 ( one way ANOVA, Dunnet's test ). Metabolites identi¢cation: DHA = dihydroandrostenedione, DHT = dihydrotestosterone, OH-testosterone = hydroxylated metabolites ( including 2K-, 2L-, 6K-, 6L- and 7K-hydroxytestosterone ); other metabolites include 5-androsten3K( L )17L-diol and an unidenti¢ed metabolite. a Data in pmol / h / mg protein. b Total testosterone metabolic rate not including aromatase activity. c Data expressed as percentage of the amount formed in control samples.

nation of organotin-cytochrome P450 interaction dif¢cult [ 26 ]. Further experiments then aimed at investigating the effects of organotin on other components of the microsomal electron transport system, viz. NADH and NADPH cytochrome c reductases. Three mollusc species were selected for the study; the mussel Mytilus galloprovincialis, the clam Ruditapes decussata, and the gastropod Thais haemastoma. The microsomal fraction of these organisms was incubated for 20 min in the presence of different concentrations of TBT or TPhT as described in Morcillo and Porte [ 26 ]. In general, TBT and TPhT differed in their speci¢city; TBT led to inhibition of both £avoprotein reductases whereas TPhT showed little effect. Important species-related differences were seen in terms of TBT effects ( Fig. 2 ). The NADH-cytochrome c reductase was more sensitive to TBT than the NADPHdependent in clams and mussels; whereas both reductases were equally inhibited by TBT in the gastropod. Clam NADH cytochrome c reductase was particularly sensitive; at a concentration as low as 50 WM TBT, a 17% inhibition was detected. Both reductases play a key role in the £ux regulation of the monooxygenase system [ 27 ]; these enzymes are electron donors of cytochrome P450 system, and inhibition of these enzymes would affect the function of the system. Subsequent experiments were addressed to determine the effect of TBT on Phase I metabolism of testosterone, a well known substrate for many vertebrate P450s. Clams Ruditapes decussata were selected for this study because their NADH-dependent reductase

was particularly sensitive to inhibition by TBT ( Fig. 2 ). Microsomal fractions isolated from digestive gland were preincubated for 20 min in the presence of 0.1 mM and 1.0 mM TBT. TBT dilutions were made in DMSO and maximal concentration of solvent in the assay was 1%. Afterwards, microsomal fractions were incubated with 7 mM [ 4-14 C ]testosterone and 0.5 mM NADPH for 1 h. The reaction was stopped on ice with 1.0 M MgCl2 , the metabolites were extracted, separated by thin layer chromatography (TLC ) and quantitated by autoradiography, with the exception of estrone and estradiol, which were originated in less quantity and they were scraped from the plates and quantitated by liquid scintillation counting as described in Morcillo et al. [ 28 ]. As shown in Table 2, TBT caused a signi¢cant decrease in the total metabolic rate of testosterone ( 28^40%), as well as a change in the pro¢le of the obtained metabolites. There was a decrease in the formation of androstenedione ( 55%) and an increase in the formation of hydroxylated metabolites ( namely 2K-, 2L-, 6K- and 6L-hydroxytestosterone ). No signi¢cant effect was seen in the formation of estrone and estradiol due to aromatase activity (Table 2 ). 3.2. In vivo studies

The effect of TBT on the cytochrome P450 system was also evaluated in vivo. To this end, post-spawning clams Ruditapes decussata were exposed to different concentrations of TBT in water ( 0.09, 0.45 and 2.27

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Wg / l of TBT as Sn ) for 7 days ( 90 randomly selected individuals per tank ). Cytochrome P450 system components ( levels of total cytochrome P450, cytochrome b5 and NAD(P )H-cytochrome c reductases ) were measured in the microsomal fraction isolated from digestive gland as described in Livingstone [ 29 ]. No statistically signi¢cant differences were found among control and treated animals, apart from NADPH cytochrome c (P450 ) reductase which increased signi¢cantly in the low dose treated animals ( data not shown ). Microsomal fractions from control and TBTexposed clams were also examined for their ability to metabolize testosterone as described above. In agreement with previous in vitro data, differences among exposed and unexposed animals were observed in terms of the pattern of metabolites formed, viz. a decrease in the formation of androstenedione in the TBT-exposed clams in conjunction with an increase in the formation of hydroxylated metabolites, although these differences were not statistically signi¢cant due to the large variability detected among samples (Table 3 ). Moreover, statistically non-signi¢cant differences among control and TBT-exposed clams were seen in terms of total metabolic rate of testosterone (Table 3 ). At this point, it is worth mentioning that organisms from the control tank ^ exposed to 4 Wl of acetone / l ( carrier ) ^ exhibited lower metabolism of testosterone than previously analysed samples (Table 2 ); thus, the effect of TBT on the metabolic rate of testosterone might be quenched by the putative interference of the carrier.

When the formation of estrone and estradiol from [14 C ]testosterone was examined, an important decrease in the formation of both metabolites was evident (Table 3 ). The amount of estradiol originated by the microsomal fraction of low, medium and high TBT-dose exposed organisms was, respectively, 93%, 14% and 11% of that formed by control organisms. Similarly, the formation of estrone was 54%, 11% and 17% of the amount formed by control organisms. These results indicate a strong decrease in the aromatization of testosterone to estrone and estradiol in TBT-exposed clams. This might be due not to direct inhibition of the enzyme, but to an indirect effect such as downregulation, since no effect was seen when microsomes were incubated in vitro with TBT (Table 2 ). Finally, tissue titres of testosterone were determined in the same samples ^ whole tissue, except digestive gland. Steroid extraction was performed with diethyl ether:ethanol as described in Bettin et al. [ 9 ], and assayed for testosterone levels using commercially available radioimmunoassay (RIA ) kits [ 28 ]. Testosterone levels in TBT-exposed clams ( 0.34^0.58 ng / g w.w. ) were signi¢cantly higher than in control organisms ( 0.10 ng / g w.w. ). These ¢ndings were consistent with previous studies with gastropods that demonstrated increases in testosterone levels together with an increase in penis length in TBT-exposed organisms [ 7,9 ], although in this case no morphological changes were seen in clams. Moreover, these results further indicate an interaction of TBT with the Phase I metabolism of testosterone and / or aromatase activity. How-

Table 3 Phase I metabolism of [14 C ]testosterone by digestive gland microsomal fractions of clams Ruditapes decussata exposed to different concentrations of TBT Metabolitea TLC metabolites Androstenedione DHA+DHT OH-Testosterone Other metabolites Total metabolitec Aromatase activity Estroned Estradiold

Control

91 ng / l TBTb

454 ng / l TBTb

2268 ng / l TBTb

188.71 þ 20.1 7.4 þ 2.6 58.3 þ 18.2 0.1 þ 0.1 254.5 þ 9.5

246.6 þ 52.7 8.7 þ 4.0 72.6 þ 19.7 0.8 þ 0.5 246.6 þ 75.8

75.0 þ 31.0 3.1 þ 1.0 64.1 þ 31.0 0.3 þ 0.1 142.6 þ 62.9

177.3 þ 48.0 7.6 þ 4.2 144.4 þ 36.8 0 329.3 þ 72.4

100 100

þ 52 þ 25

54 93

þ 19 þ 34

11 14

þ2 þ 2*

17 11

þ5 þ 4*

Values expressed in pmol / h / mg protein as mean þ S.E.M. ( n = 4 ). Signi¢cant differences vs. control indicated by *P90.05 ( one way ANOVA, Dunnet's test ). Metabolite identi¢cation as in Table 2. a Data in pmol / h / mg protein. b TBT expressed as ng of Sn. c Total testosterone metabolic rate not including aromatase activity. d Data expressed as percentage of the amount formed in control samples.

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Table 4 Measurements of imposex and organotin residues in Bolinus brandaris sampled along the Catalan Coast (Western Mediterranean ) Sampling site No. of ind.

% Female imposex

RPL index (%) VDS index

DBTa

TBTa

TPhTa

St. Carles Cambrils Vilanova Barcelona Mataroè Roses

37 100 100 100 100 100

4.5 17.1 29.5 29.8 14.2 20.8

25.2 þ 4.0 78.2 þ 9.6 22.9 þ 16.7 34.1 þ 9.6 6.4 þ 3.6 24.1 þ 8.6

27.3 þ 12.6 26.9 þ 2.5 60.5 þ 25.4 58.0 þ 13.4 n.d. 7.4 þ 3.7

4.2 þ 4.2 13.7 þ 7.2 19.5 þ 7.6 30.1 þ 10.4 n.d. 3.1 þ 1.9

30 27 44 34 24 33

0.7 3.6 3.7 4.4 3.8 3.9

a

ng / g w.w. as Sn. Values are mean þ S.E.M. ( n = 3^6 ). n.d. = below detection limit.

ever, the activity of aromatase determined in microsomal fractions of clams is very low ( 0.15 þ 0.01 pmol / h / mg protein; n = 9 ) if compared with total testosterone metabolism ( 300^400 pmol / h / mg protein ). Thus, interferences of TBT with this enzymatic activity will very unlikely be responsible for the increase of testosterone titres detected in TBTexposed clams, although it would be certainly enough to increase the testosterone / estradiol ratio and therefore interfere with hormone balance, which may in turn regulate sexual physiology [ 8 ]. In summary, these results argue for a signi¢cant interaction of TBT with androgen metabolism in the clam Ruditapes decussata. However, the signi¢cance of these changes for the physiology of the organism as well as the con¢rmation of these perturbations in ¢eldexposed organisms needs further research.

4. Imposex in the gastropod Bolinus brandaris Imposex, the imposition of male characteristics ( penis and vas deferens ) in females of some gastropod species, is one of the most reported effects of TBT in molluscs. It was ¢rst detected in the mud snail Ilyanassa obsoleta in the Eastern United States [ 30 ], and soon afterwards in several other species world-wide [ 2,3,31 ]. Occurrence of imposex has been correlated with proximity to harbours, shipping activities, and increasing TBT body burdens [ 2,32 ]. Furthermore, increases of testosterone levels and imbalance of the androgens / estrogens ratio are anticipated to be the causal mechanism [ 7^9 ]. We selected the commercial muricid Bolinus brandaris for our survey. Organisms were sampled in November 96^January 1997 from six points along the Catalan coast ( Fig. 1 ), and analysed both in

terms of organotin body residues and imposex. Organotin analyses were conducted in the digestive gland as described in Section 2. For imposex analysis, female and male penis length ( FPL and MPL ) were measured to the minimum of 1.0 mm. Relative Penis Length (RPL ) index and Vas Deferens Sequence (VDS ) index were calculated as reported in Gibbs et al. [ 33 ]. Brie£y, RPL was determined as mean length of female penis expressed as a percentage of the mean length of male penis; and VDS as the mean of all stages. Female stages were determined under a lowpower binocular microscope and de¢ned as follows. Stage 0, normal female state with no superimposition of male characteristics. Stage 1, females with a bump at the site where the penis will develop. Stage 2, females with a developed penis and maybe a very short vas deferens. Stage 3, the vas deferens extends more than halfway to the vulva. Stage 4, vas deferens reaches the vulva but overpasses it without blocking. Stage 5, the vulva is not visible and a yellow lumen is opened to the mantle cavity; egg capsules are found within the capsule gland. Results of this survey are summarised in Table 4. Symptoms of imposex were detected at all the locations sampled. Both RPL and VDS indices showed evidence that St. Carles, a mollusc farming area, is the least affected location. It was the only point where only 37% of females were penis-bearing, compared to 100% in the rest of locations. On the contrary, Barcelona and Vilanova, important commercial harbours, had the highest imposex degree, together with high TBT and TPhT residues and high TBT / DBT ratios, indicating recent inputs of the antifouling agent. Mataroè was the only point with nearly negligible organotin residues and surprisingly elevated imposex. However, it is important to consider that digestive gland is the primary site of uptake of contaminants in molluscs, therefore the analysis of this

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organ would probably re£ect recent inputs rather than chronic ones; whereas imposex is known to be an irreversible phenomenon in gastropods [ 33 ].

5. Conclusions TBT is one of the most potent androgenic compounds ever added to the environment. Several years after regulation organotin compounds are still detected in marine organisms, and the imposex phenomenon in gastropods is widely reported. However, the mechanisms underlying the imposex response, and possibly the more sensitive biochemical precursors of this phenomenon, are still to be determined. Further research will be critical for a better evaluation of the impact of organotins in coastal habitats, and for the design of environmentally-acceptable antifouling strategies.

Acknowledgements This paper is a summary of studies supported by the EC Project MAS2-CT94-0099 and the Spanish National Plan for Research (PLANYCIT ) under Project Ref. AMB95-1978-CE. Yolanda Morcillo acknowledges a predoctoral fellowship from the Catalan Government ( Generalitat de Catalunya, Conselleria d'Educacioè). Studies on the metabolism of testosterone were carried out at the Dept. of Pediatrics, UAMS / Arkansas Children's Hospital Research Institute, Little Rock, Arkansas under the supervision of Dr. Martin Ronis, whose valuable help and advice are gratefully acknowledged. The authors gratefully acknowledge Dr. Montserrat Soleè for the work on imposex analysis.

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