Ecological quality status of the Adriatic coastal waters evaluated by the organotin pollution biomonitoring

Marine Pollution Bulletin 123 (2017) 313–323

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Ecological quality status of the Adriatic coastal waters evaluated by the organotin pollution biomonitoring

MARK

A. Erdeleza, M. Furdek Turkb,⁎, A. Štambukc, I. Župand, M. Pehardaa Institute of Oceanography and Fisheries, Šetalište I. Meštrovića 63, 21000 Split, Croatia Department for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia c Department of Biology, Faculty of Science, University of Zagreb, Roosveltov trg 6, 10000 Zagreb, Croatia d Department of Ecology, Agronomy and Aquaculture, University of Zadar, Trg kneza Višeslava 9, 23000 Zadar, Croatia a

b

A R T I C L E I N F O

A B S T R A C T

Keywords: TBT pollution Imposex Hexaplex trunculus Adriatic Sea Ecological status assessment Mediterranean

The aim of this study was to evaluate the post-legislation change in tributyltin (TBT) pollution at Croatian Adriatic coast. Gastropod Hexaplex trunculus and sediments were collected, nearly 10 years after TBT based antifouling paints were banned, at 12 locations along the coast where a previous study was conducted in 2005. The study showed a decline of TBT levels over the investigated period, although all gastropods populations were highly affected by imposex meaning that prohibition did not result in the recovery of populations. The further aim was to propose the Ecological Quality Ratio (EQR) boundaries for potential use of H. trunculus as a principal bioindicator in the assessment of the ecological status of the Mediterranean regarding TBT pollution, under the Water Framework Directive (WFD). According to the proposed EQR classes, the WFD target for achieving the Good ecological status of the marine environment by 2015 was not reached.

1. Introduction Organotins, namely tributyltin (TBT), were introduced into the marine environment mainly through the application in antifouling coatings. TBT may cause various biological effects on different nontarget organisms, but the main toxic effect caused by this compound in marine ecosystem is occurrence of imposex in prosobranch gastropods (Smith, 1981; Bryan et al., 1987; Graceli et al., 2013). Imposex is defined as superimposition of male sex characteristics in females (Smith, 1971). Previous studies demonstrated that some gastropod species develop imposex even at TBT concentrations in seawater as low as 1 ng Sn l− 1 (Gibbs et al., 1988), and that imposex intensity increases as environmental TBT concentration increases and as TBT bioaccumulates (Bryan et al., 1987; Oehlmann et al., 1998a). At higher TBT concentrations advanced stages of imposex occur, severely affecting females' reproductive capabilities, population recruitment and structure (Gibbs et al., 1988). Up to date > 200 species have been proposed to monitor TBT pollution worldwide, including Hexaplex trunculus (Horiguchi, 2017). Banded dye-murex H. trunculus is a common Mediterranean gastropod. This species inhabits littoral, is locally abundant, has limited mobility, does not have planktonic larval stage and develops imposex at TBT concentrations even lower than 1 ng Sn g− 1 dry weight (d.w.) (Axiak

⁎

Corresponding author. E-mail address: [email protected] (M. Furdek Turk).

http://dx.doi.org/10.1016/j.marpolbul.2017.08.039 Received 18 January 2017; Received in revised form 24 July 2017; Accepted 16 August 2017 Available online 25 August 2017 0025-326X/ © 2017 Elsevier Ltd. All rights reserved.

et al., 1995). All these traits make this species a reliable bioindicator of TBT pollution. During the last few decades, imposex in H. trunculus has been widely investigated in relation to TBT pollution in many Mediterranean countries, e.g. Malta (Axiak et al., 1995, 2003), Italy (Terlizzi et al., 1998, 1999, 2004; Chiavarini et al., 2003; Pelizzato et al., 2004; Garaventa et al., 2006, 2007), Israel (Rilov et al., 2000), Portugal (Vasconcelos et al., 2006a), Croatia (Prime et al., 2006; Garaventa et al., 2006, 2007; Stagličić et al., 2008) and Tunisia (Lahbib et al., 2007, 2009, 2010). The majority of these studies investigated spatial distribution of imposex occurrence and some have related it to TBT pollution. Because the organotins exhibited negative effects on non-target organisms, primarily gastropods and bivalves, their use in antifouling coatings is nowadays banned in many countries worldwide, including all of Europe (Sonak et al., 2009). All antifouling coatings containing organotins are banned by the International Convention on the Control of Harmful Anti-fouling Systems on Ships (AFS Convention) since the year 2008. Croatia ratified AFS Convention in 2006, and in the same year TBT was included in the List of hazardous substances that are prohibited for use (Official gazette 17/2006), however ships containing organotins coatings have not been banned from entering Croatian ports until 2008. In EU, the prohibition of organotins on ships entered into force through Directive 2002/62/EC and Regulation EC/782/2003. In

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Fig. 1. Location of sites sampled for Hexaplex trunculus and sediments in 2015 (M1 = Marina, M2 = Milna, M3 = Biograd, H1 = Baška Voda, H2 = Rogač, H3 = Vis, B1 = Zaton, B2 = Lastovo, B3 = Crvena Luka, P1 = Brsečine, P2 = Drage, P3 = Mali Ston).

(2004) and Garaventa et al. (2006, 2007) investigated imposex indices in H. trunculus collected in Venice area (Italy) in the time period 2002–2003, the former including also coastal area around western Istria peninsula that is part of Croatia. Furthermore, Stagličić et al. (2008) analysed banded dye-murex collected in 2004 and 2006 at Kaštela bay (Croatia), while Carić et al. (2016) presented data on imposex in H. trunculus collected from several sites in Dubrovnik area in 2006. In all of these studies the investigated populations were seriously affected by imposex (VDSI ranged from 2 to 5). The first observation of imposex in H. trunculus in the central Croatian Adriatic was conducted in 2005 (Prime et al., 2006) and represents the baseline data for the evaluation of temporal trend in imposex occurrence conducted in this study. It demonstrated high levels of imposex at 12 investigated locations categorized by boating activity, although three of them were categorized as “pristine” areas. WFD set the year 2015 as the target deadline to achieve Good ecological and chemical status of the Mediterranean marine environment regarding TBT (Laranjeiro et al., 2015). The question is whether this has been accomplished? Based on the above presented review of literature data it can be stated that new data are necessary to evaluate the environmental impact of the TBT ban so this question could be answered. The aims of this study were 1) to provide the first insight on tissue burden of butyltins (TBT and its degradation products, dibutyltin (DBT) and monobutyltin (MBT)) in populations of H. trunculus in central Croatian Adriatic, and to assess the relationship between butyltin concentrations in the tissue and sediment with the imposex level; 2) to evaluate the effect of enforced law restrictions on the use of organotins in antifouling paints over a 10-year period by analysis of changes in imposex occurrence in banded dye-murex populations at 12 location in the central Croatian Adriatic; 3) to propose Ecological Quality Ratio (EQR) boundaries for the potential use of imposex in H. trunculus as a biological quality element in the assessment of the ecological status of

Annex VIII of the Water Framework Directive (WFD) 2000/60/EC organotins are declared as one of the main pollutants, and TBT is enlisted as one of the priority substances in the field of water policy (2455/ 2001/EC) that should be monitored in order to evaluate the chemical status of a water body. The presence of TBT in the environment is regulated by the prescribed maximum allowed concentration in seawater. Considering that this concentration is extremely low, 0.2 ng L− 1 (0.08 ng Sn L− 1) for all of Europe, chemical analysis of TBT at such low levels are often difficult to perform even with the most sensitive analytical methods. For this reason, the highly specific biological response to TBT, i.e. imposex in gastropods, is often used as a valuable tool in estimating the impact of TBT pollution in the marine environment. Therefore, imposex became a mandatory element under the OSPAR environmental monitoring programme (OSPAR, 2004). Furthermore, in the WFD monitoring programme it was proposed as one of the biological quality elements that could be used for evaluation and classification of ecological status of coastal waters (WFD-UKTAG, 2014). For this to be accomplished, classes of ecological status based on imposex levels should be defined as proposed by Laranjeiro et al. (2015). Several recent studies reported the widespread appearance of imposex in the Mediterranean gastropod populations, such as in Sardinia and Tunisia (Anastasiou et al., 2015; Boulajfene et al., 2015; Abidli et al., 2013), however data on temporal trends of imposex status in the Mediterranean related to the TBT ban is limited (the only data is given by Lahbib et al. (2009) for the Tunisian coast). Also, no recent data on temporal trends of imposex occurrence in the Adriatic, the northernmost arm of the Mediterranean Sea, are available. However, measurements of TBT concentrations in seawater and mussels from Croatian coast for the time period 2009–2010 showed that eastern Adriatic was polluted with organotin compounds even after the ban (Furdek et al., 2012). The first study of imposex occurrence in the Adriatic Sea was conducted by Terlizzi et al. (1998) in Brindisi area, Italy. Pelizzato et al. 314

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per each group). The samples were lyophilized and stored in the dark at − 20 °C until organotin analysis. Surface sediment samples were also lyophilized and stored in the dark at − 20 °C until organotin analysis. The extraction of butyltins (BuTs) from the tissue was performed by the method described in details by Furdek et al., 2012. Briefly, BuTs were extracted from the homogenized tissue (1–1.5 g) in 0.1 mol l− 1 HCl in methanol (10 ml) by ultrasonic stirring for 30 min. The suspension was centrifuged at 4200 rpm for 10 min and supernatant (1 ml) was used for further analysis. Then, simultaneous derivatization with NaBEt4 solution (1 ml 1% (w/v)) and extraction into hexane (1 ml) were carried out in a sodium acetate-acetic acid buffer (20 ml, pH = 4.8, c = 0.4 mol dm− 3) by mechanical shaking at 400 rpm for 30 min. The extraction of BuTs from the sediments (fraction < 2 μm) was performed by the method previously described by Milivojevič Nemanič et al. (2009) and Furdek (2015). Briefly, BuTs were extracted from the sediment (1–2 g) by acetic acid (20 ml) and ultrasonic stirring for 30 min. The simultaneous derivatization and extraction step was performed in the same manner previously described for the biological samples. The detection of BuTs (TBT, DBT and MBT) in tissue and sediments was carried out on a gas chromatograph (GC, Varian CP3800) with a pulsed flame photometric detector (PFPD, Varian). The quality control was performed by the analysis of standard reference material certified for BuTs in mussels (CE 477, ERM, Geel, Belgium) and for BuTs in marine sediments (PACS 2, Ottawa, Canada). Tripropyltin (TPrT) was used as internal standard while quantification of BuTs was performed by applying standard addition calibration method. The results obtained for BuTs were in agreement with the certified values, confirming the accuracy of the applied analytical method for the determination of butyltin compounds in the investigated samples. The limits of detection (DL) were 0.9, 1.0 and 1.9 ng Sn g− 1 d.w. for TBT, DBT and MBT in tissue, and 1.5, 2.1 and 6.1 ng Sn g− 1 d.w. for TBT, DBT and MBT in sediments, respectively.

coastal waters regarding TBT pollution in the frame of WFD directive, and to use proposed EQR boundaries to evaluate if the Good Ecological Status regarding TBT is achieved along the Eastern Adriatic Coast. 2. Materials and methods 2.1. Study area and sampling Samples of H. trunculus and sediments were collected over a 6 week period (May–June 2015) from 12 locations along the Croatian Adriatic coast (Fig. 1). The sampling locations were adopted from the study conducted in year 2005 by Prime et al. (2006). Locations were categorized by the expected intensity of the boating activity: (i) nautical marinas (M), (ii) village harbours (H), (iii) sheltered bays that were seasonally used for boating activities (B), and (iv) reference sites very rarely used for boating activities (P). For comparison of imposex between years 2005 and 2015 data from Prime et al. (2006) as well as unpublished data from that study (Peharda, unpublished data) were used. From each location 60 adult individuals (> 40 mm) were collected by snorkelling and SCUBA diving at 1–10 m depths. As the lifespan of H. trunculus is around 7–8 years (Axiak et al., 2003), and at 3 years of age this species already reaches 60 mm of shell length (Vasconcelos et al., 2006b), individuals larger than 40 mm were considered suitable for assessing the contamination status of the marine environment. The samples were transported to the laboratory, maintained in plastic tanks with flowing seawater and examined for imposex level within 2 days, as in Prime et al. (2006). The surface sediments (0–2 cm) were collected by SCUBA diving using plastic cups, and were stored at − 20 °C immediately after the sampling. 2.2. Imposex analyses The shell length (apex to aperture) was measured to the nearest 0.1 mm using Vernier callipers, the shell was cracked in a vice, and soft body was gently removed. The animals were not narcotized. After the operculum was removed, the longitudinal cut was performed on the hypobranchial gland to observe sexual features of each individual. Males were identified by the presence of penis and vas deference, and the absence of the vaginal opening and/or capsule gland. Females were identified by the presence of the vaginal opening and capsule gland. The length of straightened penis from its tip to its junction with body wall was measured for males and imposexed females with digital Vernier callipers to the nearest 0.1 mm. Three imposex indices were determined for each location: (i) the relative penis length index (RPLI, Stroben et al., 1992) defined as (mean female penis length) / (mean male penis length) × 100, (ii) the cubed relative penis size index (RPSI, Gibbs et al., 1987) defined as (mean female penis length)3 / (mean male penis length)3 × 100, and (iii) the vas deferens sequence index (VDSI) which represents the mean score of vas deferens stages of all individuals, following the general scheme for the determination of vas deferens stage (VDS) proposed by Gibbs et al. (1987) and Stroben et al. (1992), later modified by Axiak et al. (1995) and Terlizzi et al. (1999). Due to the fact that imposex stages are ordinal variables, some authors calculate VDSI as a median score (Ho and Leung, 2014). However, since VDSI is defined as a mean score of different VDS in females and this definition is used in most of the existing studies (Garaventa et al., 2007; Lahbib et al., 2008; OSPAR, 2004; WFD-UKTAG, 2014), we followed the same principle in order to be able to compare data of this study with data from literature and that given by OSPAR and WFD.

2.4. Statistical analyses Statistical analysis was performed using Statistica 8.0. The homogeneity of variances was tested by Levene's test. Difference between years 2005 and 2015 for RPLI and VDSI were tested by Mann-Whitney U test. Kruskal-Wallis test was used for: 1) comparison of imposex indices between and within different boating categories, and 2) comparison of butyltins concentrations within categories of sites according to boating activities. Spearman rank correlation analyses was preformed between imposex parameters and butyltins tissue burden. For samples with butyltins levels below the detection limit (DL), half of DL value was used. 3. Results 3.1. Spatial and temporal variation of imposex intensity Hexaplex trunculus females exhibiting imposex were observed at all sampling sites in the year 2015 (Table 1). Even at three sites that were categorized as reference sites, > 90% of collected females had certain level of imposex. The lowest VDSI, RPLI and RPSI values were recorded at reference site P3, the only site within marine protected area. At this site VDSI ranged from 0 to 3. Population from sites M1, M2 and M3 (nautical marinas) exhibited the highest RPSI (6.7–20.4%) and VDSI (> 4.7) values, showing the highest intensity of imposex. Very high levels of imposex were also observed at village harbours, especially at location H3. Populations from seasonally used sheltered bays also showed moderate to high imposex level, though to a lesser degree than in marinas and harbours. Sterile females (those with VDS stage 5) were determined at nine sites. Sterility was absent in village harbour H2, sheltered bay B3 and reference site P3. Comparison of imposex indices in populations from different types of sites showed that for RPLI (Fig. 2a) and VDSI (Fig. 2b) statistically

2.3. Chemical analyses After measurements of imposex stages, the visceral coil and the rest of the soft body of randomly selected individuals from each location were pooled into three male and three female samples (5 individuals 315

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Table 1 Summary of data for Hexaplex trunculus collected in 2015 along the Croatian Adriatic coast (shell height, percentage of females affected by imposex, percentage of sterile females, penis length ± standard deviation, relative penis length index (RPLI), relative penis size index (RPSI), vas deferens sequence index (VDSI) ± standard deviation, butyltin degradation index (BDI) ± standard deviation). Category

Nautical marinas

Location

M1 M2 M3

Village harbours

H1 H2 H3

Sheltered bays seasonally used

B1 B2 B3

Reference sites very rarely used

P1 P2 P3

Sex

M F M F M F M F M F M F M F M F M F M F M F M F

No. of ind.

20 20 20 20 20 20 20 20 20 20 20 20 15 20 20 20 20 20 20 20 20 20 20 20

Shell length (mm)

63.4 63.5 78.3 81.0 62.0 63.9 62.3 67.1 68.9 69.7 69.2 63.5 56.5 64.5 74.5 72.0 56.0 67.6 61.3 64.9 57.3 64.2 59.3 65.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.5 4.1 2.8 5.6 4.3 2.9 6.8 4.8 5.6 5.3 6.0 4.3 6.3 3.9 6.8 7.0 6.2 3.7 4.9 2.3 7.6 4.9 5.3 5.0

Females affected by imposex (%)

Sterile females (%)

100

70

100

80

100

60

100

35

100

0

100

20

100

5

100

10

100

0

100

5

100

5

90

0

Penis length (mm)

10.6 ± 2.2 5.5 ± 1.7 14.5 ± 4.1 5.9 ± 2.4 14.8 ± 2.0 8.7 ± 2.3 12.2 ± 2.8 4.9 ± 1.7 13.3 ± 2.6 6.4 ± 1.5 13.6 ± 3.4 4.4 ± 1.9 12.2 ± 2.7 4.1 ± 1.2 13.4 ± 2.4 4.1 ± 2.0 10.8 ± 2.3 3.5 ± 1.1 11.7 ± 4.9 4.3 ± 1.9 13.1 ± 1.9 4.4 ± 1.6 14.3 ± 2.3 0.9 ± 0.3

RPLI (%)

RPSI (%)

VDSI

51.6

13.8

4.7 ± 0.4

40.5

6.7

4.9 ± 0.3

58.6

20.1

4.8 ± 0.4

39.8

6.3

4.6 ± 0.4

48.1

11.1

4.1 ± 0.3

32.6

3.4

4.2 ± 0.6

33.5

3.8

4.0 ± 0.6

31.0

3.0

3.7 ± 1.0

32.3

3.4

3.9 ± 0.3

36.9

5.0

3.9 ± 0.7

33.6

3.8

4.1 ± 0.3

6.6

0.0

2.2 ± 1.2

BDI Visceral coil

Rest of the soft tissue

11.4 ± 2.4 4.8 ± 2.4 9.2 ± 3.4 8.0 ± 4.7 6.0 ± 3.8 4.6 ± 2.6 15.0 ± 2.3 / 7.7 ± 1.5 8.0 ± 4.7 / 21.2 ± 20.7 / 4.9 ± 1.8 / / 12.0 ± 8.7 / 15.2 ± 7.5 / / 4.6 ± 2.7 / /

9.6 ± 3.1 3.6 ± 0.9 6.5 ± 3.7 4.0 ± 0.6 4.5 ± 2.6 2.0 ± 0.5 5.1 ± 3.9 8.8 ± 0.9 6.5 ± 3.7 4.0 ± 0.6 7.6 ± 1.5 17.1 ± 16.1 / 8.2 ± 4.5 6.5 ± 4.4 2.4 ± 2.4 39.8 ± 23.6 12.2 ± 10.6 2.3 ± 1.6 7.5 ± 1.7 4.3 ± 1.6 7.4 ± 4.2 / /

harbour H3 (U = 123, p = 0.04) (Fig. 3b). Females with ventrally split capsule gland (caused by the growth of vas deferens tissue) with its lumen open into the mantle cavity are at VDS stage 5 (Terlizzi et al., 1999), and this females are considered as sterile females (Fig. 4). Comparison of appearance of sterile females with VDS stage 5 showed that in 2015 their percentage was much higher in all nautical marinas (M1, M2 and M3), as well as in harbour H1 (Fig. 3c). At the sheltered bay B2 and reference sites P1 and P2, sterile females were detected in 2015 even though they were not present in 2005.

significant difference can be observed between nautical marinas, village harbours and sites with seasonal or rare boating activity (RPLI: H = 58.05, p < 0.001; VDSI: H = 107.42, p < 0.001), whereas values of latter two categories did not statistically differ from each other. Imposex indices in 2015 (RPLI and VDSI) between sampling sites within the same boating category were significantly different, except for sheltered bays and VDSI for nautical marinas. Population from reference site P3, the only marine protected area site, had significantly lower RPLI values than at any other site (p < 0.007). The comparison of imposex indices in H. trunculus collected at 12 locations along the Croatian Adriatic coast in years 2005 and 2015 is presented in Fig. 3. Significantly higher values for RPLI were noted in the year 2005 at most sampling sites, except at village harbour H1 (U = 165, p = 0.34) and reference site P1 (U = 143, p = 0.12) (Fig. 3a). The comparison of VDSI in years 2005 and 2015 (Fig. 3b) showed slightly higher VDSI in 2015 at several locations (M1, M3, H1, P1 and P2) but only at two sites, nautical marina M3 and reference site P2, statistically significant increase in VDSI values is observed (U = 118, p = 0.03, and U = 92, p < 0.001, respectively). Significant reduction in VDSI values from 2005 to 2015 was observed at village

3.2. Distributions of butyltins in tissues of Hexaplex trunculus and in surface sediments Concentrations of butyltins (TBT, DBT, MBT) in tissues of H. trunculus collected at 12 sites along the Adriatic coast were determined separately in the visceral coil (digestive gland and gonads) and in the rest of the soft body, both in females and males, and the results are presented in the Fig. 5. The total BuT concentration (∑BuT = TBT + DBT + MBT) in visceral coil tissue of females in nautical marinas (73.3 ± 9.3 ng Sn g− 1 d.w.) was 2 times higher than Fig. 2. Comparison of Hexaplex trunculus imposex indices between different sites categorized by boating activity (Mnautical marinas; H-village harbours; B-sheltered bays; Preference sites) in the year 2015 according to: (a) relative penis length index (RPLI), (b) vas deferens sequence index (VDSI). Letter marks (a, b, c) denote statistically significant differences between categories (p < 0.05).

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Fig. 3. Comparison of Hexaplex trunculus imposex indices between different sites categorized by boating activity (M-nautical marinas; H-village harbours; B-sheltered bays; Preference sites) in the year 2005 and 2015 according to: (a) relative penis length index (RPLI), (b) vas deferens sequence index (VDSI) + SD, and (c) percentage of sterile females. Asterisks denote statistically significant differences between years (p < 0.05).

Fig. 5. Concentrations of butyltins (monobutyltin MBT, dibutyltin DBT, tributyltin TBT) in diverse female and male tissue types (visceral coil tissue and rest of the body tissue) of Hexaplex trunculus collected at 12 sites of different boating activity (M-nautical marinas; H-village harbours; B-sheltered bays; P-reference sites) in the central Croatian Adriatic coast in the year 2015. Mean values are presented with respective standard deviation: mean + SD. Letter marks (a, b, c, d) denote statistically significant differences in ∑ BuT between categories (p < 0.05).

in village harbours (42.5 ± 11.2 ng Sn g− 1 d.w.), 8 times higher than in seasonally used sheltered bays (8.9 ± 2.0 ng Sn g− 1 d.w.) and 18 times higher than in reference sites (4.1 ± 1.1 ng Sn g− 1 d.w.). In the rest of the soft body of females ∑BuT in nautical marinas (95.7 ± 4.6 ng Sn g− 1 d.w.) was 2 times higher than in village harbours (47.4 ± 7.4 ng Sn g− 1 d.w.), 10 times higher than in sheltered bays (9.5 ± 2.3 ng Sn g− 1 d.w.) and 5 times higher than in reference sites (19.2 ± 5.1 ng Sn g− 1 d.w.). The distribution of BuT concentrations in males showed similar trends as in females but the differences between categories were less pronounced. The highest concentrations of ∑BuT were determined in specimens from nautical marina M3 concerning all tissue types, although in the males' rest of the body tissue at this location the concentration was almost the same as at location M2. At several sites with seasonal or rare boating activity ∑ BuT was very low (< 6.0 ng Sn g− 1 d.w.) or even below detection limits, mainly in females' tissue (P1, P2, P3, B2). The measured TBT concentrations were generally low (< 15 ng Sn g− 1 d.w.), except in nautical marinas where concentration usually ranged from 5 to 60 ng Sn g− 1 d.w., while at sheltered bays and reference sites the values were always near or even

Fig. 4. Hexaplex trunculus female in VDS stage 5 indicating split capsule gland (white error) and vas deferens (black error). Scale bar 5 mm.

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presented in Fig. 7 showed sharp increase of imposex indices (VDSI) at very low levels of TBT, reaching highest VDS stages (VDSI > 4) already at TBT concentrations lower than 5 ng Sn g− 1. In particular, VDS stage 5 corresponds to mean TBT and ∑BuT concentrations of 4.3 and 41.6 ng Sn g− 1 d.w. in the visceral coil tissue, respectively. Statistically significant correlations were established between imposex indices (RPLI, RPSI, VDSI) and TBT and ∑BuT concentrations in both types of tissues (Table 2). The correlations in the visceral coil were slightly stronger between imposex indices and ∑BuT concentrations, while in the rest of the soft body stronger correlations with TBT were observed. However, imposex indices correlated stronger with ∑BuT concentrations in the visceral coil tissue than in the rest of the body tissue. Significant correlations were also observed between various imposex indices (VDSI vs RPLI, VDSI vs RPSI, RPLI vs RPSI; r = 0.67; r = 0.59; r = 0.93; respectively, p < 0.001).

below the detection limit. In most cases TBT had the lowest portion in ∑ BuT. Considering all tissue types, MBT was often the predominant butyltin specie since its proportion ranged from 27 to 87%. In order to determine the prevalence of TBT over its degradation products, and thus the occurrence of recent TBT input into the investigated environments, butyl degradation index (BDI) was calculated (BDI = (MBT + DBT) / TBT, as proposed by Diez et al. (2002) for sediments and Sousa et al. (2009) for molluscs) for the population from each location. BDI values higher than 1 indicate that no recent TBT inputs occur, while BDI values < 1 show that TBT prevail over its degradation products and therefore suggest its recent inputs into the environment. The average BDI in our study ranged from 4.6 ± 2.6 (M3) to 21.2 ± 20.7 (H3) for the visceral coil tissue, while BDI values for rest of the soft tissue were generally slightly lower ranging from 2.0 ± 0.5 (M3) to 39.8 ± 23.6 (B3). Kruskal-Wallis test confirmed that ∑BuT and TBT were significantly higher in populations from nautical marinas than in those from sheltered bays and reference sites considering all tissue types of both females and males, p < 0.05 and p < 0.01, respectively. However, there was no significant differences nor between nautical marinas and village harbours nor between sites with seasonal and rare boating activity. It is worth mentioning that concentrations of butyltins in different tissue types of males and females were not significantly different within the population from the same boating category. The only exception was TBT concentration in females' rest of the body tissue between sites H1 and H2. Spatial variations in butyltins concentrations in sediments collected at 12 locations in the central Adriatic coast in the year 2015 are shown in Fig. 6. The results indicated distinct differences among sites of different intensity of boating activities. However, due to the lack of replicates at each location those differences were not statistically significant. The ∑ BuT concentrations in marinas and harbours were comparable (104.2 in marinas vs 135.5 ng Sn g− 1 in harbours), while at sheltered bays and reference sites were below the detection limit. The highest ∑BuT concentration was found in the village harbour H2 (299.1 ng Sn g− 1), and the lowest in sediment sample from the nautical marina M2 (81.9 ng Sn g− 1). The concentrations of TBT followed the same distribution pattern as BuT. Calculated BDI values were 1.9, 2.2 and 2.1 for sites M1, M2 and H2, respectively, while they were < 1 for sites M3 (0.9) and H3 (0.7).

4. Discussion 4.1. Evaluation of TBT pollution and imposex status of H. trunculus at the Croatian Adriatic coast In order to evaluate current level of pollution of the Adriatic Coast with organotin compounds, degree of imposex was investigated in gastropod H. trunculus collected at 12 locations along the coast. To elucidate the effects of marine traffic on the ecological status of the population, the sampling locations were divided into four groups (nautical marinas, village harbours, sheltered bays and reference sites). As most of the studied populations demonstrated high degree of imposex, and none was completely free of it, it can be concluded that incidence of imposex caused by TBT pollution is still widely present along the Croatian coast and was strongly related to the intensity of boating activities - it decreased from marinas to the sites with occasional and rare boating activities (Fig. 2). These results indicate that TBT has been introduced into the marine environment after the ban of TBT based antifouling paints in 2006. The study on organotins in seawater and mussels Mytilus galloprovincialis conducted at Croatian coast during 2009–2010 also demonstrated inputs of TBT after its prohibition (Furdek et al., 2012). Notable TBT concentrations are still observed in various marine environments even years after the total ban of its use in antifouling paints (e.g. Kim et al., 2014; Pougnet et al., 2014), often related to resuspension of contaminated sediments and/or possible new inputs (e.g. Langston et al., 2015; Suzdalev et al., 2015). However, the regulatory restrictions resulted in recovery of many gastropod populations along the European Atlantic coast, including species Nucella lapillus, Tritia reticulata, Nassarius nitidus and Stramonita haemastoma, which were all previously substantially affected by imposex (e.g. Evans et al., 1996; Birchenough et al., 2002; Huet et al., 2004; Bray et al., 2011; Cuevas et al., 2014; Langston et al., 2015; Laranjeiro et al., 2015; Nicolaus and Barry, 2015), while Guðmundsdóttir et al. (2011) presented data on the increase of imposex levels near few small harbours in Iceland after intervening regulations. One of the aims of this study was to evaluate the post-legislation change in TBT pollution level and the effect that TBT ban had on H. trunculus populations. With this in mind, this study was conducted 10 years after organotins in antifouling coatings were banned in Croatia at the same sampling sites where Prime et al. (2006) conducted their research in 2005. Statistical comparisons of imposex incidence in years 2005 and 2015 (Fig. 3a, b) lead to dissimilar conclusions regarding temporal trend in imposex occurrence in Croatia. RPLI in 2015 was significantly lower for most sampling sites than in 2005, while VDSI values were similar, except at locations M3 and P2 where they were significantly higher in 2015, and location H3 where was lower. Therefore, when comparing imposex levels in years 2005 and 2015 according to RPLI values a declining trend can be observed at all sites, while VDSI index suggested that imposex level mainly remained the

3.3. Relation between imposex indices and butyltins concentration in Hexaplex trunculus Comparison of TBT and ∑ BuT tissue concentrations and VDS stages

Fig. 6. Concentrations of butyltins (monobutyltin MBT, dibutyltin DBT and tributyltin TBT) in sediments collected at 12 sites of different boating activity (M-nautical marinas; H-village harbours; B-sheltered bays; P-reference sites) in central Croatian Adriatic coast in 2015.

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Fig. 7. Relation between tributyltin (TBT) (a) and total butytin (∑ BuT) concentration and (b) VDSI in the visceral coil (vc) and in the rest of the soft tissue (rt) in Hexaplex trunculus.

reduction in TBT pollution was not sufficient to result in a recovery of H. trunculus population. Although imposex is considered as an irreversible phenomenon, when discussing a declining trend of RPLI values it is worth to mention the hypothesis of the limited reversibility of imposex, in particular FPLI, as suggested by Bryan et al. (1993) and Sousa et al. (2005, 2007). However, this should be further investigated and verify whether this applies to VDSI also. In order to elucidate the level of TBT pollution of the marine environment in the central Adriatic, in addition to imposex indices, the BuTs concentrations in the tissue of H. trunculus (visceral coil and the rest of the soft tissue) and surface sediments were determined. The ∑BuT, regardless of sex and tissue type, ranged from 0 to 348.3 ng Sn g− 1 d.w., with the highest concentrations found in marinas, somewhat less in ports, and the lowest concentrations (often near or below the detection limits) in the sheltered bays and reference sites (Fig. 5). Even though the difference in BuTs concentrations between those categories was not statistically significant, this study nevertheless demonstrated that BuTs body burden in H. trunculus is related to the degree of boating activity, the same as found for imposex indices. It should be emphasized that imposex represents a dose-dependent but irreversible response to the total TBT accumulated throughout the life of an individual (Laranjeiro et al., 2015; Axiak et al., 2003), and therefore the degree of imposex determined in this study could be partly due to the past exposure to TBT. Since the depuration time of TBT in molluscs is usually described by the half-life of one to several months (Gomez-Ariza et al., 1999; Sousa et al., 2009), the determined BuTs concentrations in the tissue of H. trunculus undeniably indicated that the investigated populations were exposed to TBT pollution over the last few months. However, despite the possible time discrepancy between imposex development and TBT accumulation, rather weak but still statistically significant correlations were found between imposex indices (VDSI, RPLI and RPSI) and TBT concentration (Table 2). This is in agreement with observation of many studies that the intensity of imposex in gastropods is related to the levels of accumulated TBT (Axiak et al., 1995; Pellizatto et al., 2004; Ho and Leung, 2014). An even stronger correlation was observed between imposex indices and ∑BuT concentrations, particularly in the visceral coil. This is due to the fact that ∑BuT concentrations more reliably reflect the total uptake of the TBT. Tributyltin is bioaccumulated through feeding and initially accumulates in the visceral coil where it undergoes dealkylation to DBT and MBT through the P-450 dependent mixed-function oxidase system. Later, all three are transported to other body organs (Axiak et al., 1995; Pellizzato et al., 2004). The results of our study are in agreement with this observations as the statistically significant correlations were found between all TBT, DBT and MBT in the visceral coil (Spearman rank correlation; TBT vs DBT: r = 0.76; DBT vs MBT: r = 0.85; TBT vs MBT: r = 0.70; p < 0.05), as well as between TBT, DBT and MBT concentrations in the visceral coil (vc) and in the rest of the soft tissue (rt) (Spearman rank correlation; TBTvc vs TBTrt: r = 0.79; DBTvc vs DBTrt: r = 0.69; MBTvc vs MBTrt: r = 0.52; p < 0.05). Many published papers reported different distribution patterns of BuTs in the H. trunculus

Table 2 Spearman rank order correlations between imposex indices and butyltins concentrations (TBT, ∑ BuT) in visceral coil tissue and rest of the soft body tissue. Different p-levels are applied.

Visceral coil tissue Rest of the soft tissue ⁎⁎⁎ ⁎⁎

VDSI vs ∑ BuT

RPLI vs ∑ BuT

RPSI vs ∑ BuT

VDSI vs TBT

RPLI vs TBT

RPSI vs TBT

⁎⁎⁎

⁎⁎⁎

⁎⁎⁎

⁎⁎⁎

⁎⁎⁎

⁎⁎⁎

⁎⁎

⁎⁎

⁎⁎

⁎⁎⁎

⁎⁎⁎

⁎⁎⁎

0.65

0.48

0.70

0.52

0.59

0.48

0.58 0.58

0.66 0.64

0.59 0.60

p < 0.001. p < 0.01.

same over a 10-year period. It is therefore questionable which imposex parameter is more reliable for assessing the contamination and ecological status of the marine environment regarding TBT pollution, especially taking into account that both of them correlate well with TBT concentration in H. trunculus (Table 2). Stroben et al. (1992) suggested that VDSI is the most reliable indicator for imposex in T. reticulata because of the seasonal changes in the penis length, while Pelizzato et al. (2004) preferred female penis size (RPSI) when comparing contaminated sites because of the larger range of variations in RPSI than in VDSI. Whereas both of the studies were conducted in the same time of the year (May–July; fresh and not narcotized animals), penis length indicators are considered comparable, in particular because RPSI and RPLI were below 100%. However, according to OSPAR, WFD and many other studies, the key imposex parameter to be monitored for evaluation of TBT pollution is VDSI because of its biological implication as it reveals the reproductive capability of population (OSPAR, 2004; WFDUKTAG, 2014; Oehlmann et al., 1998b; Laranjeiro et al., 2015). The conclusion based on determined VDSI values, that no declining trend in imposex levels is observed, is further supported by the percentage of sterile females (those with VDS stage 5) which was higher at most locations in 2015 (7 out of 12; Fig. 3c). Having in mind that VDS provides information on reproductive capability of a population and risk of its extinction, we can undoubtedly conclude that the prohibition of TBT based antifouling paints in Croatia did not result in the recovery of H. trunculus population. However, it should be mentioned a model experiment on the imposex development in H. trunculus exposed to different levels of TBT concentration (5 and 50 ng l− 1) described by Abidli et al. (2009). This study demonstrated that female penis length was significantly higher in individuals exposed to higher TBT concentration after 6 months of exposure, while VDSI was the same for both exposure levels. According to this research, penis length in H. trunculus better indicates the level of TBT concentrations in the environment since the highest VDS stage develops even at lower concentration. Therefore, the difference between imposex indices observed in our study could be interpreted as follows; RPLI decline from 2005 to 2015 suggests that the level of TBT pollution in the marine environment decreased along this period, while VDSI demonstrates that apparent 319

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concentrations in sediments at sites categorized as nautical marinas and harbours were 302.3 and 296.2 ng Sn g− 1, and in those categorized as small ports and reference sites were 63.1 and 53.1 ng Sn g− 1, respectively. Due to lack of replicate samples in our study, and the fact that these two studies were not conducted at the same sites, the reliable comparison of the results is not possible, but nevertheless a slight decrease in ∑BuT concentrations in marinas and harbours, as well as in ports and reference sites, could be observed. However, taking into account that average sedimentation rate in the coastal area is around 0.5–1 cm per year, it is difficult to determine temporal changes in surface sediments (0–5 cm) over the period shorter than 5 years. After all, on the basis of the presented results, both BuTs concentrations in H. trunculus and sediments, we can conclude that TBT pollution rather decreased during the last 10 years in Croatia, confirming the effectiveness of the ban. However, the decreased TBT input into the marine environment did not result in the decrease of imposex level in H. trunculus and the recovery of this population because the concentrations of TBT in the environment are still higher than those that cause advanced stages of imposex in the local gastropod populations. Among all gastropod species investigated so far, H. trunculus is considered as the most sensitive with respect to its biological response to TBT (Axiak et al., 1995; Abidli et al., 2009). This is further supported in our study as the highest VDS stage was reached at TBT and ∑BuT concentrations below 5 and 50 ng Sn g− 1 d.w., respectively. These values are significantly lower than those reported by Pelizzato et al. (2004) and Garaventa et al. (2007) who reported that VDS stage 5 corresponds to approximately 90 and 235 ng Sn g− 1, respectively. This corroborates the conclusion made by Garaventa et al. (2007), that correlation analyses of VDSI and butyltins tissue concentration could be unreliable at high concentrations because investigated populations usually carried body burdens of butyltins which are above the saturation limits.

tissue. Some authors stated significantly higher concentrations in the visceral coil tissue (Axiak et al., 1995, 2003; Pellizzato et al., 2004; Garaventa et al., 2006), while others found higher load of organotins in the rest of the body tissue (Garaventa et al., 2007). In our study significant difference in the accumulation of BuTs in the visceral coil and in the rest of the body tissue was not observed. Distribution of BuTs in the investigated populations showed the highest proportion of MBT and the lowest of TBT (MBT > DBT > TBT) thus showing that TBT is faster metabolized to DBT and MBT and eliminated from the organism, than is being accumulated from the environment. This, together with the BDI values calculated for H. trunculus tissue which were higher than 1 at all locations (Table 1), strongly suggests that there was no recent inputs of TBT into the investigated environments, at least not in the last several months. However, it should be mentioned that high BDI values in gatropods could be also explained by raised availability of MBT and DBT as a result of TBT degradation in surface sediments (Ruiz et al., 2008). This is supported by the statistically significant correlations that was found in our study between MBT and DBT concentrations in sediments and H. trunculus tissue (Spearman rank correlation; DBTsed vs DBTvc: r = 0.62; p < 0,001; DBTsed vs DBTrt: r = 0.62; p < 0.05; MBTsed vs MBTvc: r = 0.47; p < 0,05). Correlation between TBT in sediment and tissue was not found. The observation of BuTs tissue concentrations in H. trunculus over investigated time period was not possible because our study represented the first data on BuTs tissue burden in populations of H. trunculus for the central part of the Croatian Adriatic. When comparing the mean BuTs concentrations in our study to those of previous study on H. trunculus in the Northern Adriatic for the period 2002–2003 (∑ BuT ranged between 192 and 4233 ng Sn g− 1 d.w., Garaventa et al., 2007), it can be noticed that concentrations are lower in our study conducted in 2015. They are also lower than TBT concentrations in H. trunculus from other areas in the Mediterranean in the period 1992–2003 (Axiak et al., 1995; Pelizzato et al., 2004). Distribution of BuTs concentrations in sediments also showed expected differences between different categories of sites regarding the intensity of boating activity. The ∑BuT concentration in marinas and harbours were rather similar (average concentrations are 104.2 and 135.5 ng Sn g− 1 d.w., respectively), while in sediments from sites with rare marine traffic (bays and reference sites) BuTs were below the detection limits. Tributyltin bioaccumulated in H. trunculus at these sites was also low (ranged from 0.9 to 3.3 ng Sn g− 1 d.w.), confirming low BuTs levels in these environments. It is important to underline that sample from harbour H1 was gravel-grained sediment which could explain low level of BuTs determined at this site since this type of sediment has very low ability to adsorb TBT (Furdek et al., 2016). Many published papers demonstrated that some sediments, as a consequence of high affinity of TBT for the adsorption onto particulate matter and very slow TBT degradation in sediments (half lives ranged from several years to decades), represent a long-term storage of TBT (Omae, 2003). Due to resuspension of contaminated surface sediments, its desorption back into the water column may occur, which results in a new threats to biota (e.g. Ruiz et al., 2008; Kim et al., 2014; Pougnet et al., 2014; Langston et al., 2015; Suzdalev et al., 2015). Although H. trunculus lives in the direct contact with sediments, the significant correlations between TBT concentration in sediments and tissue or imposex indices were not confirmed in our study. A reason for absence of such correlations could be the influence of the sediment characteristics on the TBT adsorption. Some of the sediment samples (from locations M2, H1, B1, B2, B3, P1, P2) were mainly sandy and gravel sediments which usually have low amount of organic matter and thus low capacity to adsorb TBT. On the contrary, sediments containing more fine particles have higher affinity for adsorption of organic matter and TBT, resulting in much slower TBT degradation in these sediments which consequently represent a long-term source of pollution (Furdek et al., 2016). The recent study conducted in the period 2009–2013 along the Croatian Adriatic coast (Furdek, 2015) demonstrated that mean ∑ BuT

4.2. Evaluation of ecological status classes for Water Framework Directive based on imposex levels in Hexaplex trunculus According to the EU Water Framework Directive (WFD, Anex V, 2000/60/EU), one of the biological quality elements for the classification of ecological status of surface waters is composition and abundance of benthic invertebrate fauna. Although not specifically referred to in the WFD, the imposex assessment could be used as a metric within the benthic invertebrate biological quality element since it represents a specific effect of a particular contaminant on a sensitive indicator species (WFD-UKTAG, 2014; Laranjeiro et al., 2015; Ruiz et al., 2017). To perform evaluation of the ecological status according to WFD, each biological quality element should be evaluated by comparing the measured conditions (the observed value) against that described for reference conditions (minimally disturbed), and this is reported as an Ecological Quality Ratio (EQR). The evaluation of the ecological status of the environment is performed by determining the EQR values which define five ecological status classes (High, Good, Moderate, Poor and Bad), in accordance to the effects of anthropogenic disturbance in the biological community. The detailed description of each ecological status class, based on the information from OSPAR (2004) and WFD (Annex V, 2000/60/EC), is given by Laranjeiro et al. (2015). When imposex is used as a biological quality element, the EQR values should be calculated by converting the determined VDSI values into EQR, according to Eq. (1) (WFD-UKTAG, 2014; Laranjeiro et al., 2015):

EQR =

(M − O) M

(1)

(M is maximum VDSI that a population may attain, and O is the observed VDSI). Imposex in species N. lapillus is already proposed as a tool for WDF ecological status assessment of coastal waters in the UK (WFD-UKTAG, 320

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environmental data relating to the of imposex levels in H. trunculus (VDSI < 1), the EQR value and the corresponding VDSI are difficult to establish for this category. For this category Laranjeiro et al. (2015) adopted the VDSI value (VDSI < 0.3) for N. lapillus and T. reticulata set by OSPAR (2004). The Good status class is defined by the absence of the risk for population extinction, which, when using imposex as s parameter for ecological status assessment, requires at least absence of sterile females. The data available for H. trunculus in the Mediterranean, presented in Fig. 8, indicate that sterile females appear in populations with VDSI > 3.0 (confidence interval p = 0.95). However, some sterile females were also observed in a few populations with 1.8 < VDSI < 3.0. Therefore, the Good status class, which indicates lack of sterile females in the population, should be defined with the interval VDSI < 1.8. The Moderate status implies low risk of population extinction, meaning that sterile females may appear, while Poor status corresponds to a major deviation in population abundance caused by high female sterility. According to Laranjeiro et al. (2015), the VDSI boundary between the Moderate and the Poor class is related to distinction between low and high risk of population extinction and therefore corresponds to the incidence of > 50% of sterile females. The relationship between VDSI and percentage of sterile female presented in Fig. 8 demonstrated that 50% of sterile females corresponds to approximately VDSI 4.5 so this should be the proposed value for the boundary between Moderate and Poor class. The Bad status refers to population extinction and the absence of specimen individuals due to extremely high TBT pollution. The VDSI values that were proposed as the classes boundaries were converted into EQR boundaries for ecological status classes using Eq. (1), and are presented in Table 3. EQR boundaries for N. lapillus and T. reticulata (Laranjeiro et al., 2015), the gastropods most often used for imposex monitoring at the European Atlantic coast, are shown for comparison. However, the proposed boundaries represent only the initial step in establishing the EQR classes for imposex in H. trunculus and should be further validated and confirmed on the basis of a much larger set of data. Further studies of imposex appearance at very low TBT levels when VDSI is ≤ 1 are particularly needed. According to the proposed EQR classes for H. trunculus presented above, the ecological status of the marine environment in the Central Adriatic for the year 2015 was evaluated. The results indicated that none of the surveyed sites reached the classification of Good status (not even the protected marine areas). All investigated nautical marinas (M1, M2, M3) and harbour H1 are evaluated as the environments of Poor ecological status, while other village harbours, sheltered bays and reference sites are categorized as Moderate class environments. Therefore, the data presented in this study showed that WFD target for achieving the Good ecological status by the year 2015 is not reached. If the same classification of ecological status is applied using the data from 2005, it can be concluded that the legislative restriction of TBT based antifouling paints did not result in the recovery of the investigated environments during the last 10 years.

2014), while Laranjeiro et al. (2015) proposed ecological status classes for N. lapillus, T. reticulata and Littorina littorea. Because various gastropod species have different sensitivity to TBT and a different path of imposex development (Axiak et al., 1995), the proposed ecological status classes for those species, and the corresponding EQR values are not applicable for H. trunculus. Furthermore, none of these species is widely distributed in the Mediterranean and therefore cannot be used in WFD monitoring programme in this area. Due to its ubiquity in the Mediterranean, and a very sensitive response to TBT contamination, H. trunculus could be the principal bioindicator for the purpose of WFD monitoring programme in the Mediterranean. For this to be realized, the appropriate EQR values for the ecological status classes should be defined. Therefore, one of the aims of this work was to propose such values on the base of the available data for imposex indices (VDSI and percentage of sterile females (%S)) in H. trunculus adopted from the literature (this study; Axiak et al., 1995; Terlizzi et al., 1998; Rilov et al., 2000; Prime et al., 2006; Garaventa et al., 2007; Stagličić et al., 2008; Lahbib et al., 2011), following the approach described in the work of Laranjeiro et al. (2015). Considering that the principal aim of WFD is to preserve diversity and abundance of each population, the occurrence of female sterility can be one of the factors in the evaluation of boundaries of the ecological status classes regarding imposex. It is also very important to notice that endocrine modulators can have extensive consequences for the population fitness beyond the merely inducing sterility in subset of individuals. TBT has been previously shown to affect traits related to Darwinian fitness, such as growth, mortality and fecundity of H. trunculus already at environmentally relevant concentrations (Abidli et al., 2009; Lahbib et al., 2012). Thus further studies should aim to reveal the extact impact on the population fitness at TBT concentrations lower than those causing female sterility. The relationship between the percentage of sterile females and the corresponding VDSI determined in 105 different populations from 9 studies is presented in Fig. 8 (this study-12 populations; Axiak et al. (1995) – 9 populations; Terlizzi et al. (1998) – 15 populations; Rilov et al. (2000) – 16 populations; Prime et al. (2006) – 12 populations; Garaventa et al. (2007) – 8 populations; Stagličić et al. (2008) – 7 populations; Lahbib et al. (2008) – 20 populations; Lahbib et al. (2011) – 6 populations). High ecological status class corresponds to undisturbed environmental conditions and is therefore achieved if no (or minor) anthropogenic alteration occurs. This status equals to extremely low TBT concentrations in water, usually close to zero and often below detection limits of the most sensitive analytical methods. Due to the lack of

5. Conclusion The prohibition on use of TBT in antifouling coatings resulted in a decline of BuT levels in the central Croatian Adriatic. However, all investigated populations of Hexaplex trunculus are still highly affected by imposex, meaning that the prohibition of TBT based antifouling paints did not result in the recovery of this population. The level of imposex in the investigated populations correlates with TBT and total butyltins body burden. Ecological Quality Ratio (EQR) boundaries for the potential use of imposex in H. trunculus as a tool for the ecological status assessment of coastal waters regarding TBT pollution, in the frame of WFD, were proposed. According to proposed EQR boundaries, WFD target for achieving the Good ecological status by the year 2015 is not reached at Eastern Adriatic Coast.

Fig. 8. The relationship between the percentage of sterile females (%S) and VDSI determined in 105 different populations of Hexaplex trunculus from the Mediterranean: polynomial fit equation VDSI = 3.0258 + 0.0501 ∗ x − 0.0003 ∗ x2 with confidence interval (conf.int.) p = 0.95.

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Table 3 Proposed EQR values defined for use of imposex as biological quality element for the ecological status assessment in the frame of WFD. Ecological status class

High Good Moderate Poor Bad

Nucella lapillus (Laranjeiro et al., 2015)

Tritia reticulata (Laranjeiro et al., 2015)

Hexaplex trunculus (proposed in this study)

Proposed EQR classes

Corresponding VDSI

Proposed EQR classes

Corresponding VDSI

Proposed EQR classes

Corresponding VDSI

1.00–0.95 0.95–0.50 0.50–0.25 0.25–0.00 Population absence

0.0–0.3 0.3–3.0 3.0–4.5 4.5–6.0

1.00–0.93 0.93–0.80 0.80–0.40 0.40–0.00

0.0–0.28 0.28–0.8 0.8–2.4 2.4–4.0

na 0.64 0.64–0.10 0.10–0.00 Population absence

na < 1.8 1.8–4.5 4.5–5.0

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