INTRODUCTION
Pollutants such as tributyltin (TBT), polyaromatic hydrocarbons (PAHs), organochlorinated compounds and trace metals are present in areas with intense maritime activity in Patagonian coasts (Gil et al., Reference Gil, Harvey and Esteves1999, Reference Gil, Torres, Harvey and Esteves2006; Commendatore et al., Reference Commendatore, Esteves and Colombo2000; Esteves et al., Reference Esteves, Commendatore, Nievas, Massara Paletto and Amín2006; Commendatore & Esteves, Reference Commendatore and Esteves2007; Massara Paletto et al., Reference Massara Paletto, Commendatore and Esteves2008; Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009). Aquatic invertebrates, and molluscs in particular, are widely used as bioindicators of polluted environments (Meador et al., Reference Meador, Stein, Reichert, Varanasi and Ware1995; Kim et al., Reference Kim, Oh, Shim, Lee, Yim, Hong, Shin and Lee2002; Antizar-Ladislao, Reference Antizar-Ladislao2008), while biomarkers are powerful tools to detect environmental damage and risk status (Dahlhoff, Reference Dahlhoff2004). Pollutants could affect living organisms by inducing reactive oxygen species (ROS) formation (Winston & Di Giulio, Reference Winston and Di Giulio1991; Cheung et al., Reference Cheung, Zheng, Li, Richardson and Lam2001; Leonard et al., Reference Leonard, Harris and Shi2004; Nicholson & Lam, Reference Nicholson and Lam2005). Oxidative stress is the result of the imbalance between the generation and neutralization of ROS by antioxidant mechanisms (Davies, Reference Davies1995). Oxidative stress responses (e.g. antioxidant enzyme activities and/or oxidative damage to lipids) have been used as biomarkers in molluscs to test and quantify the toxic effects of pollutants in the aquatic environment (de Almeida et al., Reference de Almeida, Miyamoto, Bainy, de Medeiros and Di Mascio2004; Belcheva et al., Reference Belcheva, Zakhartsev, Dovzhenko, Zhukovskaya, Kavun and Chelomin2011; Sabatini et al., Reference Sabatini, Rocchetta, Luquet, Guido and Ríos de Molina2011a). The increased activity or de novo synthesis of antioxidant enzymes to mitigate oxidative damage has been considered as an adaptation of organisms to stress conditions (Young & Woodside, Reference Young and Woodside2001). Among these enzymes are superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) which protect ROS scavenging cells (Karakoc et al., Reference Karakoc, Hewer, Phillips, Gaines and Yuregir1997; Borković et al., Reference Borković, Šaponjić, Pavlović, Blagojević, Milošević, Kovačević, Radojičić, Spasić, Žikić and Saičić2005) and glutathione S-transferase (GST) as well, a phase II detoxifying enzyme, exhibiting a protective mechanism against oxidative stress (Prohaska, Reference Prohaska1980; Sheehan & Power, Reference Sheehan and Power1999; Doyen et al., Reference Doyen, Vasseur and Rodius2005). Moreover, aquatic organisms also present non-enzymatic antioxidant defences (e.g. vitamin E, reduced glutathione-GSH-, between others) contributing to minimize oxidative damage (Sayeed et al., Reference Sayeed, Parvez, Pandey, Bin-Hafeez, Haque and Raisuddin2003; Wang et al., Reference Wang, Yan, Liu, Li and Wang2008).
In molluscs, the digestive gland is the principal site for bioaccumulation and detoxification of pollutants and the main target of oxidative disruption (Malanga et al., Reference Malanga, Estevez, Calvo and Puntarulo2004). In several bivalve species exposed to pollutants, oxidative damage and an increased/decreased activity of antioxidant enzymes have been registered (Bainy et al., Reference Bainy, Almeida, Muller, Ventura and Medeiros2000; Sabatini et al., Reference Sabatini, Rocchetta, Luquet, Guido and Ríos de Molina2009, Reference Sabatini, Rocchetta, Luquet, Guido and Ríos de Molina2011a, Reference Sabatini, Rocchetta, Nahabedian, Luquet, Eppis, Bianchi and Ríos de Molinab; Giarratano et al., Reference Giarratano, Duarte and Amin2010, Reference Giarratano, Gil and Malanga2013; Di Salvatore et al., Reference Di Salvatore, Calcagno, Ortíz, Ríos de Molina and Sabatini2013).
In marine gastropods from the Argentinean coast, the imposex phenomenon (penis or vas deferens neoformation) in females exposed to tributyltin (TBT) (Gibbs & Bryan, Reference Gibbs and Bryan1986) has been detected in all the harbour areas (Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009). Many gastropod species have been affected by imposex in Argentina, while the nassarid Buccinanops globulosus showed high sensibility to TBT (Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009). It has been demonstrated that TBT could induce imposex, shell malformation (Chagot et al., Reference Chagot, Alzieu, Sanjuan and Grizel1990; Alzieu, Reference Alzieu2000; Bigatti & Carranza, Reference Bigatti and Carranza2007; Márquez et al., Reference Márquez, González and Bigatti2011) and also causes oxidative stress (Huang et al., Reference Huang, Chen, Zhao, Zuo, Chen and Wang2005; Wang et al., Reference Wang, Chen, Li, Wei and Yu2005; Jia et al., Reference Jia, Zhang, Wang, Lin, Zou, Huang and Wang2009; Zhou et al., Reference Zhou, Zhu and Cai2010).
The gastropod Buccinanops globulosus inhabits sandy or muddy bottoms of shallow waters (Pastorino, Reference Pastorino1993) in Patagonian coasts, and most of the time lives buried in the sediment (Scarabino, Reference Scarabino1977). It is distributed along the South-western Atlantic Ocean (Pastorino, Reference Pastorino1993). Buccinanops globulosus is dioecious, with internal fertilization. Females attach the egg capsules to their own shells (Penchaszadeh, Reference Penchaszadeh1971), and are larger than the males. In general, the populations from Patagonia have shown variability in biological parameters such as growth, shell shape and ageing (Narvarte et al., Reference Narvarte, Willers, Avaca and Echave2008; Avaca et al., Reference Avaca, Narvarte and Martín2013; Bökenhans, Reference Bökenhans2014; Primost et al., Reference Primost, Bigatti and Márquezin press). This species is edible and is part of an expanding artisanal fishery (Narvarte et al., Reference Narvarte, Willers, Avaca and Echave2008; Averbuj et al., Reference Averbuj, Rocha and Zabala2014). Sublethal effects and bioaccumulation of TBT and other pollutants (trace metals, hydrocarbons) have been detected in harbour areas (Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009; Torres et al., Reference Torres, Commendatore, Primost and Bigatti2013; Primost, Reference Primost2014). While signalling by retinoid X receptors (RXR) (Nishikawa et al., Reference Nishikawa, Mamiya, Kanayama, Nishikawa, Shiraishi and Horiguchi2004) could be involved on the imposex development in gastropods, although the induction mechanisms are under study, the determination of oxidative stress responses in imposex-affected gastropods still remains inconclusive in Argentina.
The aim of this study was to evaluate oxidative stress responses associated to maritime traffic contamination in imposex-affected B. globulosus from Nuevo Gulf, Argentina.
MATERIALS AND METHODS
Study area and imposex incidence
The study was performed in three sites of Nuevo Gulf, with decreasing maritime activity: harbour area at Luis Piedra Buena harbour (LPB) (42°43′‘57″S 65°1′53.9″W), Punta Cuevas beach (PC) (42°46′45″S 64°59′34″W) and Cerro Avanzado beach (CA) (42°49′37.66″S 64°51′29.19″W) (Figure 1). In the LPB site activity of large vessels is frequently present (~720 vessels per year) (APPM, 2013); in this area 100% imposex was reported in gastropods since 2000 (Bigatti & Penchaszadeh, Reference Bigatti and Penchaszadeh2005; Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009; del Brío, Reference del Brio, Castro, Fillmann, Commendatore, Gomes Costa and Bigatti2011; Primost, Reference Primost2014), while moderate pollution by PAHS, trace metals and TBT were previously recorded in sediments and molluscs (Gil et al., Reference Gil, Harvey and Esteves1999; Massara Paletto et al., Reference Massara Paletto, Commendatore and Esteves2008; Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009). The PC site is a recreational public area frequently presenting diving vessels, where low pollution by TBT and trace metals was measured (Primost, Reference Primost2014) and lesser imposex parameters were reported (Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009; Primost, Reference Primost2014). The CA beach is a recreational area where very low or null imposex incidence was reported as well as no detectable TBT pollution (Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009; del Brío, Reference del Brio, Castro, Fillmann, Commendatore, Gomes Costa and Bigatti2011; Primost, Reference Primost2014); in this area there is low maritime traffic and sport vessels are present only occasionally. Table 1 summarizes the pollution levels previously detected in the sampling sites.
Location of sampling sites in Nuevo Gulf, Patagonia, Argentina.
Maximum values of different pollutants detected in gastropods (whole tissues) and sediments from sampling sites in Nuevo Gulf.
Nd: Non detectable.
Adult female gastropods Buccinanops globulosus (25 approximately at each site) were collected using baited traps. The sex was determined in situ by presence or absence of the ventral pedal gland (only present in females) used to fix egg capsules on its own shell. Total shell length (TSL) and body weight relative to size (BW) was recorded in the laboratory. Incidence of imposex (% I) was considered as the percentage of females with a penis or vas deferens development; correspondingly mean female penis length (FPL) was estimated only in females with penis development and using 0.1 mm precision digital caliper.
Oxidative stress parameters
In a subsample of nine females per site, oxidative stress parameters were determined. The digestive gland was carefully dissected, weighed (with a digital scale 0.01 g) and frozen at −80°C for later oxidative stress determinations.
Digestive glands were homogenized with 0.154 M KCl (1:5 w v−1) containing 0.5 mM phenylmethylsulphonyl fluoride (PMSF) and 0.2 mM benzamidine (protease inhibitors) to study oxidative stress parameters. The homogenates were centrifuged at 12,000 × g during 30 min (4°C) and the supernatants were stored for later determinations.
Total soluble protein content was measured by the method of Bradford (Reference Bradford1976), using bovine serum albumin as standard. The results were expressed as μg of total protein per mL.
Superoxide dismutase (SOD, EC 1.15.1.1) activity was assessed by inhibition of photoreduction of NBT (nitro blue tetrazolium) and monitoring absorbance at 560 nm according to Beauchamp & Fridovich (Reference Beauchamp and Fridovich1971). The standard assay mixture contained 5, 10 and 15 µL enzymatic sample, 0.1 mM EDTA, 13 mM DL-methionine, 75 µM NBT and 20 µM riboflavin, in 50 mM phosphate buffer (pH 7.5), to a final volume of 3 mL. Samples were exposed for 15 min to intense cool-white light, and then kept in the dark until absorbance was measured at 560 nm. Results were expressed as U per mg protein. A SOD unit was defined as the enzyme amount necessary to inhibit the reaction rate by 50%.
Glutathione S-transferase (GST, EC1.11.1.9) activity was measured by monitoring the absorbance at 340 nm using 1-chloro-2 4-dinitrobenzene (CDNB) (100 mM) as substrate according to Habig et al. (Reference Habig, Pabst and Jakoby1974). Briefly, we mixed 10 µL of glutathione (GSH) (100 mM in phosphate buffer) and 20 µL of sample in 960 µL of 100 mM phosphate buffer (pH 6.5) and 10 µL CDNB. One GST Unit was defined as the amount of enzyme needed to catalyse the formation of 1 µmoL of GS-DNB per minute at 25°C.
Reduced GSH levels were determined monitoring the absorbance at 412 nm after 30 min incubation at room temperature following the Anderson (Reference Anderson1985) procedure. Briefly, 100 µL supernatant from the 11,000 × g sample was acidified with 50 µL of 10% sulphosalicylic acid. After centrifugation at 8000 × for 10 min, supernatant (acid-soluble GSH) aliquots were mixed with 6 mM 5,5-dithiobis-(2-nitrobenzoic) acid (DTNB) in 0.143 M buffer sodium sulphate (pH 7.5) (containing 6.3 mM EDTA). Results were expressed as nmol GSH per mg of protein.
Lipid peroxidation was determined measuring thiobarbituric acid reactive substances (TBARs) according to Vavilin et al. (Reference Vavilin, Ducruet, Matorin, Venediktov and Rubin1998). Briefly, the 11,000 × g supernatant (175 µL) from total homogenate was mixed with thiobarbituric acid (TBA) (26 mM) solution and incubated at 95–100°C for 45 min. After cooling, the reaction mixture was centrifuged and the supernatant absorbance was determined at 535 nm. TBARs concentration was estimated using an extinction coefficient of 156 mM−1 cm−1 and absorbance determination at 535 nm. Results were expressed as μmol TBARs per mg of protein.
Statistical analysis
Normality and homogeneity of variances were tested by Lilliefors’ and Bartlett's tests, respectively (Sokal & Rohlf, Reference Sokal and Rohlf1979). Results from size, weight and oxidative stress parameters were analysed by one way ANOVA followed by a Tukey's post hoc test. Results for imposex analysis were compared between sites by Kruskal–Wallis followed by a Dunn post hoc test. Differences were considered significant with P < 0.05. Statistica7 software was used for statistical analysis. A DistLM multiple correlations was performed using PRIMER software (Clarke & Gorley, Reference Clarke and Gorley2006) to compare the effect of stress parameters (as co-variable) on penis length (as response variable). Prior to analysis, variables were transformed by Z-score using R software (https://www.r-project.org/).
RESULTS
Imposex incidence
A total of 66 females of Buccinanops globulosus were analysed for imposex incidence and a subsample of 27 females (nine per site) was used for the determination of oxidative stress parameters. Total shell length (TSL) and body weight (BW) were significantly different between sites (TSL: F = 27.306, P < 0.0001, df = 2, N = 66; BW: F = 46.006, P < 0.0001, df = 2, N = 66). In both cases, the highest values were obtained in the LPB site (Table 2).
Total shell length, body weight (means ± SD) and imposex parameters in Buccinanops globulosus.
FPL: female penis length.
* Significant differences between sites (LPB, PC and CA).
The imposex incidence was 100% in LPB (Table 2) and significant differences in female penis length (FPL) between LPB vs. PC sites were observed (U = 450.000, P < 0.0001, N = 43). In CA site, the FPL was not calculated because only one female showed imposex development (with a small incipient penis).
Antioxidant defences
In order to analyse the antioxidant defences, results firstly showed that total protein content in the digestive gland did not differ between sampling sites (F = 0.220, P = 0.804, df = 2, N = 27) (data not shown). Therefore, all measured variables were standardized as a function of protein content.
Gastropods collected from the harbour area (LPB site) showed higher superoxide dismutase (SOD) activity than those from the other two sites (PC and CA) (F = 13.277, P = 0.0001, df = 2, N = 27) (Figure 2A). Also the reduced glutathione content (GSH) revealed a similar pattern, showing the highest values in the LPB site (F = 8.148, P = 0.002, df = 2, N = 27) (Figure 2C).
Superoxide dismutase (SOD) (A), glutathione-S-transferase (GST) (B) activities expressed as U mg−1 prot and reduced glutathione (GSH) (C) levels expressed as nmol mg−1 prot, in digestive gland of Buccinanops globulosus. Results are expressed as mean ± SD (N = 9). Letters a and b indicate significant differences between sampling sites (LPB, PC and CA).
On the other hand, glutathione-S-transferase (GST) activity in digestive gland did not show significant differences between sampling sites (F = 2.342, P = 0.118, df = 2, N = 27) (Figure 2B).
Oxidative damage
In relation to oxidative damage, significant differences in lipid peroxidation among sites were obtained (TBARs: F = 6.357, P = 0.006, df = 2, N = 27); individuals collected in the LPB site showed the highest values (Figure 3).
Lipid peroxidation, expressed as μmol TBARS mg−1 prot, in digestive gland of Buccinanops globulosus. Results are expressed as mean ± SD (N = 9). Letters a and b indicate significant differences between sampling sites (LPB, PC and CA).
Stress parameters and imposex response
Significant differences were obtained in DistLM for SOD, GSH and TBAR variables on penis length (as response variable). These results showed that 61.5% of variability in penis length was explained by stress parameters (Table 3).
Results from DistLM multiple correlations between stress parameters (co-variable) and penis length (response variable).
Best solution: R 2 = 0.6152; No Vars = 4; Selections = All. * indicates significant differences at P < 0.05 between sites (LPB, PC, CA) for these variables.
DISCUSSION
The imposex incidence and female penis length (FPL) recorded in this work for Buccinanops globulosus could be related to maritime traffic and levels of TBT reported previously in Nuevo Gulf (Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009; del Brío, Reference del Brio, Castro, Fillmann, Commendatore, Gomes Costa and Bigatti2011). Pollutants could be bioaccumulating in aquatic organisms and affecting their defence mechanisms (Regoli & Principato, Reference Regoli and Principato1995; Chandran et al., Reference Chandran, Sivakumar, Mohandass and Aruchami2005; Chen et al., Reference Chen, Jia, Cai, Lin and Ma2011). In the LPB area, del Brío and colleagues detected butyltin levels (TBT + dibutyltin-DBT- + monobutyltin-MBT-) up to 265.8 ng (Sn) g−1 dry weight (dw) in sediments and up to 567.8 ng (Sn) g−1 (dw) in the tissues of the marine gastropod Odontocymbiola magellanica, the gonads and digestive gland being the organs with the highest TBTs concentration (del Brio et al., Reference del Brio, Castro, Fillmann, Commendatore, Gomes Costa and Bigattiin press). Also polyaromatic hydrocarbons such anthracene, benzo(b)fluoranthene (Torres et al., Reference Torres, Commendatore, Primost and Bigatti2013) and trace metals such as copper (Cu), iron (Fe), lead (Pb) and zinc (Zn) were detected in B. globulosus (Primost, Reference Primost2014) in the LPB area confirming the capacity of these gastropod species to bioaccumulate different pollutants and potentially form reactive oxygen species (ROS).
ROS formation and changes in the oxidative balance have been observed as a result of exposure to environmental levels of TBT in bivalves (Huang et al., Reference Huang, Chen, Zhao, Zuo, Chen and Wang2005; An et al., Reference An, An and Choi2009) and gastropods (Jia et al., Reference Jia, Zhang, Wang, Lin, Zou, Huang and Wang2009; Gopalakrishnan et al., Reference Gopalakrishnan, Huang, Wang, Wu, Liu and Wang2011). Imposex in B. globulosus was associated with TBT presence (Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009) in the LPB area. In this work, the oxidative stress responses registered in imposexed gastropods could be attributed to TBT and other contaminants detected at the LPB harbour area such as trace metals and PAHs (Gil et al., Reference Gil, Harvey and Esteves1988, Reference Gil, Harvey and Esteves1999, Reference Gil, Torres, Harvey and Esteves2006; Commendatore et al., Reference Commendatore, Esteves and Colombo2000; Di Salvatore et al., Reference Di Salvatore, Calcagno, Ortíz, Ríos de Molina and Sabatini2013; Torres et al., Reference Torres, Commendatore, Primost and Bigatti2013; Primost, Reference Primost2014).
It is well known that a wide range of pollutants enhance enzymatic and non-enzymatic antioxidants in marine invertebrates to protect cells against oxidative damage (Livingstone, Reference Livingstone2001; Brown et al., Reference Brown, Galloway, Lowe, Browne, Dissanayake, Jones and Depledge2004; Valavanidis et al., Reference Valavanidis, Vlahogianni, Dassenakis and Scoullos2006). Our results show highest SOD activities and highest concentrations of reduced glutathione in the digestive gland of gastropods from the LPB site. However, the glutathione-S-transferase (GST) activity showed a different pattern compared with these former antioxidant responses, where no significant differences in its activity were observed among the three sampling sites. Glutathione-S-transferase is a biotransformation enzyme which catalyses the conjugation of electrophilic pollutants with reduced glutathione (GSH). The resulting conjugates increase their water solubility favouring the excretion processes (Armstrong, Reference Armstrong1997; Hayes et al., Reference Hayes, Flanagan and Jowsey2005). In molluscs, the activity of GST usually increases in relation to detoxification processes (Almeida et al., Reference Almeida, Bainy, Dafre, Gomes, Medeiros and Di Mascio2005; Huang et al., Reference Huang, Chen, Zhao, Zuo, Chen and Wang2005). However in 2005, Huang and colleagues determined that GST activity in the fish Meretrix meretrix may be increased or inhibited depending on high or low TBT concentrations in water, respectively (Huang et al., Reference Huang, Chen, Zhao, Zuo, Chen and Wang2005). Our results shows that pollution present in the PC site would induce a low increase in GST activity in the digestive gland of B. globulosus, while the pollutant presence in CA environment was not enough to produce changes in GST activity. In the present work, antioxidants (SOD and GSH) increased in the proximity of the harbour area, which was in concordance with higher imposex levels and penis length. Former studies, in the same sampling area, related to oxidative stress responses in the bivalve Aulacomya atra have detected seasonal changes in the antioxidant defences in relation to trace metal exposure and environmental pollution (Di Salvatore et al., Reference Di Salvatore, Calcagno, Ortíz, Ríos de Molina and Sabatini2013; Giarratano et al., Reference Giarratano, Gil and Malanga2013). In both studies, animals from the harbour area were the most affected, showing an increase in the antioxidant defences and also suffering higher oxidative damage to lipids. Meanwhile, a study in the fish Sebastiscus marmoratus exposed to TBT also revealed an increase in SOD activity in the liver (Wang et al., Reference Wang, Chen, Li, Wei and Yu2005). In this sense, SOD increasing in B. globulosus probably could be related to TBT contamination detected recently in the area (Bigatti et al., Reference Bigatti, Primost, Cledón, Averbuj, Theobald, Gerwinski, Arntz, Morriconi and Penchaszadeh2009; del Brío, Reference del Brio, Castro, Fillmann, Commendatore, Gomes Costa and Bigatti2011).
Lipid peroxidation has also been reported as a principal cause of cellular damage induced by oxidative stress conditions (Valavanidis et al., Reference Valavanidis, Vlahogianni, Dassenakis and Scoullos2006). Membrane alterations in molluscs are the major target of cellular damage in organisms exposed to trace metals and other toxic substances (Viarengo et al., Reference Viarengo, Canesi, Pertica, Poli, Moore and Orunesu1990, Reference Viarengo, Canesi, Pertica and Livingstone1991). In the present work, B. globulosus showed a marked increment in lipid peroxidation in the digestive gland of gastropods collected from the harbour area compared with animals from CA and PC sites. In addition, our results are in accordance with those reported by Zhou et al. (Reference Zhou, Zhu and Cai2010), where TBT exposures increase lipid peroxidation (measured as malondialdehyde (MDA) levels) in the abalone Haliotis diversicolor supertexta. Similar results were also observed in laboratory studies where rats exposed to repeated TBT doses showed incremental MDA levels (Liu et al., Reference Liu, Wang, Lian and Xu2006); Bernat and colleagues also reported the same effect in the filamentous fungus Cunninghamella elegans exposed to TBT (Bernat et al., Reference Bernat, Gajewska, Szewczyk, Słaba and Długoński2014).
Our results suggest that the differences in terms of oxidative stress responses and high imposex incidence observed in B. globulosus at the harbour site indicate a negative effect on its physiological state due to the presence of pollutants in the aquatic environment. The possible relationship between the induction mechanism of imposex and oxidative stress should be tested in controlled experiments exposing normal and imposexed individuals to TBT, followed by comparative measurement of oxidative stress parameters in experimental groups.
CONCLUSIONS
In conclusion, both oxidative stress responses and imposex incidence were increased in gastropods inhabiting the harbour area. While B. globulosus suffers an increase of the antioxidant defences (SOD activity and GSH content), an oxidative damage to lipids (TBARs levels) was still observed.
This is the first study on oxidative stress responses associated with marine pollution in an edible gastropod affected by imposex in Argentina. Although TBT is not the only pollutant present in the harbour area, further integrated studies are necessary to evaluate the role of oxidative stress responses in Buccinanops globulosus as biomarkers of TBT presence.
ACKNOWLEDGEMENTS
Special thanks to Dr E. Marzinelli, Dr L. Arribas, Dr M. Lozada and Dr Pitu Mendez.
FINANCIAL SUPPORT
This work was supported by grants: PICT 1232, PICT 2929, PICT 1491, PICT 1476 and PIP 067 (MP and GB), UBACYT20020100300055 and ANPCYTPICT-2010-0260 (SES), UBACYT20020100100985 (MCRM).
