Organotin compounds in seawater and Mytilus galloprovincialis mussels along the Croatian Adriatic Coast

Marine Pollution Bulletin 64 (2012) 189–199

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Organotin compounds in seawater and Mytilus galloprovincialis mussels along the Croatian Adriatic Coast Martina Furdek a,⇑, Mitja Vahcˇicˇ b, Janez Šcˇancˇar b, Radmila Milacˇicˇ b, Goran Kniewald a, Nevenka Mikac a a b

- Boškovic´ Institute, Bijenicˇka 54, 10000 Zagreb, Croatia Division for Marine and Environmental Research, Ruder Department of Environmental Sciences, Jozˇef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia

a r t i c l e

i n f o

Keywords: Organotin compounds (OTCs) Tributyltin (TBT) Seawater Mussels Croatian Adriatic Coast

a b s t r a c t In this work, data on the level of organotin compounds (OTCs) in seawater and mussels collected along the entire Croatian Adriatic Coast are presented. The samples were collected in 2009 and 2010 at 48 locations representing different levels of maritime activities, including marinas, ports and reference sites. Butyltins (BuTs) were found in all analyzed samples, representing >97% of OTCs, and ranged from 0.46 to 27.98 ng Sn L 1 in seawater and from <6 to 1675 ng Sn g 1 in mussels. The results indicate a recent input of TBT, with the highest concentrations of BuTs found in the marinas. It appears that the Adriatic coast is still polluted with TBT despite the fact that TBT-containing antifouling paints have been banned in Croatia since 2008. It is questionable how much TBT pollution decreased since 2005, when a high incidence of imposex was established in the same area. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The biocidal properties of mainly trisubstituted organotin compounds (OTCs) were discovered in the late 1950s. Since then, OTCs have had wide applications as biocides in agrochemicals, antifouling paints, wood preservation or material protection. Disubstituted organotin compounds are mostly used as PVC stabilizers in packaging and coating materials, foils and various types of piping (Hoch, 2001). The biological effects of organotin compounds on organisms depend on both the nature and the number of the organic groups bound to the Sn cation in the OTCs; therefore, the highest level of toxicity is shown for the trisubstituted compounds (Omae, 2003). Among all OTCs, the most toxic is tributyltin (TBT), which is used as a biocide in antifouling paints for ships and is therefore directly introduced into the marine environment. Areas impacted by intensive shipping and industrial activities, such as ports and shipyards, usually show the highest degree of pollution by TBT (Hoch, 2001). Tributyltin and triphenyltin (TPhT) may cause various biological effects on different non-target organisms, such as a reduction of growth in mussels (Salazar and Salazar, 1996), shell thickening and growth anomalies in oysters (Champ and Seligman, 1996), a reduction of the dogwhelk population (Hoch, 2001), immunological dysfunction in fishes (Hoch, 2001), and an increased mortality rate of larval fish (Fent and Meier, 1992). However, the most severe effect of these compounds is via their activity as endocrine disruptors, leading to imposex, which is de⇑ Corresponding author. E-mail address: [email protected] (M. Furdek). 0025-326X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2011.12.009

fined as the superimposition of male characteristics in female gastropod species (Gibbs and Bryan, 1986). As a result of imposex, females can become sterile, and the affected population may become locally extinct. Imposex is induced at very low concentrations of tin (as low as 1–2 ng L 1) (Omae, 2003; Champ and Seligman, 1996). For this reason, TBT is defined as one of the most toxic anthropogenic pollutants to be purposely introduced into the marine environment (Goldberg, 1986; Roper et al., 2001). It has been included in the European list of priority pollutants (Commission Directive, 2000) and in the group of organic compounds on the OSPAR list of chemicals for priority action (OSPAR, 2008). Due to these findings, many countries worldwide have banned the application of TBT-based antifouling paints, beginning with France in 1982 and followed by the UK in 1986. In the USA, TBT was banned in 1988, and between 1987 and 1990, it was banned in Europe, Canada, Australia, New Zealand and Japan (Garaventa et al., 2007). Recognizing the harmful environmental effects of OTCs, the International Maritime Organization (IMO) adopted an International Convention on the Control of Harmful Antifouling System on Ships and invited member countries to ratify it. Croatia was one of the contracting states and ratified the Convention in December 2006. According to the AFS Convention, starting 1st January 2003, no OTCs acting as biocides should be applied on ships, and starting 1st January 2008, antifouling systems containing organotin compounds must be removed from ships (AFS Convention, 2003). The legislation was adopted in Croatia in January 2008 and is applied to all ships, except military and public ships. However, these restrictions cannot immediately solve the problem of the pollution of the marine environment with OTCs due to their

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persistence in sediments and continuous release back into the water column (Hoch and Schwesig, 2004). Organotins will probably cause problems long after their ban, remaining a matter of concern and requiring monitoring for years to come. There are no data on the pollution of the Croatian Adriatic Coast with OTCs, except for a few studies that report the occurrence of imposex in a population of gastropoda Hexaplex trunculus (Garaventa et al., 2006, 2007; Prime et al., 2006); however, there are only a few measurements of the concentration of OTCs in this species (Garaventa et al., 2007). Imposex can be a reliable biological indicator of organotin contamination because the concentration of accumulated tributyltin in gastropods is positively related to the degree of imposex (Omae, 2003; Garaventa et al., 2007). In 2002– 2003, Garaventa et al. (2006, 2007) found imposexed females at all four of the investigated stations along the North Adriatic Coast (Istria) and much higher concentrations of butyltin and phenyltin derivatives in the Croatian than in the Italian part of the North Adriatic (Garaventa et al., 2007). Prime et al. (2006) reported a high level of imposex in populations of H. trunculus collected in 2005 at 12 locations along middle and southern Croatian coast. These results indicated that OTC pollution exists along the entire Croatian Adriatic Coast, requiring the urgent determination of OTC concentration. The Croatian coast is a well-known tourist destination and is intensively exposed to marine and shipping activities, with more than 60 marinas along the 800 km of coastline. The evaluation of the degree of pollution with OTCs is an essential prerequisite for the development of adequate environmental management and protection plans of the coastal area and the effective implementation of regulations. According to the existing regulations in all European countries, including Croatia, TBT pollution of the marine environment should be monitored by measuring the concentration of butyltin compounds in seawater. The ubiquitous mussel Mytilus galloprovincialis (and some other bivalve species) is also used as a biomonitoring organism due to its filter-feeding habits, ability to bioaccumulate different pollutants (heavy metals, organic compounds, OTCs), sessile lifestyle and wide geographical distribution (Magi et al., 2008). The analysis of seawater provides information on the concentration of OTCs in a selected sampling period at that moment only, as this concentration varies greatly over time due to water circulation, inputs of fresh water (run-off) or accidental spills (Milivojevic Nemanic et al., 2009). Alternatively, the concentration of OTCs in mussels is a result of the accumulation of OTCs over longer periods of time and therefore reflects an average pollution at the investigated site. Therefore, the comparison of the results obtained by the simultaneous determination of organotin concentrations in seawater and mussels is a reliable indicator of the level of pollution in a certain area. The aim of this work was to determine for the first time the level of organotin pollution along the Croatian part of the Adriatic Coast. For this purpose, samples of both seawater and mussels from 48 select locations along the entire coast (from north to south) were analyzed for the presence of OTCs. To elucidate the influence of maritime activities on the level of OTC pollution, different types of locations in terms of the intensity of maritime activities were selected and grouped as marinas, ports and reference locations.

2. Materials and methods 2.1. Study area and sampling Samples of seawater and M. galloprovincialis mussels were collected at 48 sampling sites located along the entire Croatian Adriatic Coast during 2009 and 2010 (Fig. 1). The sampling locations were chosen to elucidate the influence of maritime activities and

shipping traffic on the contamination of the marine environment by organotin compounds. Depending on the expected intensity of marine traffic, which is the main source of OTC pollution, sampling stations were divided into three groups. The first group comprised marinas, which are greatly influenced by traffic and tourist activities and are usually located in enclosed bays with a limited efficiency of water exchange. The second group included ports and were located in bigger or smaller towns, handling the traffic of larger ships or used by local residents for mooring of small boats. The third group consisted of reference sites, representing areas that are distant from a direct anthropogenic influence and minimally exposed to marine traffic. The sampling was performed in September 2009 and in March and September 2010. A total of 88 samples, 47 seawater samples and 41 mussel samples, were collected. The sampling sites included 12 marinas, 22 ports and 14 reference sites, and their locations are shown in Fig. 1. The water samples were taken at a depth of 0.5 m with dark glass bottles (1 L) previously rinsed with seawater. The samples were acidified with concentrated HNO3 to a pH of 3 immediately after sampling and stored in the dark at 4 °C until analysis. The extraction of OTCs was performed within 7 days of the sampling. The mussels were collected from the docksides, rocky shores or boat strings, according to their availability. At some locations where seawater samples were taken, mussels were not available at the moment of sampling due to, for example, regular cleaning procedures in marinas or natural events such as periods of low salinity of estuarine waters. Approximately 40 specimens were collected from each sampling site, placed into a clean plastic bag and transported to the laboratory in a refrigerated container. Generally, the length of the collected mussels varied between 35 and 80 mm, but at each particular location, an effort was made to collect samples of similar sizes. Directly after sampling, the shells were removed, and the soft tissue was homogenized in a blender and stored at 20 °C. The samples were lyophilized and stored at 20 °C until analysis, which was performed within 3 months of the date of sampling. 2.2. Reagents and standards Monobutyltin trichloride (MBTCl3, 95%), dibutyltin dichloride (DBTCl2, 97%), tributyltin chloride (TBTCl, 96%), monophenyltin trichloride (MPhTCl3, 98%), diphenyltin dichloride (DPhTCl2, 96%), triphenyltin chloride (TPhTCl, 95%) and tripropyltin chloride (TPrTCl, 98%) were obtained from Strem Chemicals (Newburyport, MA, USA). Monooctyltin trichloride (MOcTCl3, 99%) and dioctyltin dichloride (DOcTCl2, 99%) were purchased from LGC Promochem (Wesel, Germany), and trioctyltin chloride (TOcTCl, >90%) was from Fluka (Buchs, Switzerland). The organotin standard stock solutions (1000 mg Sn L 1) were prepared in methanol and stored in the dark at 4 °C for 6 months. The working standard solutions were prepared from the stock standard solutions by dilution in MilliQ water (weekly for 10 lg Sn mL 1 and daily for 0.100 lg Sn mL 1). Anhydrous sodium acetate was purchased from Kemika (Zagreb, Croatia). Acetic acid, methanol and hexane were obtained from Merck. Sodium tetraethylborate (NaBEt4) was purchased from Sigma–Aldrich (Steinheim, Germany). Ethylating solution (1% w/ v) was prepared daily by dissolving an appropriate amount of NaBEt4 in MilliQ water. The accuracy of the analytical procedure for the mussels was checked by the analysis of the certified reference material CE 477 (ERM, Mussel Tissue, European Commission, Geel, Belgium). 2.3. Instrumentation The analyses of OTCs in the mussel samples were carried out on a Varian CP-3800 gas chromatograph (GC) coupled to a pulsed

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Fig. 1. Map of the Croatian Adriatic Coast with the sampling locations indicated (M – marinas; P – ports; R – reference sites).

flame photometric detector (PFPD) (Varian) and fitted with 1177 split/splitless injector (Varian) and 8400 auto sampler (Varian). The GC was equipped with a capillary column CP-Sil 5 CB (30 m  0.32 mm i.d.) coated with a 100% dimethyl polysiloxane (0.25 lm film thickness). Helium was used as carrier gas (2.0 mL min 1). The following temperature program was applied for the separation of OTCs: the column temperature was held at 50 °C for the first minute, raised to 80 °C at a heating rate of 5 °C min 1, then to 200 °C at heating rate of 10 °C min 1, and finally to 280 °C at 35 °C min 1, with a final hold of 5 min. The injector port was kept at 275 °C. A split ratio of 1/20 (after injection) and an injection volume of 1 lL were used. The detector, fitted with the OG 590 interference filter, was held at 300 °C. The gas flow rates were 17, 18 and 17 mL min 1 for Air1, Air2 and H2, respectively, and the gate delay, gate width and trigger level were set to 3 ms, 2 ms and 400 mV, respectively. The determination of OTCs in seawater was carried out on an Agilent 6890 gas chromatograph (Agilent Technologies) equipped with an Agilent 6890 Series Autosampler. The injector was coupled

to an Agilent 7500ce ICP-MS (Agilent Technologies) via a heated transfer line and fitted with a 15 m  0.25 mm DB-5MS capillary column (film thickness 0.25 lm) coated with 5% phenyl-methylpolysiloxane (Agilent J&W Scientific). For the separation of OTCs on the 15-m column, the following GC temperature program was applied: at the start, the column temperature was held at 50 °C for 0.8 min, then raised to 200 °C at heating rate of 20 °C min 1 and held there for 2 min, then raised to 220 °C at heating rate of 40 °C min 1 and held there for 0.5 min and, in final step, raised to 280 °C at heating rate of 50 °C min 1 and held there for 2 min. The inlet temperature was held at 240 °C and the transfer line at 280 °C. Helium at a flow rate of 1 mL min 1 was used as a carrier gas, the injection mode was splitless, and the injection volume was 2 lL. The operating conditions of the ICPMS and other details of the method are described in Vahcic et al. (2011). A KS-15 mechanical shaker (Edmund Bühler) was used for the derivatization/extraction step for both seawater and mussel samples, and the centrifugation of sample extracts was performed with a Sigma 3-16 laboratory centrifuge (Fisher Bioblock scientific). The

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ultrasonic-assisted extraction was performed in a Cole-Parmer 8891 Ultrasonic bath. The mussel samples were lyophilized in the Labconco FreeZone 2.5. 2.4. Analytical procedures 2.4.1. Mussels The analytical procedure was adopted from the literature (Milivojevicˇ Nemanicˇ et al., 2009), with slight modifications. Approximately 0.5 g of lyophilized mussel sample was placed in a centrifuge tube and spiked with 50 lL of the internal standard solution of TPrT (10 lg Sn mL 1). The extraction was carried out in 10 mL 0.1 mol L 1 HCl in methanol in the ultrasonic bath for 1 h. After extraction, the suspension was centrifuged at 4200 rpm for 15 min. The simultaneous derivatization and extraction step was performed as follows: 1.0 mL sample extract was added to 20 mL sodium acetate/acetic acid buffer (c = 0.4 mol dm 3, pH = 4.8). Sodium tetraethylborate (NaBEt4) was used for derivatization of OTCs into fully alkylated volatile compounds. Thus, 1 mL 2% (w/v) NaBEt4 solution was added, followed by the addition of 1.0 mL hexane and a pH adjustment to 4.8 with ammonia (25%). The mixture was mechanically shaken at 400 rpm for 30 min. Phase separation was achieved after 10 min. The organic phase was then collected into 2-mL vials and centrifuged for 15 min at 4200 rpm to obtain a clear hexane extract. The upper hexane layer was transferred into another 2-mL vial, and 1 lL was injected into the GC-PFPD. The same procedure, with the exception that no sample was added, was used to determine procedural blanks. The quantification of OTCs was performed by applying the standard addition calibration method. 2.4.2. Seawater The analytical procedure is described elsewhere (Vahcˇicˇ et al., 2011). Briefly, 300 mL acidified seawater sample was added to 100 mL sodium acetate/acetic acid buffer (c = 0.4 mol dm 3, pH = 4.8) and spiked with 50 ll of the internal standard solution of TPrT (100 ng Sn mL 1). Then, 0.5 mL 2% (w/v) NaBEt4 solution was added to the spiked samples for the ethylation of organotins, followed by the addition of 1 mL hexane to extract the ethylated OTC species. After adjusting the pH to 4.8, the samples were mechanically shaken for 30 min at 400 rpm. The organic phase was collected into 2-mL vials, and 1 lL was injected into the GC– ICP-MS. Blank samples were spiked with the internal standard (TPrT) and subjected to the same analytical procedure. 2.4.3. Quality control The accuracy of the analytical procedure for the determination of OTC concentrations in mussels was checked by analyzing the certified reference material for mussels CE 477 (ERM, European Commission, Geel, Belgium). The results obtained for butyltin compounds were in agreement with the certified values, confirming the accuracy of the applied analytical method for the determination of butyltin compounds in mussels. Because CE 477 is certified only for butyltin compounds, it was spiked with known amounts of phenyl compounds. Recoveries between 75% and 115% were obtained for phenyl compounds and between 80% and 120% for octyltin compounds. The limits of detection were 6–10 ng Sn g 1 for butyltins, 11–16 ng Sn g 1 for phenyltins and 16–25 ng Sn g 1 for octyltins. A certified reference material for the determination of OTCs in seawater does not exist. Therefore, an uncontaminated seawater sample was spiked with known amounts of all nine of the OTCs analyzed. The recoveries of the spiked target compounds in samples ranged from 85% to 99% for butyltins, from 87% to 109% for phenyltins and from 103% to 126% for octyltins. The obtained limits of detections ranged from 0.11 to 0.45 ng Sn L 1 for butyltin

compounds, from 0.11 to 0.16 ng Sn L 1 for phenyltin compounds, and from 0.07 to 0.10 ng Sn L 1 for octyltin compounds. The differences between the average OTC concentrations for the defined groups of locations were evaluated statistically using Student’s t-test (test of mean values). Pearson’s correlation coefficient was used for the evaluation of the correlation between the concentrations of different organotin compounds. 3. Results and discussion The concentrations of butyltins, phenyltins and octyltins were determined in seawater and mussel samples to evaluate the extent of the pollution of the marine environment with organotin compounds along the Croatian Adriatic Coast. The samples were collected at 48 sampling locations, which were divided into three groups according to the level of exposure to marine traffic and consequent degree of OTC contamination. The concentrations of the measured organotin compounds are given for each group (marinas, ports and reference sites) separately and are expressed as ng Sn L 1 for seawater and as ng Sn g 1 dry weight (d.w.) for mussels. In addition, the total butyltin concentrations in each sample are given because they better reflect the original input of TBT from antifouling paints. 3.1. The distribution of OTCs in seawater Organotin compounds were detected in all analyzed seawater samples. Their concentrations (average values and ranges) are shown in Table 1. Butyltins represented 96.4% of the OTCs, and the distribution of their individual concentrations in all analyzed samples is shown in Fig. 2. Phenyltins, which were found only in 7 out of the 47 samples analyzed, represented 3.6% of the total OTCs. Octyltins were not detected in any seawater sample. The total P BuT concentration ( BuT = TBT + DBT + MBT) ranged from 0.46 to 27.98 ng Sn L 1. The average BuT concentration found in the marinas was 9.60 ± 5.20 ng Sn L 1, and it was higher than those determined in the ports (7.72 ± 5.36 ng Sn L 1) and reference sites (6.02 ± 2.08 ng Sn L 1). The concentrations within each group varied considerably because each sampling group covered a wide range of different locations with variable intensity of marine traffic. The TBT levels varied between 0.21 and 15.41 ng Sn L 1, with the highest concentration detected at the sampling station P18. In general, the highest TBT concentrations were found in the marinas, where the average TBT concentration was 3.92 ± 3.42 ng Sn L 1. In the ports and reference sites, the average TBT concentrations were 1.5 and 7 times lower, respectively, than those determined in the marinas, as expected. These results confirm that the marinas were, in general, the locations with the most exposure to TBT pollution coming from the use of TBT-based antifouling paints. In the case of the marinas, the average concentrations of DBT and MBT, which are products of TBT degradation, were 3.31 ± 2.12 and 2.37 ± 2.11 ng Sn L 1, respectively (Table 1), and were lower than their parent compound. The higher concentrations of TBT in comparison with its degradation products suggests a recent input of TBT into the marine environment because TBT degrades rapidly in seawater with a half life of few days up to few months (Cima et al., 2003; Seligman et al., 1988). Once introduced into the water column, TBT is subjected to degradation to less alkylated compounds by microbial and photolytic processes. Environmental conditions such as light, temperature and ambient water quality (including microbial population) influence the TBT degradation rate, resulting in the different reported half lives of TBT (Seligman et al., 1996). The recent input of TBT is even more apparent when the data for each butyltin compound are presented as a percentage P of the sum of butyltin concentrations (e.g., % of TBT/ BuT, Table 2).

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Table 1 P Summarizing statistic for butyltins (MBT, DBT, TBT), total butyltin ( BuT) and phenyltin (TPhT) concentrations in seawater and mussels collected in marinas, ports and reference sites along the Croatian Adriatic Coast in 2009 and 2010. Location type and number of samples

Marinas (n = 14 for seawater, n = 13 for mussels)

Ports (n = 21 for seawater, n = 18 for mussels)

Reference sites (n = 12 for seawater, n = 10 for mussels)

Samples from all locations (n = 47 for seawater, n = 41 for mussels)

a

Seawater (ng Sn L

Mean SD Min Max Median Mean SD Min Max Median Mean SD Min Max Median Mean SD Min Max Median

1

Mussels (ng Sn g

1

TPhT

MBT

DBT

TBT

P

0.28 0.12 0.13 0.36 0.35 1.26a – – – – <0.11 – – – – 0.42 0.39 0.13 1.26 0.35

211 83 128 393 188 117 74 <10 253 124 75 67 <10 231 46 155 99 <10 393 141

282 157 142 704 235 114 92 <5 391 101 41 30 <5 87 47 191 184 <5 704 134

349 246 112 1045 301 148 129 35 462 93 47 27 <6 110 41 223 224 <6 1045 170

842 371 444 1676 712 380 276 34 1031 316 163 113 – 384 129 570 450 – 1676 448

)

MBT

DBT

TBT

P BuT

2.37 2.11 0.33 6.15 1.42 3.41 2.09 0.27 7.26 3.78 3.74 2.00 0.38 6.75 4.03 2.74 2.11 0.27 7.26 3.03

3.31 2.12 0.64 7.05 2.57 1.83 1.03 0.10 5.32 1.71 1.43 0.64 0.21 1.97 1.54 2.38 2.07 0.10 7.41 1.80

3.92 3.42 1.11 10.26 2.65 2.49 3.43 0.67 15.41 1.22 0.86 0.32 0.21 1.34 0.96 3.31 4.06 0.21 15.41 1.30

9.60 5.20 2.58 18.94 7.62 7.72 5.36 0.47 27.98 7.39 6.02 2.80 0.73 10.06 6.58 8.43 5.95 0.46 27.98 7.44

(d.w.)) BuT

TPhT 90 18 71 126 80 84 25 64 112 77 <11 – – – – 88 18 64 126 80

Detected only in one sample.

30

TBT DBT

ng Sn L-1

25

MBT

20 15 10 5 0

MARINAS Fig. 2. The distribution of butyltin compounds in seawater samples (ng Sn L 2010).

PORTS 1

REFERENCE SITES ⁄

) collected along the Croatian Adriatic Coast ( September 2009;

In the case of the marinas, TBT represented 50.3 ± 20.9% of the total BuTs and was the prevailing compound, whereas the average proportions of DBT and MBT were 31.7 ± 10.2% and 17.9 ± 14.6%, respectively. Highly significant correlations were found between TBT and DBT (r = 0.8920, p < 0.001), DBT and MBT (r = 0.9552, p < 0.001), and TBT and MBT (r = 0.8454, p < 0.001). This implies a constant input of fresh TBT into the water column followed by its degradation to less alkylated compounds. The high correlations between butyltin species also indicate that there is no other significant input of MBT and DBT except from TBT degradation (Seligman et al., 1996). Relatively high variations in the BuT concentrations were found between the different marinas (Fig. 2). However, these variations were not related to the marina size (defined by the number of berths) but rather to the microlocation of the marina (the degree of sheltering from wind and wave action) and the efficiency of the water exchange. For example, in the largest marina M2, with 1200 berths, a low concentration of BuTs was found, as this marina

⁄⁄

March 2010;

⁄⁄⁄

September

is located in a large and relatively open bay. In contrast, high concentrations of BuTs were found in two small marinas: M1 (300 berths) and M7 (300 berths), located within small and sheltered bays with shallow water and limited rates of water exchange. The sampling location within the marina also played an important role, as the sample collected directly in front of a crane in marina M10 showed a very high level of BuTs. It is worthwhile to mention that in two marinas (M6 and M8) in which sampling was performed in 2009 and 2010 (Fig. 2), lower concentrations were obtained in 2010 (also in mussels, see Section 3.3), possibly indicating a decrease of the TBT inputs. In the case of the ports, the average TBT concentration of 2.49 ± 3.43 ng Sn L 1 corresponded to 32.3 ± 20.8% of total BuTs, whereas the degradation products DBT and MBT represented 24.8 ± 6.1% and 42.9 ± 20.5% of the total BuTs, respectively (Tables 1 and 2). It is obvious that recent inputs of TBT also existed in the port areas; however, the high proportion of MBT indicates that it was not as continuous as in the case of the marinas. The

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Table 2 P Summarizing statistic for percentages of butyltin species (MBT, DBT and TBT) towards the total butyltin ( BuT) in seawater and mussels collected in marinas, ports and reference sites along the Croatian Adriatic Coast in 2009 and 2010. Location type and number of samples

Marinas (n = 14 for seawater, n = 13 for mussels)

Ports (n = 21 for seawater, n = 18 for mussels)

Reference sites (n = 12 for seawater, n = 10 for mussels)

Samples from all locations (n = 47 for seawater, n = 41 for mussels)

Mean SD Min max Median Mean SD Min Max Median Mean SD Min Max Median Mean SD Min Max Median

P BT species/ BuT in seawater (%)

BT species/

MBT

DBT

TBT

MBT

DBT

TBT

17.9 14.6 5.5 56.0 11.7 42.9 20.5 10.6 66.9 54.8 57.9 10.4 38.3 70.5 59.2 34.3 22.7 5.5 70.5 33.4

31.7 10.2 22.1 48.5 30.1 24.8 6.1 17.2 40.6 24.4 24.9 5.9 19.0 38.4 23.8 28.0 8.8 17.2 48.5 26.0

50.3 20.9 10.2 72.4 58.4 32.3 20.8 12.1 72.2 19.4 17.2 7.5 10.1 35.7 15.6 37.8 22.8 10.1 72.4 31.2

27.5 10.0 11.9 56.1 26.7 31.6 11.3 0.0 55.3 30.7 44.2 12.4 21.8 60.2 41.7 31.8 12.2 0.0 60.2 29.8

33.9 9.4 22.2 45.9 34.3 29.3 8.9 0.0 39.8 30.9 23.8 12.1 0.0 43.4 25.3 30.5 10.2 0.0 45.9 31.0

38.7 13.7 21.7 63.0 34.7 39.1 12.8 21.4 100.0 36.8 32.0 8.9 17.3 44.8 34.8 37.7 12.7 17.3 100.0 35.0

concentrations determined in the ports were unexpectedly high, with the average TBT concentration barely 1.5 times lower than that determined in the marinas. This finding suggests that the local population probably still uses the TBT-based antifouling paints for their boats, as the use of these paints is more economical than other antifouling paint formulations. The distribution of BuT concentrations in the ports was more uniform than in the marinas (Fig. 2), except for one location (P18), which had the highest concentration among all seawater samples (27.98 ng Sn L 1). However, this concentration can be explained by the fact that it is a little port with very shallow water (0.5–1 m depth), and reconstruction of the port, including some dredging operations, was carried out approximately one year before the sampling. Therefore, the desorption of the BuT compounds from contaminated sediments is the most likely reason for the highest BuT concentration found at this location. Sediments are considered to be a sink for BuT compounds and a source of renewed pollution of the water. The adsorption of OTCs to the solid particles is a reversible process, and these compounds can be released back into the water column by the resuspension of the sediment (Hoch et al., 2003; Hoch and Schwesig, 2004). The transfer of organotins from the sediment to the overlaying water has been estimated to be from 50 to 790 nmol m 2 y 1 (Cima et al., 2003). The average concentrations of the BuT compounds found at the reference sites were 0.86 ± 0.32 ng Sn L 1 for TBT, 1.43 ± 0.64 ng Sn L 1 for DBT and 3.74 ± 2.0 ng Sn L 1 for MBT (Table 1), representing 17.2 ± 7.5%, 24.9 ± 5.9% and 57.9 ± 10.4% of the total BuTs, respectively (Table 2). Because both degradation products, especially MBT, were higher than TBT at all stations, it was concluded that there was no significant recent input of TBT. This finding was as excepted, as these areas are located far from the intense marine traffic. However, the TBT concentrations and the total BuT concentrations detected in most of the samples were comparable with those detected in the ports, which indicated that even these areas were exposed to TBT input. The lowest TBT concentrations were determined at the sampling stations R10 (0.33 ng Sn L 1) and R11 (0.21 ng Sn L 1), which are areas quite distant from the marine traffic. In addition to butyltins, phenyltin species were also detected in some seawater samples, accounting for 3.57% of the total organotins. They were found mainly in the samples from the marinas

P BuT in mussels (%)

and in the sample from port P18, where the highest TBT concentration was also found. The concentrations of TPhT varied from 0.13 to 0.36 ng Sn L 1, whereas DPhT and MPhT were not detected in any sample. Triphenyltins in the marine environment are from the same source as tributyltins, as they are commonly used as co-biocides in antifouling paints, although at much lower concentrations. Consequently, their concentrations in seawater were lower than the concentrations of TBT (as also reported by Omae, 2003 and Milivojevicˇ Nemanicˇ et al., 2009). There are also other possible sources of TPhT (i.e., they are used in agricultural insecticides and pesticides), but these sources of pollution are less important in the areas investigated in this study. Despite the fact that the average total BuT concentration diminished from the marinas to the ports to the reference stations (Table 1), these differences were not as significant as what was expected (t-test, p > 0.05) considering the large differences in the exposure of these types of locations to the marine traffic. A factor that could have contributed to the higher BuT levels at locations that were only sometimes exposed to TBT input could be the slow degradation rate of MBT to inorganic Sn and its accumulation in the water column. It has been reported by some studies that there is no apparent degradation of MBT even after 280 days, indicating a much longer half-life of MBT in comparison with those for TBT and DBT (Seligman et al., 1996). The decrease of the average MBT concentration from reference locations to ports and marinas supports this finding (Table 1). Because the sampling season could also play a role, it is worth mentioning that the majority of the samples from the ports were collected at the end of the winter period (March), whereas the samples from the marinas were collected at the end of the summer (September). Some authors have reported higher TBT concentrations in the winter as a consequence of dry-docking activities (Zanon et al., 2009; Tang et al., 2010) and the slower TBT degradation rates in water during the winter due to weaker solar irradiation and lower microbial activity at winter temperatures (Maguire et al., 1986; Seligman et al., 1996; Langston et al., 1987). Furthermore, in a study by Seligman et al. (1996), it was shown that faster degradation rates in water from areas of high ambient TBT concentrations are possible. They suggest that these areas have higher concentrations of TBT-degrading microbes, which can represent an adapted or acclimated population with a faster metabolism of TBT. Consequently, TBT was introduced into

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the water column degrade rapidly, which causes lower concentrations despite recent input. As already mentioned, the permanent input of TBT in the marinas was confirmed by the highly significant correlation found between TBT and DBT (r = 0.8920, p < 0.001), which also indicated that there was no other significant input of DBT than that resulting from TBT degradation. A less significant correlation was found for the reference stations (r = 0.5238, p < 0.1), whereas there was no correlation in the case of the ports (r = 0.2755, p > 0.1). The lack of correlation between TBT and its degradation product may indicate that impact of local municipal wastewaters on the DBT concentrations in ports cannot be overlooked. Some authors have reported that municipal wastewaters can represent an additional source of DBT and MBT in the marine environment (Tang et al., 2010; Diez et al., 2005; Hoch et al., 2003). 3.2. The distribution of OTCs in mussels The average values and concentration ranges of OTCs in mussels collected at 31 locations along the Croatian Adriatic Coast are presented in Table 1. Butyltins represented 97.98% of the total organotins, and the distribution of these concentrations is shown in Fig. 3. The highest total BuT concentration detected was 1676 ng Sn g 1, and BuT compounds were below the detection limit of the analytical method in only one sample. Phenyltins were detected in 17 out of 41 samples, and they were responsible for only 2.02% of the total organotins, whereas octyltins were not found in any of the analyzed samples. The average BuT concentration determined for the mussels from marinas was 842 ± 371 ng Sn g 1, approximately 2.5 times higher than that determined in ports (380 ± 276 ng Sn g 1) and more than 5 times higher than that at reference sites (163 ± 113 ng Sn g 1) (Table 1). The difference in the concentrations of BuTs between the marinas and the other types of locations was much more pronounced in mussels than in seawater with regard to both the average concentrations (Table 1) and individual values (Figs. 2 and 3). The concentrations within each group varied considerably, similar to the seawater samples. The average concentrations of TBT followed the same distribution pattern as the total BuT concentrations. The highest average TBT concentration was determined in the marinas (349 ± 276 ng Sn g 1); it was lower in the ports (148 ± 129 ng Sn g 1) and was lowest at reference sites (47 ± 27 ng Sn g 1). Among all the samples, the highest concentration of TBT was measured in marina M6 in September 2009

(1045 ng Sn g 1), when a high concentration in seawater was also found. In the case of the marinas, TBT represented 38.7 ± 13.7% of the total BuTs (Table 2) and was the prevailing compound in 9 out of 18 samples, whereas the average proportions of DBT and MBT were 33.9 ± 12.8% and 27.5 ± 10.0%, respectively. One of the reasons for the higher TBT concentrations than those of DBT is a recent input of TBT in the environment, when the intake rate was higher than the metabolism rate in mussels; therefore, TBT did not have enough time to decompose to DBT (Magi et al., 2008; Diez et al., 2005; Boscolo et al., 2004). Furthermore, many authors have reported that M. galloprovincialis mussels and other bivalves efficiently accumulate TBT due to their limited ability to metabolize TBT to DBT and MBT as a consequence of the lack of an efficient detoxification system for TBT (Lee, 1996; Roper et al., 2001; Chandrinou et al., 2007; Diez et al., 2005). The average proportions of TBT, DBT and MBT obtained in the ports were 39.1 ± 12.8%, 29.3 ± 8.9% and 31.6 ± 11.3%, respectively (Table 2). In general, the TBT concentrations and the total BuT levels were 2.5 lower in the ports than in the marinas, as expected, as ports have lower marine traffic intensity. As previously mentioned, almost all of the samples from the ports were collected at the end of the winter (March), when shipping activities were not as intensive as in the summer. The comparatively high concentrations of BuTs in the seawater from ports can be explained by a less efficient degradation of BuT compounds under winter conditions. Additionally, the accumulation of organotin compounds from the seawater to mussels could also be less efficient under these conditions. Some authors have reported that at lower winter temperatures, the mussels have slower metabolisms and, consequently, a lower accumulation rate (Chandrinou et al., 2007; Salazar and Salazar, 1996) and a slower filtration rate, which can decrease the uptake of TBT (Kobayashi et al., 1997; Tang et al., 2010). In some of the ports, the concentrations of TBT were as high as 460 ng Sn g 1, which is the same level of contamination as in marinas. The highest concentrations were found at the sampling station P18, where the highest seawater concentration was also found. At this location, two samplings were performed (in March 2009 and September 2010), and very high concentrations of BuTs were obtained in both samples, both in seawater and mussels, indicating a permanent input of TBT into the water column. In the case of the reference sites, the concentrations of butyltins were significantly lower than in the marinas and ports. In all of the samples, MBT was the prevailing compound (Fig. 3), representing 44.2 ± 12.4% of the total BuTs (Table 2). This was as expected, as

1800

ng Sn g-1 (d.w.)

1600

TBT DBT

1400

MBT

1200 1000 800 600 400

MARINAS

PORTS

2* R * 4* * R 5 R * 7 R ** 7* * R * R 8* 11 R ** 12 R ** 13 R ** 14 **

R

M 1 M *** 2* M ** 3* * M * 4* M M 6* 6* * M * M 8* 8 M *** 9* M ** 10 M *** 1 M 1** 11 M *** 12 **

0

P 2* P * 3* P * 4* P * 5* P * 6* P * 7 P ** 10 P * 13 P ** 1 P 4** 14 P *** 16 P ** 17 P ** 18 P ** 18 P *** 19 P ** 20 P ** 21 P ** 22 **

200

REFERENCE SITES

Fig. 3. The distribution of butyltin compounds in Mytilus galloprovincialis mussels collected along the Croatian Adriatic Coast (⁄September 2009; ⁄⁄March 2010; ⁄⁄⁄September 2010).

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these areas are not directly influenced by marine traffic. However, the average percentage of TBT was also significant (32.0 ± 8.9% of the total BuT), which was almost the same as in the marinas and ports. Additionally, it was significantly higher than the percentage of TBT in the seawater at the same locations. This could be explained by a limited ability of the mussels to metabolize TBT once it is accumulated from the seawater. Among all of the samples, the only one in which BuT compounds were not detected was the sample collected at the mussel farm (location R5), confirming that this farm is located in an unpolluted area. This result is in accordance with those from other studies showing that the concentrations of butyltins in aquacultured mussels at farms located offshore are lower than those in mussels from the coastal areas under anthropogenic influences (Hong et al., 2002; Chandrinou et al., 2007). Among all of the phenyltins, only TPhT was detected, in concentrations ranging from 64 to 126 ng Sn g 1, with an average concentration of 88 ± 18 ng Sn g 1 (Table 1). The determined concentrations were 2–10 times lower than those of TBT, depending on the sample location. The lower concentrations of TPhT in comparison with those of TBT were also found in other studies (Barroso et al., 2004; Zanon et al., 2009; Regoli et al., 2001; Omae, 2003; Albalat et al., 2002) as a consequence of a lower TPhT concentration in seawater. The concentrations of MPhT and DPhT were below the detection limits in all of the analyzed samples, supporting findings that TPhT in biological samples is more stable than TBT and does not metabolize easily to DPhT and MPhT (Fent and Hunn, 1991; Chandrinou et al., 2007). Some authors have found correlations between TPhT and TBT levels in mussels (Sousa et al., 2009a), but such a correlation was not established in our study, as was also reported by Bortoli et al. (2003). When we looked for a relationship between particular BuT compounds among all the mussel samples, we noticed that a significant correlation existed between TBT and DBT (r = 0.8605, p < 0.001) and between DBT and MBT (r = 0.7733, p < 0.001), indicating that there was no other source of DBT and MBT than that generated by TBT degradation in mussel tissue (Sousa et al., 2009a; Ruiz et al., 2005). The correlation between the concentrations of TBT and MBT was lower (r = 0.4082, p < 0.01), suggesting that some DBT could be discharged from the mussel before being decomposed to MBT (as suggested in the model of TBT metabolism in mussels proposed by Suzuki et al. (1998)) or suggesting a slower rate of DBT metabolism (Albalat et al., 2002; Suzuki et al., 1998). We also looked for a correlation of the TBT accumulated in mussels and its concentration in seawater at the same location, as seawater is considered to be the major source of TBT in mussels. However, such a correlation was not found in our study. The lack of a correlation could be a consequence of the time period needed for mussels to reflect the ambient TBT concentration. Furthermore, bioaccumulation is influenced by many factors, such as growth rate, temperature, tidal exchange, heavy rainfall, current speed, oxygen content, salinity, food and suspended particle supply and the exposure to various kinds of other pollutants (Salazar and Salazar, 1996; Gomez-Ariza et al., 1999), making bioaccumulation processes very complex and difficult to predict. The accumulation of TBT in the mussel M. galloprovincialis involves a number of processes, such as the uptake from the water column (absorption), the distribution within the organism, the metabolism (degradation) to DBT and MBT and the elimination of these compounds back to the water (depuration). The final concentration in the organism is reached when all these processes are balanced and when a steady state has been established (Gomez-Ariza et al., 1999). An understanding of TBT reaction kinetics, including the degradation rate of TBT and the time required to reach steady state, is necessary to define the period over which the mussels reflect the ambient TBT bioavailability. Some studies have reported that the mussel

M. galloprovincialis, when exposed to high concentrations of TBT, reaches a steady state after 60–90 days (Salazar and Salazar, 1996; Gomez-Ariza et al., 1999). However, because of the lack of such experiments at the TBT concentrations that are close to those usually found in the marine environment (low ng L 1 level), the time required for TBT to reach the steady state in mussel tissue in the environment is still not well defined (Tang et al., 2010). Because mussels show the time-averaged concentrations of OTCs in the marine environment, they are better indicators of the level of environmental OTC pollution in comparison with seawater, which only shows the momentary contamination level. The results from this work support this conclusion, as the differences in the OTC concentrations between marinas, ports and references sites were significant (t-test, p < 0.05) and much more pronounced for mussels than for seawater. The distribution of OTCs in mussels clearly shows that the marinas were more contaminated than the ports and, in particular, the reference sites, which was expected based on the exposure of these locations to marine traffic. 3.3. The evaluation of the degree of TBT pollution along the Croatian Adriatic Coast The use of TBT-based antifouling paints has been banned in Croatia since January 2008. According to the Croatian regulation, which defines the maximum allowed concentrations of priority pollutants in natural waters, the maximum single allowed concentration of TBT is 0.62 ng Sn L 1 (1.5 ng TBT L 1), while the yearly average TBT concentration must not be higher than 0.08 ng Sn L 1 (0.2 ng TBT L 1). Our results show that in 44 out of 47 seawater samples collected (93.6% of all sampling locations), the measured TBT concentrations exceeded the maximum single concentration allowed, reaching values up to 25 times higher. Of note, 10 out of the 12 water samples from the reference sites also had higher concentrations than allowed. In 2004, the OSPAR commission (OSPAR, 2004) updated the Ecotoxicological Assessment Criteria (EAC) values for TBT in water, sediments and biota, according to the most recent knowledge on the TBT concentrations that cause possible adverse effects in mollusk populations. The lower EAC value is the concentration needed for the protection of all marine species from chronic effects, including the most sensitive species, whereas the upper EAC value is defined as the highest concentration that is expected to not cause acute toxic effects. Between the lower and the upper EAC values, biological effects are possible (e.g., biomarker response, reproduction, impaired growth), whereas above the upper EAC value, longterm biological effects are likely, and acute biological effects on the survival of the population are possible. The OSPAR commission proposed a lower EAC value of 0.04 ng Sn L 1 (0.1 ng TBT L 1) and an upper EAC value of 0.62 ng Sn L 1 (1.5 ng TBT L 1) for seawater, and these values for mussels were set to 4.91 ng Sn g 1 (12.0 ng TBT g 1) and 71.7 ng Sn g 1 (175 ng TBT g 1), respectively (OSPAR, 2004). The upper EAC limit for seawater corresponds to the maximum concentration allowed by Croatian law. Because the measured TBT concentrations at all the sampling locations were above the lower EAC value for both seawater and mussels and were higher than the upper EAC value in 91.5% of seawater samples and 61.0% of mussel samples, it can be concluded that some biological effects for the most sensitive species at all the locations investigated in this study are highly probable. Imposex, which is the most adverse effect caused by TBT in gastropods and appears at a concentration of 1 ng L 1, could therefore occur at almost all sampling locations. The US Environmental Protection Agency (USEPA) set a slightly higher criterion (3.0 or 7.4 ng L 1) for the protection of seawater aquatic life from chronic toxic effects. According to this criterion, mollusk populations from 25% of the investigated sampling locations could be affected.

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M. Furdek et al. / Marine Pollution Bulletin 64 (2012) 189–199 Table 3 Literature data on tributyltin (TBT) and total butyltin (BuT) concentrations in seawater (ng Sn L

a

1

or ng Sn g

1

)

1

) and mussels (ng Sn g BuT (ng Sn L

1

1

) reported in the last decade in Europe.

or ng Sn g

1

Country

Year

TBT (ng Sn L

)

Ref.

Seawater Greece (Saranikos Gulf) Corsica Portugal Slovenia Italy (Venice Lagoon) Spain (Bay of Castropol) Portugal (NW) Spain (NW, Gijon) Slovenia Slovenia Croatia

1998–1999 1999 1999–2000 2000–2002 2003 2005 2005 2005 2005–2006 2009 2009–2010

8–70 0.8–82 <3.14–29 10–586 10–586 0.1–6.2 0.9–18.6 0.3–80 <0.47–90 <0.15–16.5 0.2–15.4

16–132 1.6–97 23–79 15–721 85–725a 0.8–7.4 – 2.5–101 10–135 0.5–21.2 0.5–28.0

Thomaidis et al. (2007) Michel et al. (2001) Diez et al. (2005) Milivojevic Nemanic et al. (2009) Berto et al. (2007) Uveges et al. (2007) Sousa et al. (2007) Rodriguez-Gonzalez et al. (2006) Milivojevic Nemanic et al. (2009) Vahcic et al. (2011) This paper

Mussels Italy (Venice Lagoon) Portugal Spain (Galicia) Portugal Slovenia Slovenia France (Arcachon Bay) Italy (Venice Lagoon) Italy (Venice) Croatia (Hexaplex trunculus) Italy (Genova) Spain (Bay of Castropol) Portugal (NW) Slovenia Portugal Croatia

1999–2000 1999–2000 2000 2000 2000–2002 2001–2002 2001–2002 2001 and 2003 2003 2003 2005 2005 2005 2005–2006 2006 2009–2010

<0.12–1840 <5.71–489 221–1856 11–789 50–6434 240–7900 27–1621 <4.9–2732 51–244 102–333 570 52–96 24–500 50–500 0.9–720 <6–1045

2–3670 25–548 286–5193 11–1739 150–9991 440–9270 40–2191 <4.9–5907 88–309a 216–3099 1042 86.2–131 35–732 100–980 2.9–1120 <6–1676

Bortoli et al. (2003) Diez et al. (2005) Ruiz et al. (2005) Barosso et al. (2004) Milivojevic Nemanic et al. (2009) Scancar et al. (2007) Devier et al. (2005) Zanon et al. (2009) Boscolo et al. (2004) Garaventa et al. (2007) Magi et al. (2008) Uveges et al. (2007) Sousa et al. (2007) Milivojevic Nemanic et al. (2009) Sousa et al. (2009a) This paper

Without MBT concentration.

There are three studies that investigated the occurrence of imposex in H. trunculus populations along the Croatian Adriatic Coast (Prime et al., 2006; Garaventa et al., 2006, 2007). Garaventa et al. (2006, 2007) determined the degree of imposex and TBT concentration in the gastropod H. trunculus at 4 locations in the Istrian region (located near station R1 in our study) from 2002 to 2003. They found imposexed females at all stations and a high percentage of sterile females (up to 83.3%) at some stations. The TBT concentrations in the gastropods were of the same order of magnitude as the TBT concentrations in the mussels at location R1 in our study, but these data are not directly comparable because it is known that different marine organisms have different TBT accumulation abilities. Prime et al. (2006) investigated the degree of imposex in H. trunculus collected in 2005 at several locations in the central and southern part of the Croatian coast. As we did in this study, they classified sampling locations into several groups regarding the level of boating activities (marinas, small harbors, sheltered bays and pristine areas) and found corresponding differences in imposex levels. The degree of imposex was high in the marinas and harbors, moderate in the sheltered bays, and could be detected even in the pristine areas. These findings are in agreement with our results and confirm that even areas distant from direct anthropogenic influence are polluted with TBT compounds to some extent. Because the concentrations of TBT in several gastropod samples collected in 2003 were similar to the ones measured in mussels sampled in the period 2009–2010, it seems that the degree of the pollution of Croatian coast with TBT did not decrease significantly from 2003 to 2010, despite of ban of TBT-based antifouling paints in 2008. Mussels are commonly consumed seafood by which TBT can be transferred to humans via dietary uptake. Therefore, it would be interesting to calculate the possible intake of TBT by contaminated mussels from the Adriatic Coast. The Tolerable Daily Intake (TDI) of TBT for humans was established to be 0.25 lg (kg body weight) 1 per day based on immune function studies, and this value was

adopted by the World Health Organization (WHO-IPCS, 1999; Antizar-Ladislao, 2008). Thus, a 70-kg individual should have a maximum dietary intake of 17.5 lg TBT per day. From the results on the TBT concentrations in mussels presented in this study and taking into account the average dry/wet weight ratio of 0.15 for the mussels, we can calculate the amount of mussels that this person could consume without exceeding their given TDI. For the mussels from marinas, it would be approximately 70 g dry or 460 g fresh mussels; for mussels from the ports, it would be approximately 215 g dry or 1440 g fresh mussels; and for mussels from the reference sites, it would be approximately 400 g dry or 2600 g fresh mussels. Therefore, if we take into account that in Croatia only approximately 10 kg/year (27 g/day) of seafood is consumed per capita, the calculated quantities indicate that the consumption of mussels generally does not present a health threat. To elucidate if the Croatian coast is more polluted with TBT than coastal areas in other countries, with a focus especially on Europe, we compared the measured concentrations in seawater and mussels with the TBT concentrations from the literature published in the last 10 years (Table 3). With respect to European countries, it is clear that TBT concentrations in both seawater and mussels were significantly higher in 1999–2003 than in 2005–2006. The maximum concentrations in the first period were, both in seawater and mussels, 5–10 times higher than in the second period. Notably, the concentrations measured in 2005–2006 in all European countries were still higher than the AEC values defined by OSPAR, and those areas can be considered to be contaminated with TBT. The TBT concentrations in the samples from the Adriatic Coast from 2009 to 2010 (this study) fell well within the range of concentrations measured in European countries from 2005 to 2006. We did not find any data on the TBT levels in seawater and mussels in European countries in samples collected after 2006, except for two studies in which the OTC concentrations in seawater from Slovenia collected in 2009 (Vahcˇicˇ et al., 2011) and from Spain collected from 2008 to 2009 (Marti et al., 2011) were reported. There-

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fore, we cannot conclude if the Croatian coastal waters are currently more polluted with TBT than those in other European countries. Our conclusion that the TBT contamination is of a recent origin is made based on the high proportion of TBT in the seawater and mussel samples. This suggests that the ban of TBT-based antifouling paints is not efficient and that these paints are still in use in Croatia. The slow removal of TBT from the marine environment due to its persistence in sediments can be an additional reason for the elevated TBT levels. It would be beneficial to have data on the TBT levels in marine samples collected from other European countries after the total ban in 2008 to elucidate how efficient this ban is in other European countries. These data would also show the time period that is required for the marine environment to recover from the heavy pollution with TBT. Data on the OTC levels from the Slovenian coast (Vahcˇicˇ et al., 2011), which showed much higher values than permitted, suggest that, at least in Slovenia, TBT pollution in 2009 was still a great concern. The data on the TBT concentrations in Mediterranean coastal waters of Spain reported by Marti et al. (2011) are not relevant for comparison with the data from this study because samples were not taken from ports or marinas. However, TBT was detected at some locations and reached the value of 11 ng Sn L 1 at one location, indicating its recent input at that sampling site. The most recent study on the imposex level in Portugal demonstrated that, although a decline in TBT pollution was apparent in 2003–2008, the Portuguese coast is still extensively affected by imposex, showing that fresh input of TBT still continues to occur (Sousa et al., 2009b; Galante-Oliveira et al., 2011). A very recent study from Island (Guomundsdottir et al., 2011) also showed that although the degree of imposex decreased significantly from 1992 to 2003, a further decrease from 2003 to 2008 was much slower. In addition, the same authors also observed a notable increase in imposex at several locations in 2008, implying the incessant input of organotins. Acknowledgements The work was supported by the Ministry of Science, Education and Sport of the Republic of Croatia under the Grant No. 0980982934-2715 and by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia within the research program P1-0143. The work was performed in the frame of the bilateral Croatian–Slovenian collaboration supported by the Croatian and Slovenian Ministries of Sciences. The authors are grateful to Dr. Renato Batel for his help in collecting seawater and mussel samples. References Albalat, A., Potrykus, J., Pempkowiak, J., Porte, C., 2002. Assessment of organotin pollution along the Polish Coast (Baltic Sea) by using mussels and fish as sentinel organisms. Chemosphere 47, 165–171. Antizar-Ladislao, B., 2008. Environmental levels, toxicity and human exposure to tributyltin (TBT)-contaminated marine environment. A review. Environ. Int. 34, 292–308. Barroso, C.M., Mendo, S., Moreira, M.H., 2004. Organotin contamination in mussel Mytilus galloprovincialis from portuguese costal waters. Mar. Pollut. Bull. 48, 1145–1167. Berto, D., Giani, M., Boscolo, R., Covelli, S., Giovanardi, O., Massironi, M., Grassia, L., 2007. Organotins (TBT and DBT) in water, sediments, and gastropods of the southern Venice lagoon (Italy). Mar. Pollut. Bull. 55, 425–435. Bortoli, A., Troncon, A., Dariol, S., Pellizzato, F., Pavoni, B., 2003. Butyltins and phenyltins in biota and sediments from the Lagoon of Venice. Oceanologia 45, 7–23. Boscolo, R., Cacciatore, F., Berto, D., Marin, M.G., Giani, M., 2004. Contamination of natural and cultured mussels (Mytilus galloprovincialis) from the nothern Adriatic Sea by tributyltin and dibutyltin compounds. Appl. Organomet. Chem. 18, 614–618. Champ, M.A., Seligman, P.F., 1996. An introduction to organotin compounds and their use in antifouling paints In: Champ, M.A., Selignam, P.F., Organotin – Environmental Fate and Effects, Chapman and Hall, London, pp. 1–25.

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