Tributyltin (TBT) has been largely used as a biocide in ship antifouling paints over the last four decades and, consequently, TBT pollution became a widespread problem around the world. Estuaries stand amongst the most polluted areas since they usually

1
Imposex levels in Hydrobia ulvae (Pennant, 1777)
to evaluate spatial and temporal changes of
Tributyltin (TBT) pollution in Ria de Aveiro (NW,
Portugal) from 2003 to 2007 Galante-Oliveira
S1, Oliveira I, Barroso C1 1CESAM Department
of Biology, University of Aveiro, 3810-193
Aveiro, Portugal. susana.oliveira_at_ua.pt
1. Introduction
Tributyltin (TBT) has been largely used as a
biocide in ship antifouling paints over the last
four decades and, consequently, TBT pollution
became a widespread problem around the world.
Estuaries stand amongst the most polluted areas
since they usually enclose ports, dockyards and
marinas. In these areas, sediments may act as a
major reservoir of TBT and this account for the
persistence of this compound in the environment
even several years after its usage prohibition.
Restrictive measures to ban the use of TBT based
antifouling paints on small vessels (lt25 m in
length) and aquaculture equipment were introduced
in most European countries by the EC Directive
89/677/EEC. It was adopted in Portugal in 1993
but was insufficient to reduce TBT pollution
(Barroso and Moreira, 2002). Later, in 2001, the
International Maritime Organization (IMO) adopted
the International Convention on the Control of
Harmful Antifouling Systems (AFSs) on Ships (AFS
Convention). This resolution called for a
worldwide prohibition on the application of
organotins (OTs) as biocides in AFSs on ships by
the effective date of 1 January 2003, and a
complete banishment by 1 January 2008. However,
the Convention could just enter into force 12
months after 25 States representing 25 of the
world's merchant shipping tonnage have ratified
it. Until then, the legal effect of 1 January
2003 would be suspended (IMO, 2001). Meanwhile,
the EU adopted the Regulation No.782/2003 on the
prohibition of TBT coatings on Member States
national mercantile fleets and on ships operating
under their authority from 1 July 2003. The AFS
Convention entry into force date was met on 17
September 2007 with the 25th State ratification,
representing a total of 38 of the worlds
merchant shipping tonnage. As a result, the TBT
international ban is finally stated for 17
September 2008 (IMO, 2007). It is, therefore,
important to monitor imposex levels evolution
because is a way to check the legislation
efficiency reducing TBT pollution and TBT
persistence in sediments. The aim of this study
is to evaluate spatial and temporal evolution of
TBT pollution in Ria de Aveiro using Hydrobia
ulvae imposex as a biomarker of sediment
contamination.
2. Sampling strategy
3. Biological analysis

• In the laboratory, animals where maintained in
  aquaria for a maximum period of 9 days.
• About 60 adult animals were analyzed per station
  (Stn.) after a 45 narcotization period with 35
  MgCl2.
• Specimens were measured using a graduated
  eyepiece in a stereo microscope, to the nearest
  0.14mm.
• After shells removal, animals were sexed and
  parasitized specimens were discarded from the
  analysis (Schulte-Oehlmann et al., 1997).
• The following imposex indices were determined
• FPLI mean female penis length index
• MPLI mean male penis length index
• RPLI relative penis length index
  (FPLIx100/MPLI)
• VDSI vas deferens sequence index
• I percentage of affected females (incidence)
• Penis length was measured with a graduated
  eyepiece in a stereo microscope providing an
  accuracy of 0.03mm. VDSI was classified according
  to the scoring system proposed by
  Schulte-Oehlmann et al. (1997) for H. ulvae.

• About 100 animals were collected by hand at the
  intertidal shore, except where there was an
  insufficient available number of specimens.
• For spatial monitoring
• Animals were collected at Stns.1-10 (Fig. 1)
• These sites were proposed in 1998 by Barroso et
  al. (2000) as the best approach for TBT pollution
  monitoring in Ria the Aveiro.
• For temporal monitoring
• Three sampling occasions were considered
  regarding TBT usage restrictive measures
• 2003, moment when the Regulation No.782/2003 was
  applied
• 2004, one year after that partial ban in EU
  Member States
• 2007, one year before the total ban.

A. Coastal fishing port B. Chemical port C. North
commercial port D. Deep-sea fishing
port E. Aveiro dockyards F. South commercial
port G. S. Jacinto dockyard H. Costa Nova
marina I. Gafanha marina J. Torreira
marina K. Ovar marina
Figure 1. Ria de Aveiro and adjacent coastal area
map indicating the main TBT pollution sources
(AJ on the left) and sampling sites location,
code and name (110 on the right).
4. Results
Table 1. Hydrobia ulvae. Number of analyzed
specimens (n) at each site in 1998 by Barroso et
al. (2000), 2003, 2004 and 2007 (current study),
with the indication of mean shell heights (mm)
for males and females. Standard deviations (SD)
are given next to the mean value in the format
mean(SD). For statistical comparisons from 2003
to 2007 the one way ANOVA and one way ANOVA on
ranks (Kruskal Wallis test) were applied,
depending on the assumptions of normality or/and
homogeneity of variance were confirmed or not,
respectively. Post-hoc tests for
multi-comparisons were also applied the Tukey
test after each parametric ANOVA and the Dunns
test after the non parametric ones.
Multi-comparisons significance is indicated next
to the last year of the tested pair p lt 0.05
p lt 0.01 p lt 0.001 - animals not
found. For additional data on sites location
compare with Fig.1.
1998 1998 2003 2003 2004 2004 2007 2007 2003 2004 2007
Stn. code and name ? n ? Shell height ? n ? Shell height ? n ? Shell height ? n ? Shell height MPLI MPLI MPLI
1. Aveiro, Pêga 31 6.2(0.4) 44 5.6(0.4) 41 5.6(0.4) 29 5.3(0.4) 2.9(0.6) 3.5(0.5) 2.8(0.7)
2. Ponte Ílhavo 30 5.5(0.5) 20 5.1(0.4) 20 5.6(1.3) 28 4.5(0.2) 2.0(0.4) 3.7(0.4) 3.1(0.5)
3. Ponte Ermida 31 6.4(0.7) 40 5.4(0.5) 33 5.5(0.4) 19 3.7(0.3) 2.3(0.5) 2.3(1.0) 2.7(0.7)
4. Ponte Barra, Gramata 28 5.6(0.5) 20 5.3(0.5) 26 5.6(1.0) 21 5.2(0.5) 1.9(0.3) 2.9(0.8) 3.7(0.7)
5. Juliões 28 5.9(0.4) 20 5.2(0.4) 21 5.4(0.7) 21 5.4(0.6) 1.6(0.2) 2.9(0.5) 3.6(0.8)
6. Muranzel 27 6.5(0.6) 20 5.1(0.4) 9 4.0(0.6) 20 4.3(0.3) 1.9(0.2) 2.2(0.6) 2.7(0.5)
7. Torreira 33 6.0(0.4) 15 5.0(0.5) 20 6.1(0.6) 13 4.5(0.4) 1.7(0.3) 4.3(0.5) 3.3(0.6)
8. Ponte Varela 26 6.6(0.4) 20 5.0(0.3) 20 4.9(0.5) 20 5.3(0.4) 1.6(0.2) 3.1(0.6) 3.3(0.5)
9. Ovar marina 27 5.8(0.4) 20 5.2(0.4) - - 1 3.0(0.0) 2.1(0.3) - 2.8(0.0)
10. Murtosa 26 6.2(0.2) 20 5.3(0.5) 20 6.2(0.5) - - 2.0(0.2) 3.1(0.6) -
? n ? Shell height ? n ? Shell height ? n ? Shell height ? n ? Shell height FPLI FPLI FPLI
1. Aveiro, Pêga 33 6.0(0.8) 48 5.5(0.4) 52 5.4(0.5) 37 5.2(0.3) 0.4(0.4) 0.3(0.4) 0.3(0.2)
2. Ponte Ílhavo 31 5.4(0.4) 40 5.0(0.6) 33 6.4(0.6) 36 4.6(0.3) 0.4(0.4) 1.3(1.2) 0.2(0.1)
3. Ponte Ermida 33 6.5(0.6) 59 5.5(0.3) 41 5.7(0.4) 51 3.7(0.3) 0.2(0.2) 0.2(0.1) 0.2(0.1)
4. Ponte Barra, Gramata 29 5.7(0.5) 40 5.3(0.5) 33 5.2(1.2) 41 5.4(0.7) 0.3(0.4) 0.3(0.4) 0.2(0.2)
5. Juliões 28 5.8(0.4) 40 5.0(0.4) 40 5.2(0.6) 41 5.4(0.6) 0.3(0.4) 0.1(0.1) 0.4(0.3)
6. Muranzel 28 6.6(0.8) 40 5.1(0.4) 20 4.5(0.8) 27 4.3(0.3) 0.2(0.2) 0.1(0.1) 0.1(0.1)
7. Torreira 33 6.1(0.5) 45 5.1(0.4) 40 6.2(0.9) 40 4.4(0.3) 0.3(0.3) 0.4(0.4) 0.2(0.1)
8. Ponte Varela 27 5.9(0.6) 40 5.1(0.5) 40 5.1(0.4) 42 5.6(0.5) 0.1(0.1) 0.2(0.2) 0.2(0.2)
9. Ovar marina 27 5.7(0.4) 40 5.3(0.6) - - 3 3.4(0.3) 0.3(0.3) - 0.1(0.1)
10. Murtosa 28 6.2(0.2) 40 5.0(0.6) 41 6.3(0.9) - - 0.3(0.3) 0.3(0.4) -

5. Discussion and Conclusions

• Imposex indices vary with animal age and shell
  size (Silva et al., 2003 Galante-Oliveira et
  al., 2005) for that reason, temporal comparisons
  were just performed at sites where no significant
  differences in shell heights occurred.
• No animals were found at Stn. 9 in 2004 and at
  Stn. 10 in 2007.
• No significant differences in RPLI and I were
  registered from 2003 to 2007 (Fig.2) however, a
  general increasing tendency in I was observed.
• Increases in imposex indices were statistically
  confirmed for VDSI (Fig.2) and FPLI (Table1) at
  Stn.5 (near 2 contamination hotspots Fig.1).
• There is a global stabilization of TBT pollution
  levels in Ria de Aveiro from 2003 to 2007 TBT
  pollution persists sediments are probably acting
  as a deposit of the compound, namely near places
  known in the past as the main TBT sources.

Figure 2. Hydrobia ulvae. Temporal trend of
imposex levels (VDSI, RPLI and I) exhibited by
specimens collected at 10 common sites in Ria de
Aveiro (NW Portugal) in 1998 (Barroso et al.,
2000), 2003, 2004 and 2007 (current study). For
statistical comparisons from 2003 to 2007 the one
way ANOVA on ranks (Kruskal Wallis test) was
applied since normality or/and homogeneity of
variance were not confirmed. The only significant
difference was in VDSI from 2003 to 2007. In this
case, the significance of the Dunns test for
multi-comparisons is indicated next to the
respective site (Stn.5) in the map corresponding
to the last year of the tested pairs (2007) p
lt 0.05. For additional data on sites location
compare with Fig.1.
References
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Marine Ecology Progress Series 201,
221-232. Barroso, C.M., Moreira, M.H. 2002.
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veira, S., Cardoso, M., Oliveira, I., Barroso, C.
M. 2005. CICTA 2005. Cadiz Spain p.194. IMO,
2001. International Convention on the Control of
Harmful Anti-Fouling Systems on Ships. (Ref. No.
AFS/CONF/26). International Maritime
Organization, London UK. IMO, 2007. Summary of
Conventions as at 30 November 2007 (on line,
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http//www.imo.org International Maritime
Organization, London UK. Schulte-Oehlmann, U.,
Oehlmann, J., Fioroni, P., Bauer, B. 1997. Marine
Biology 128, 257-266. Silva, J. G.,
Galante-Oliveira, S., Cardoso, M. N., Barroso, C.
M., Moreira, M. H. 2003. CICTA 2003. Porto
Portugal p.231.