Ecological Indicators 93 (2018) 966–974

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Ecological Indicators
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Original Articles

Environmental quality status of the Portuguese coast regarding TBT T

pollution – Recommendations for considering imposex monitoring within
the scope of the Marine Strategy Framework Directive
⁎
Filipe Laranjeiroa, , Paula Sánchez-Marínb, Isabel Benta Oliveiraa, Susana Galante-Oliveiraa,
Carlos Barrosoa
a
Biology Department and CESAM, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
b
Department of Animal Biology and Ecology, University of Vigo, E-36310 Vigo, Spain

A R T I C LE I N FO A B S T R A C T

Keywords: The recent years witnessed an increasing society consciousness on subjects concerning aquatic pollution. In
Tributyltin Europe, several legislative pieces have been implemented to improve and protect the aquatic environment like
Biomarker the Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD), as well as the
Water Framework Directive specific strategies adopted by Regional Sea Conventions. Here we assess the quality status of the Portuguese
OSPAR
coastal and transitional waters regarding TBT pollution, using imposex as biomarker, aiming its integration as an
Nassarius reticulatus
Nucella lapillus
indicator of environmental quality assessment under the MSFD. Imposex data available for two gastropod
bioindicators, Nucella lapillus and Nassarius reticulatus, are used for that purpose, applying the classification
previously proposed for the WFD (described elsewhere) and the OSPAR Assessment Criteria. A good quality
status was achieved in most of the stations located in coastal waters while a worse ecological status was per-
ceived in stations located in transitional water bodies, due to the proximity of TBT contamination sources. The
comparison between the WFD classification proposal and the OSPAR Assessment Criteria shows a similar
temporal trend in the study area however, slight differences can be perceived and are discussed. Both the WFD
and OSPAR should be considered on the MSFD implementation but they differ in their objectives regarding TBT
pollution. Therefore, this work allows the integration of imposex monitoring within the MSFD and recommends
the use of the WFD assessment criteria also in this directive.

1. Introduction Directive (WFD, Directive 2000/60/EC). This last was implemented in

In Europe, the implementation of an integrated policy for the pro-
tection of the marine environment has been attempted over the last
decades. Four regional sea conventions were created during the second
half of the last century – the Convention for the Protection of the
Marine Environment of the North-East Atlantic (OSPAR Convention),
the Convention on the Protection of the Marine Environment of the
Baltic Sea Area (HELCOM Convention), the Convention for the
Protection of the Mediterranean Sea Against Pollution (Barcelona
Convention), and the Convention on the Protection of the Black Sea
Against Pollution (Bucharest Convention; only since 1992). These
conventions aim to protect the marine environment, involving coun-
tries sharing the same region to work together and converge in the same
environmental objectives. At the European Union (EU) policy level,
marine environment was only partially considered in directives such as
the Habitats Directive, the Birds Directive, and the Water Framework
2000 and was a vital step for the protection of EU water bodies, in-
cluding transitional and coastal waters. Finally, in 2008, the im-
plementation of the Marine Strategy Framework Directive (MSFD) gave
an integrated policy regarding the protection of the marine environ-
ment to the EU member states. Both these directives and regional sea
conventions pay special attention in reducing the aquatic chemical
pollution and improving environmental quality.
Tributyltin (TBT) is a well-known chemical pollutant due to its use
as a biocide in antifouling paints, which contributed in the past for an
overall spread of this substance throughout the aquatic environment,
causing a variety of adverse effects to non-target organisms at very low
concentrations (Matthiessen and Gibbs, 1998; Alzieu, 2000). Probably
the most well-known adverse effect caused by TBT is imposex, a female
masculinization phenomenon already described for more than 260
gastropod species worldwide (Titley-O'Neal et al., 2011). Due to this
and other deleterious effects, a global ban of TBT antifouling paints was

⁎
Corresponding author.
E-mail address: flaranjeiro@ua.pt (F. Laranjeiro).

https://doi.org/10.1016/j.ecolind.2018.05.054
Received 23 February 2018; Received in revised form 21 May 2018; Accepted 24 May 2018
1470-160X/ © 2018 Elsevier Ltd. All rights reserved.
F. Laranjeiro et al.
implemented in 2008 by the International Maritime Organization
(IMO) through the International Convention on the Control of Harmful
Anti-Fouling Systems on Ships (AFS Convention), which was preceded
by a similar action initiated in 2003 in the EU (Regulation 782/2003/
EC). Nevertheless, TBT contamination and imposex are still a global
issue despite the global and regional restrictions on its use (Castro et al.,
2018; Lahbib et al., 2018; Laranjeiro et al., 2018). This chemical is also
included in the list of priority substances in the WFD, OSPAR, and
HELCOM.
The WFD requires member states to perform a holistic monitoring
assessment integrating several elements of quality (biological, hydro-
morphological, and physico-chemical). The biological elements’ as-
sessment is based on the population and community condition, eval-
uated by means of Ecological Quality Ratios (EQR), while a good
chemical status is achieved when Environmental Quality Standards
(EQS) defined for each priority substance are respected. However, there
is a growing movement for the integration of aquatic effect-based
monitoring tools as an essential bridge between the chemical and bio-
logical elements’ assessment (Hagger et al., 2008; Martinez-Haro et al.,
2015; Beiras, 2016). The need for this approach is also evidenced in a
technical report on aquatic effect-based monitoring tools produced by
the European Commission (Wernersson et al., 2015). This opening was
already foreseen in the later MSFD, especially on its descriptor 8
“Concentrations of contaminants are at levels not giving rise to pollu-
tion effects” (Law et al., 2010; Lyons et al., 2010; Wernersson et al.,
2015), which ultimately, and very recently, considered the use of bio-
markers or other endpoints, such as mortality rates or population re-
productive capacity, in monitoring programs under this descriptor
(Commission Decision (EU) 2017/848). In this sense, due to its high
specificity and sensitivity to TBT pollution, imposex should be con-
sidered as a useful biomarker in monitoring programs under the MSFD,
as well as in the WFD, as it is already mandatory under OSPAR and
HELCOM monitoring programmes. OSPAR developed specific guide-
lines for imposex monitoring with several bioindicator species, in-
cluding the dog-whelk Nucella lapillus and the netted-whelk Nassarius
reticulatus, and defined six classes of ecological quality based on the Vas
Deferens Sequence Index (VDSI; parameter used to grade imposex ex-
pression intensity, see Gibbs et al. (1987) for N. lapillus and Stroben
et al. (1992a) for N. reticulatus, (OSPAR, 2004)). Through this imposex-
based classification is possible to understand if a site reaches the Eco-
logical Quality Objective (EcoQO), when organisms are exposed to TBT
concentrations in water below the Environmental Assessment Criteria
(EAC) set at 0.1 ng/L.
In order to commit imposex monitoring within the WFD objectives,
in previous studies (Laranjeiro et al., 2015b, 2017) we recommended
the adoption of the same strategy of OSPAR’s Coordinated Environ-
mental Monitoring Programme (CEMP) into this directive. This would
allow the harmonization of environmental policies in Europe and would
provide the WFD with robust and resourceful instruments to assess the
quality status of water bodies. However, considering that the focus of
the WFD for the general protection of the aquatic ecosystems is to keep
the diversity and abundance of species undisturbed, it was proposed
different assessment classes based on VDSI levels (Laranjeiro et al.,
2015b). In fact, it was suggested that the VDSI boundaries should better
reflect the risk of population decline or extinction due to TBT pollution,
and so it was proposed five ecological classes – as required by the WFD
– that are slightly different from OSPAR’s. Consequently, the quality
objectives of OSPAR and the proposed within the WFD are met at dif-
ferent VDSI values. It should also be noted that the TBT chemical ob-
jective differs slightly between OSPAR (0.1 ng TBT/L) and WFD (0.2 ng
TBT/L) (OSPAR, 2004; EC, 2008).
Regional sea conventions and EU directives have a fundamental role
on the MSFD implementation with their experience, dataset, and tools
(Borja et al., 2013; van Hoof et al., 2014; Vethaak et al., 2017). Under
the previously described descriptor 8, a good environmental status
(GES) achieved under the MSFD should always be in agreement with
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Ecological Indicators 93 (2018) 966–974
WFD’s EQS and assessed in coordination with member states that share
the same marine region, with regional sea conventions playing a central
role for this purpose. Still, as previously described, not always exists a
total agreement between the WFD and regional sea conventions re-
garding TBT objectives.
The aim of this work is to assess the environmental status of the
Portuguese coast, regarding TBT pollution, and its temporal evolution
under two different perspectives: one based on the imposex assessment
criteria defined by OSPAR, and the other based on the imposex as-
sessment criteria, previously suggested by our group (Laranjeiro et al.,
2015b), within the WFD. This analysis provides a better evaluation of
this proposal and its applicability within the MSFD for the assessment of
GES. The data set used for this analysis corresponds to imposex mon-
itoring surveys performed with the bioindicators N. lapillus and N. re-
ticulatus in the Portuguese coast between 2000, the year of the WFD
implementation, and 2014, a crucial year that precedes the date by
which WFD environmental objectives must be met (2015). The Portu-
guese coast represents a good scenario for this study because it is
subjected to intense naval traffic related to commercial, fishing, and
shipyard activities, and, to a lesser extent, recreational navigation. On
the other hand, some of the world’s major maritime shipping routes
cross Portuguese jurisdictional waters, namely the ones connecting
Africa and North America with Northern and Southern Europe (through
the Mediterranean). Hence, coastal ecosystems in this country are fre-
quently exposed to contamination derived from this anthropogenic
activity, making Portugal a good example to track environmental
quality status, regarding TBT pollution trends along the years, and to
compare OSPAR and WFD classifications.
2. Methods
2.1. Study area
The study area extends along the Portuguese mainland coast, from
station (stn.) 1 – Vila Praia de Âncora – in the North, to stn. 45 – Alvor –
in the South-East (Fig. 1). As Portugal is a signing member of the
OSPAR Convention, this sampling area is included in the OSPAR region
IV (Bay of Biscay and Iberian Peninsula). This geographical range be-
longs to the same sub-region defined by the MSFD as part of the wider
marine region of North-East Atlantic Ocean, and is also under the jur-
isdiction of the WFD. The sampling of gastropods for imposex assess-
ment was performed along the coastline in sandy and rocky shores
covering 11 out of 15 coastal water bodies defined by WFD in Portugal.
Sampling also occurred inside estuarine areas on rocky and sandy-
muddy substrates with 7 transitional water bodies being monitored.
Historically, the major hotspots of TBT pollution are located inside
these transitional waters as they harbour commercial and fishing ports,
shipyards, marinas, and moorings (Santos et al., 2000; Barroso et al.,
2002; Sousa et al., 2009).
2.2. Imposex data
The evolution analysis of the Quality Status, regarding TBT pollu-
tion, along the Portuguese coast between 2000 and 2014 involved
imposex data presented in the following studies: regarding the year
2000 for both Nucella lapillus and Nassarius reticulatus imposex data was
taken from Barroso and Moreira (2002) and Barroso et al. (2002); for
the years 2003, 2006, 2008 and 2011, N. lapillus data was taken from
Galante-Oliveira et al. (2006, 2011) and Oliveira et al. (2012) while for
N. reticulatus it was taken from Sousa et al. (2005), Rato et al. (2009),
Sousa et al. (2009) and Laranjeiro et al. (2018). By means of grading the
masculinization level of females and assessing the impact of TBT pol-
lution on the ability of females to reproduce, as a proxy to predict
population abundance and perpetuity, only the data on the Vas De-
ferens Sequence Index (VDSI) were considered in the present work. The
VDSI was classified according to the scoring system proposed by: (i)
F. Laranjeiro et al.
Fig. 1. Map of the Portuguese coast showing sampling sites location (1–45),
main harbours (black dots) and respective vessel-related activities (indicated by
square symbols according to the bottom legend).
Gibbs et al. (1987) for N. lapillus and (ii) Stroben et al. (1992a) for N.
reticulatus. This index was chosen for the analysis because it is less af-
fected by confounding factors such as shell size, season of sampling,
distance to capsules clusters (Galante-Oliveira et al., 2010) or the pre-
sence of other contaminants (Oehlmann et al., 2000; Santos et al.,
2008), than other commonly used imposex indices such as those based
on Penis Length or Percentage of Imposex. In addition, VDSI allows a
direct evaluation of the population reproductive capacity which is of
foremost relevance within the European directives context (Laranjeiro
et al., 2015b).
2.3. Environmental quality classification
The temporal evolution of the environmental quality status re-
garding TBT pollution along the Portuguese coast was assessed by
comparing two different imposex-based criteria. Both classification
systems are presented on the top of Fig. 2. One was established by
OSPAR after imposex was considered a mandatory element under OS-
PAR’s CEMP (OSPAR, 2004). Assessment classes were defined based on
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Ecological Indicators 93 (2018) 966–974
intervals of VDSI values observed in N. lapillus, i.e., sites are classified
according to the degree of imposex severity evidenced, integrating the
biological effects with predicted TBT concentrations in the environ-
ment. According to OSPAR, the EcoQO is achieved when N. lapillus
presents VDSI < 2.0 (Class A or B), which indicates exposure to TBT
concentrations below the EAC defined as 0.1 ng TBT/L. This means that
adverse effects in the more sensitive taxa caused by chronic exposure to
TBT are unlikely to occur if concentrations are below the EAC. The
EcoQO is not achieved for VDSI ≥ 2.0, a situation that comprises the
following classes: i) Class C (2.0 ≤ VDSI < 4.0) represents gastropod
exposure to environmental levels of TBT higher than the defined EAC,
which means that there is a risk of adverse effects in the more sensitive
taxa; ii) Class D (4.0 ≤ VDSI < 5.0) where a dog-whelk population
with this VDSI level has its reproductive capacity severely affected in-
dicating high TBT concentrations in the environment; iii) Class E
(VDSI ≥ 5.0) indicates that N. lapillus populations are unable to re-
produce due to female sterility; iv) Class F classifies areas where the
more sensitive gastropod species are absent due to TBT pollution
(OSPAR, 2004). These classes were also established for other species
(ex.: N. reticulatus), taking into account VDSI regressions between these
species and that of dog-whelks collected at the same locations (see
Fig. 2). The use of two common bioindicators allows a more robust
spatial assessment as N. lapillus might be absent at some sites either due
to pollution or to habitat characteristics.
The alternative WFD criteria was proposed in a previous work
(Laranjeiro et al., 2015b) in order to integrate imposex monitoring
assessment within the WFD, since TBT is also regarded as a priority
hazardous substance under this directive. This proposal is based on the
previous criteria for N. lapillus but with some alterations to meet WFD
demands and objectives. Therefore, to proceed according to the WFD,
imposex values are computed to EQR through the equation:
(M − O)
EQRimposex = M , where M is the maximum VDSI score that a po-
pulation may attain and O is the observed VDSI value for a population
at one given site. This will render a value between 0 and 1 as demanded
by the WFD to guarantee comparability between EU member states in
all biological monitoring results. According to our proposal, the VDSI
criteria for the WFD should be centred in the impact of pollution on
population abundance and community diversity, and be regarded as
operational criteria to decide if a member state action is required or not.
Therefore, a “High” status is achieved for VDSI < 0.3, whilst a “Good”
ecological status under the WFD may be reached for VDSI as high as 3.0
in N. lapillus, which means that TBT has a negligible impact on the
population abundance due to the absence or scarcity of sterile females.
Moderate status correspond to 3.0 ≤ VDSI < 4.5, which means that
pollution is causing female sterilisation but there is still a high pro-
portion of non-sterilised females (> 50%) in the population that breed
and guarantees recruitment of new individuals; in this situation the
population presumably does not face a risk of extinction but the
abundance may eventually be reduced, which impels the member state
to take an action. The “Poor” status is defined for 4.5 ≤ VDSI ≤ 6 when
more than 50% of the females are sterilised, which will probably reduce
population abundance and, in extreme cases, will certainly drive the
population to extinction due to lack of breeding and recruitment. Fi-
nally, the “Bad” ecological status refers to a situation where N. lapillus
or other most sensitive species became extinct due to TBT pollution. As
can be seen, the number of Ecological Quality classes is reduced to five,
instead of six as recommended by OSPAR (see Fig. 2). Our previous
study also included the use of other bioindicators besides N. lapillus in
order to cover a wider diversity of habitats and to expand the mon-
itoring area. Therefore, a correspondence between N. lapillus and N.
reticulatus VDSIs was established through the same methods used by
OSPAR (see Fig. 2). For an extended description of the rationale used to
set each Ecological Class limits see Laranjeiro et al. (2015b).
F. Laranjeiro et al. Ecological Indicators 93 (2018) 966–974

Fig. 2. Classification of the ecological quality status of the Portuguese coast, regarding TBT pollution, in 2000 and 2014 using two gastropod bioindicator species –
Nucella lapillus and Nassarius reticulatus – and the two different criteria shown in the top table – the OSPAR Assessment Criteria (OSPAR, 2004) and the WFD proposed
Ecological Quality classification by Laranjeiro et al. (2015b). “At least Good” means that can be either High or Good status, while “At best Poor” means that can be
either Poor or Bad status (it is not possible to distinguish using N. reticulatus).

969
F. Laranjeiro et al.
3. Results
Comparison of imposex severity in 2000 and 2014 clearly shows a
decreasing trend (Fig. 2), as already reported in a previous study
(Laranjeiro et al., 2018), which indicates the improvement of the
quality status along the Portuguese coast; however, when comparing
the two quality criteria – OSPAR and WFD – some differences are de-
picted, as seen in Fig. 2. Classifications presented in this figure were
based on the VDSI and EQR data obtained at each station that can be
found in Supplementary material (Table 1). For N. lapillus, in 2000 the
OSPAR’s EcoQO is only achieved at 2 stations (27 and 43) with the
others varying between assessment classes C (6 sites) and D (6 sites). In
2014, the EcoQO is reached at all the stations, except in stn. 39 clas-
sified as C. According to our previous proposal for WFD, most EQRs
determined for N. lapillus in 2000 are below 0.5 (Table 1), indicating a
major deviation from undisturbed conditions caused by TBT. Similarly
to the OSPAR classification, only 2 stations (27 and 43) reached the
“Good” environmental status with the remainder presenting “Mod-
erate” status. In 2014, EQR values are close to 1 at all surveyed sites
implying a “Good” status condition (“High” status at stn. 17), except
stn. 39 that maintains the “Moderate” status.
Regarding N. reticulatus, in 2000 and according to the OSPAR
classification, only 2 stations (14 and 43) reached the EcoQO, with the
others portraying worse classification: 5 stations in Class C; 11 in D; 16
in E. An improvement in the populations’ condition was registered in
2014, as 18 stations reached the EcoQO while the others were classified
as Class C (10 sites), D (5 sites), and E (3 sites). A similar picture is
obtained with the WFD classification though, in 2000, 4 stations (14,
37, 39 and 43) had already met WFD objectives by presenting “Good”
or “at Least Good” environmental status. The “at Best Poor” status, the
worse classification given by this species, was found at 24 stations,
whilst the “Moderate” status was diagnosed at other 6. An improvement
in the environmental status is observed in 2014: “Good” or “at Least
Fig. 3. Distribution of the classification classes given by N. lapillus and N. reticulatus for sites where they live sympatrically, according to (A) OSPAR Assessment
Criteria (OSPAR, 2004) and (B) WFD proposal by Laranjeiro et al. (2015b). N = number of stations analysed.
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Good” status is met at 28 stations, while 8 failed to reach this objective,
with 6 of them still presenting the “at Best Poor” status.
Fig. 3 shows the distribution of the quality classes according to
OSPAR and WFD defined by each species at sites where they both live
sympatrically. By the analysis of this figure it is possible to observe that
the netted-whelks indicate Good Ecological Status in 2014 for all sites
analysed, whilst some sites fail to reach the OSPAR EcoQO. Moreover, it
is also perceived some differences between species classification: im-
posex levels in N. lapillus decrease before the N. reticulatus ones; at the
end, in 2014, all stations reach “Good” in the WFD classification ac-
cording to the nassariid, but some fail this objective according to the
muricid. Considering all the years, both species retrieve the same
classification for a given site 53% of the times with OSPAR classifica-
tion, and 56% of the times with WFD classification (in this analysis the
two upper WFD classes – “High” and “At Least Good” – were grouped as
“Good”). However, bigger difference can be depicted between classifi-
cations regarding the agreement on attaining the quality objectives and
it seems to be a better accordance when using the WFD proposal: while
both species, collected at the same site at a given moment, achieve the
OSPAR EcoQO in 39% of the times, bioindicators reach simultaneously
the good quality objective in 53% of the times along the years.
4. Discussion
TBT pollution has been a concerning issue all over the world
(Alzieu, 1991; Horiguchi et al., 1995; Gibbs and Bryan, 1996), a por-
trait that can be perceived at the Portuguese coast in the year 2000
(Fig. 2). By then, both N. lapillus and N. reticulatus evidenced a wide
dissemination of TBT pollution throughout that area, and at con-
centrations that generally produced low environmental quality status.
This situation was facilitated by the inefficacy of the legal restrictions
on TBT usage along the Portuguese coast at that time (EEC directive 89/
677), as this chemical could still be used in ships > 25 m in 2000
F. Laranjeiro et al.
(Barroso and Moreira, 2002; Santos et al., 2002). However, later re-
strictions banning TBT in all ships seemed to finally allow a reduction of
TBT pollution (Sousa et al., 2009; Galante-Oliveira et al., 2011;
Laranjeiro et al., 2018), which is confirmed by the imposex levels de-
cline registered in both species till 2014, improving the quality status
according to both OSPAR and WFD criteria. The data obtained for N.
lapillus – mainly distributed along the open coast – indicate that only
one station (stn. 39) did not reach the OSPAR EcoQO or the WFD Good
Quality Status regarding TBT pollution in 2014. The data obtained for
N. reticulatus retrieve a slightly different picture for the same geo-
graphical area: the EcoQO/Good Ecological Status is not achieved in
several stations in 2014. Even if different, this picture does not con-
tradict the status evidenced by N. lapillus, since the N. reticulatus im-
posex levels registered at the open coast also reached the EcoQO/Good
Quality Status. However, due to its distribution, the netted-whelk can
be found near the hotspots of TBT pollution already identified along the
Portuguese coast where N. lapillus does not occur, which allows the
assessment of a wider and diversified geographical area.
The WFD demands that waters reach a minimum of “Good status”
by the end of 2015, otherwise a member state action is required to
improve the condition of water bodies through ‘River Basin
Management Plans’. The current work identified many sites along the
Portuguese coast where bioindicators exhibit high levels of imposex,
indicating that the “Good status” was not reached at all sampled loca-
tions, according to the proposed WFD classification. These sites, parti-
cularly fishing ports and marinas, still register unacceptable levels of
TBT pollution, and so compulsory measures are needed to achieve the
“Good status” target quickly. Fresh inputs due to illegal use of TBT
antifouling paints may still occur – as it is probably the case of
Zambujeira do Mar, Porto das Barcas (stn. 39) – and must be inspected
and controlled by national authorities. However, one possible ubiqui-
tous source of TBT could be the sediments. As sediment acts as a long-
term sink of TBT and represents a source of pollution, new methods for
pollution control must target this compartment clean-up. These may
include: (i) mapping TBT levels in sediments by chemical methods and
evaluate bioavailability by specific bioassays (Laranjeiro et al., 2015a,
2017); (ii) localization of “hotspots” of contamination; (iii) removal of
contaminated sediments by dredging; (iv) decontamination of sedi-
ments by physical/biological processes. Following these remedial
dredging operations, monitoring must continue to assess the effective-
ness of the clean-up action. Clean-up procedures could be harmonised
with conventional dredging operations that are frequently performed in
ports and marinas to restore navigable depths.
It seems evident that imposex monitoring assessment will be more
robust when more species are considered due to their different dis-
tribution (Barroso et al., 2000; Laranjeiro et al., 2015b, 2018). How-
ever, it is observed that, when collected at the same sites, both of our
bioindicators may not retrieve exactly the same classification. This
discrepancy was also recently observed in Galicia (Spain) (Ruiz et al.,
2017) and in Ria de Aveiro (Laranjeiro et al., 2015b). Nevertheless,
what can be seen as a flaw in these imposex-based classifications might
be, in fact, a reflection of the natural variability induced by multiple
environmental and biological factors. For instance, the regressions used
to convert VDSI levels of one species into another, which are the basis
for the definition of the quality classes VDSI boundaries in N. reticulatus,
may need improvement in future studies but in all circumstances will
always comprise residual errors (Ruiz et al., 2017; Laranjeiro et al.,
2018). Additionally, TBT concentration in the water column, the en-
vironmental matrix to which N. lapillus is exposed to, may had evolved
along the years at different rates than in sediments, the preferred sub-
strate for the netted-whelk, since this matrix can act as a sink of TBT
(Sarradin et al., 1995; Langston et al., 2015), which will produce the
inter-species discrepancies observed. These species sensitivity to TBT
could be in fact similar when considering its relationship with OTs
tissue concentrations (Rial et al., 2018). However, it is the greater
build-up of TBT body burden by N. lapillus (e.g., due to the higher level
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Ecological Indicators 93 (2018) 966–974
of contamination of their food), previously described by Stroben et al.
(1992b), that will cause a greater response by the dog-whelk (Rial et al.,
2018), and therefore in imposex monitoring it is expected that N. la-
pillus will show higher imposex levels than N. reticulatus for the same
locations, rendering the muricid a more sensitive monitoring tool. De-
spite the cited limitations, more than 50% of the sites retrieve the same
classification using both species and, thus, the multispecies monitoring
still seems to be the best approach to monitor vast marine areas.
When comparing the OSPAR and WFD imposex-based classifica-
tions, it is important to understand their main differences. As previously
written, the major differences rely on the number of classes, and on the
premises and objectives that are behind the quality class definition.
Regarding the number of classes, OSPAR criteria for N. lapillus com-
prises six assessment classes, while for the WFD only five are defined.
This fact will consequently grant OSPAR classification more power to
discriminate TBT pollution levels among stations, as we can see in
Fig. 2. For instance, in the year 2000, when using N. lapillus, several
sites are classified as C or D according to OSPAR, but only as Moderate
according to the WFD. Another interesting difference between both
classifications, as seen in Fig. 3, is that a WFD’s Good Status is generally
achieved before the OSPAR EcoQO when using both species. This
makes OSPAR classification more conservative than the WFD, regarding
TBT pollution, which is in good agreement to the TBT chemical quality
objectives by OSPAR (EAC = 0.1 ng/L) and WFD (EQS = 0.2 ng/L).
These differences between classifications are then reflected in the
agreement rate between the two species environmental classifications.
Here, our proposal seems to display a better agreement than the OSPAR
classification in both attaining the quality objectives and retrieving the
same environmental classification, as shown in the results section.
Aiming the integration of TBT pollution monitoring into the MSFD,
as the main objective of the current work, it is better to first understand
the similarities and divergences between the different legislative pieces,
regarding TBT pollution. The OSPAR Convention defines TBT as a
priority substance and requires a comprehensive monitoring of the
chemical as it defines TBT’s EACs for water, sediment and biota, and
uses imposex as a mandatory tool under its CEMP; moreover, mon-
itoring should be performed in transitional waters and territorial seas of
the Contracting Parties, as well as i) the sea beyond and adjacent to the
territorial sea under the jurisdiction of the coastal state, and ii) the high
seas. In its turn, the WFD also defines TBT as a priority substance. This
directive sets an EQS for water only, and does not require any effect-
based monitoring tool; therefore, TBT monitoring is only mandatory
under the chemical quality elements in surface waters, which comprises
rivers, lakes, transitional and coastal waters (up to a distance of 1
nautical mile), and underground waters. Finally, only very recently the
criteria and methods to be used on the evaluation of the environmental
status under the MSFD were defined (Commission Decision (EU) 2017/
848). The hazardous substances assessment under descriptor 8
“Concentrations of contaminants are at levels not giving rise to pollution
effects” should primarily respect the EQS defined in the WFD within
coastal and territorial waters. However, unlike the WFD, the Decision
(EU) 2017/848 suggests the use of biomarkers, which should be agreed
at regional and subregional levels. Therefore, imposex monitoring, as a
mandatory element in OSPAR or HELCOM monitoring programs, has
the best predicates to be used in the definition of the GES under the
MSFD. The main objective of the MSFD is to achieve and maintain the
GES in all European marine regions. Still, the main question should be
which boundary should be applied to define the GES in the case of
imposex: the OSPAR assessment criteria (VDSI ≤ 2.0 for N. lapillus and
≤0.3 for N. reticulatus) or the WFD criteria proposed by our group
(Laranjeiro et al., 2015b) (VDSI ≤ 3.0 for N. lapillus and ≤0.8 for N.
reticulatus). This is of foremost importance since economic and en-
vironmental issues may arise if member states do not comply with the
same ecological objectives. Accounting the spatial overlap between the
WFD and the MSDF in coastal areas, and the already initiated effort by
OSPAR to align their assessment methodologies and objectives with the
F. Laranjeiro et al.
WFD, like the OSPAR’s EAC with WFD’s EQS (OSPAR, 2012), the ap-
plication of a more WFD-oriented classification would be more practical
and desirable for a better accordance between EU directives. The
boundary for good status proposed by Laranjeiro et al. (2015b) is in
better agreement with the EQS of 0.2 ng TBT/L for chemical monitoring
than the more restrictive OSPAR assessment criteria (0.1 ng TBT/L).
Moreover, the results presented in this work show a better agreement
between species when using the WFD proposal. Therefore, we re-
commend that a multi-species approach using imposex (or intersex) as a
biomarker of TBT pollution should be mandatory under the MSDF as
well, applying our proposal (Laranjeiro et al., 2015b) for the classifi-
cation of the environmental quality status. This would not suppose any
major changes on the methods employed by OSPAR, on which this
proposal is based; the only difference would be the criteria for the en-
vironmental status classification and the EQS for TBT that would have
to be aligned with the WFD’s. Still, it is important to note that, con-
sidering a literal assumption of descriptor 8, the MSDF share the fun-
damental objective of all legislative frameworks to finally eradicate TBT
pollution. However, it is important to understand that there is the need
to define more holistic and realistic ecological objectives. With this in
mind, one should not lessen the intent to eradicate TBT pollution, but as
it seems that this contaminant may still remain in the environment for
decades (Langston et al., 2015) more practical objectives are needed. In
fact, a VDSI ≤ 3.0 in N. lapillus as here proposed to be used under the
MSFD seems to be protective for three main reasons: i) it corresponds to
a TBT environmental concentration that would not utterly affect the
marine ecosystems (Laranjeiro et al., 2015b); ii) it is based on the
safeguard that TBT pollution is decreasing and populations will recover
with time; iii) it has enough power to impel member states to act im-
mediately if this boundary is exceeded.
The implementation of the MSFD has been seen as a difficult chal-
lenge, mainly due to the great dissimilarities between marine regions
that will probably make member states to differ on their environmental
objectives (Bertram and Rehdanz, 2013; Bellas, 2014; Crise et al.,
2015). The case here proposed is not an exception since TBT pollution
monitoring has been implemented for years in the Baltic Sea (HELCOM)
and North East Atlantic (OSPAR) marine regions, but are only now
recommended to be used in the Mediterranean Sea (MEDPOL) (UNEP/
MAP 2015, 2016), while no information is available for the monitoring
programs involving TBT in the Black Sea, under the Black Sea Com-
mission. For these regions, the implementation of imposex monitoring
under the MSFD would require additional work at the regional sea
conventions’ level to find adequate bioindicators. There are several
reports of imposex in the Mediterranean, with the most important
species being Hexaplex trunculus (Axiak et al., 1995; Terlizzi et al.,
1999), Bolinus brandaris (Solé et al., 1998; Chiavarini et al., 2003),
Stramonita haemastoma (Rilov et al., 2000; Chiavarini et al., 2003) and
Nassarius nitidus (Berto et al., 2007; Lahbib et al., 2011). For the Black
Sea, Rapana venosa seems to be one of the few bioindicators available
(Micu et al., 2009). However, no intercalibration between these species
and the ones used by OSPAR or HELCOM were found, i.e., the assess-
ment of imposex levels with these species living sympatrically in order
to obtain a regression between their VDSI levels, which would define
their TBT response relationship. Still, some studies evidenced that is
possible to find these species sympatrically: Hexaplex trunculus and
Nassarius reticulatus (Gibbs et al., 1997); Nassarius nitidus and Nassarius
reticulatus (Rodríguez et al., 2009). Regarding these last species,
Cacciatore et al. (2016) has already proposed an ecological classifica-
tion to be used under the WFD based on imposex expression in N. ni-
tidus. Even if this classification was developed based on different
principles from the ones proposed in Laranjeiro et al. (2015b), the
authors reported that the EQRs obtained for N. nitidus are in accordance
with this proposal for N. reticulatus, which would be expected due to
their similar sensitivity in terms of the Relative Penis Length (Rodríguez
et al., 2009). Very recently, other WFD-based ecological classifications
were proposed for Hexaplex trunculus (Erdelez et al., 2017; Cacciatore
972
Ecological Indicators 93 (2018) 966–974
et al., 2018) but they differ in their boundaries. Thus, it is evident that
intercalibration studies in the Mediterranean and the Black Sea, and
between these and North Atlantic species, are needed to define similar
quality objectives. Additionally, other regions outside Europe, with
high levels of TBT contamination (Mattos et al., 2017; Castro et al.,
2018; Lahbib et al., 2018), could benefit with the development of an
ecological based classification for a more effective TBT pollution
monitoring. In this sense, some works were already developed and
propose ecological classifications for the Northern Coast of Africa
(Lahbib et al., 2018) and Brazil (da Costa et al., 2017). But again, it is
our opinion that an intercalibration between species is essential to
achieve a correct and global imposex-based ecological classification.
5. Conclusions
The classification previously proposed to define the quality status of
the water bodies under the WFD is proved to be applicable under the
MSFD monitoring programs, since both directives share common ob-
jectives and geographical areas. Moreover, the possibility to use bio-
markers within MSFD, as well as the great experience in applying im-
posex as a biomarker for TBT pollution under the regional sea
conventions, makes this a suitable tool to be rapidly integrated within
the MSFD monitoring programs. Such an approach shows that the en-
vironmental status of the Portuguese coast, concerning TBT pollution,
improved considerably from 2000 to 2014, proving the effectiveness of
the latest legislation banning the use of TBT-based antifoulants.
Nevertheless, there are some sites where the Good status was still not
achieved in 2014, thus requiring proper action - such as the clean-up of
sediments or the control of illegal use of TBT - if the situation prevails.
Additionally, this work corroborates the need of using a multi-spe-
cies approach for imposex assessment as a consistent tool in TBT pol-
lution monitoring, as it retrieves a wider picture of pollution scenarios.
Nevertheless, further work on the calibration of imposex values be-
tween bioindicator species is needed in order to find a common ground
between all the European marine regions and, thus, properly assess its
environmental quality status under the MSFD.
Acknowledgments
Thanks are due for the financial support to CESAM (UID/AMB/
50017 – POCI-01-0145-FEDER-007638), to FCT/MCTES through na-
tional funds (PIDDAC), and the co-funding by the FEDER, within the
PT2020 Partnership Agreement and Compete 2020. Filipe Laranjeiro,
Isabel Benta Oliveira and Susana Galante-Oliveira were funded by FCT
and POPH/FSE funds through the research grants SFRH/BD/72298/
2010 (F.L.), SFRH/BD/71271/2010 (I.B.O.), SFRH/BPD/70368/2010
and SFRH/BPD/107813/2015 (S.G.-O.). Paula Sánchez-Marín was
supported by a postdoctoral grant from “Consellería de Cultura,
Educación e Ordenación Universitaria” of Xunta de Galicia.
References
Alzieu, C., 1991. Environmental problems caused by TBT in France: assessment, regula-
tions, prospects. Marine Environ. Res. 32 (1–4), 7–17.
Alzieu, C., 2000. Impact of tributyltin on marine invertebrates. Ecotoxicology 9 (1–2),
71–76.
Axiak, V., Vella, A.J., Micallef, D., Chircop, P., Mintoff, B., 1995. Imposex in Hexaplex
trunculus (Gastropoda: Muricidae): first results from biomonitoring of tributyltin
contamination in the Mediterranean. Mar. Biol. 121 (4), 685–691.
Barroso, C.M., Moreira, M.H., Gibbs, P.E., 2000. Comparison of imposex and intersex
development in four prosobranch species for TBT monitoring of a southern European
estuarine system (Ria de Aveiro, NW Portugal). Marine Ecol.-Progr. Ser. 201,
221–232.
Barroso, C.M., Moreira, M.H., 2002. Spatial and temporal changes of TBT pollution along
the Portuguese coast: inefficacy of the EEC directive 89/677. Mar. Pollut. Bull. 44 (6),
480–486.
Barroso, C.M., Moreira, M.H., Bebianno, M.J., 2002. Imposex, female sterility and orga-
notin contamination of the prosobranch Nassarius reticulatus from the Portuguese
coast. Marine Ecol.-Progr. Ser. 230, 127–135.
Bellas, J., 2014. The implementation of the Marine Strategy Framework Directive:
F. Laranjeiro et al.
shortcomings and limitations from the Spanish point of view. Marine Policy 50 (Part
A), 10–17.
Beiras, R., 2016. Assessing ecological status of transitional and coastal waters; current
difficulties and alternative approaches. Front. Marine Sci. 3.
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 (10–12), 425–435.
Bertram, C., Rehdanz, K., 2013. On the environmental effectiveness of the EU Marine
Strategy Framework Directive. Marine Policy 38, 25–40.
Borja, A., Elliott, M., Andersen, J.H., Cardoso, A.C., Carstensen, J., Ferreira, J.G.,
Heiskanen, A.-S., Marques, J.C., Neto, J.M., Teixeira, H., Uusitalo, L., Uyarra, M.C.,
Zampoukas, N., 2013. Good Environmental Status of marine ecosystems: What is it
and how do we know when we have attained it? Mar. Pollut. Bull. 76, 16–27.
Cacciatore, F., Noventa, S., Antonini, C., Formalewicz, M., Gion, C., Berto, D., Gabellini,
M., Brusà, R.B., 2016. Imposex in Nassarius nitidus (Jeffreys, 1867) as a possible in-
vestigative tool to monitor butyltin contamination according to the Water Framework
Directive: a case study in the Venice Lagoon (Italy). Ecotoxicol. Environ. Saf. 148,
1078–1089.
Cacciatore, F., Boscolo Brusà, R., Noventa, S., Antonini, C., Moschino, V., Formalewicz,
M., Gion, C., Berto, D., Gabellini, M., Marin, M.G., 2018. Imposex levels and butyltin
compounds (BTs) in Hexaplex trunculus (Linnaeus, 1758) from the northern Adriatic
Sea (Italy): ecological risk assessment before and after the ban. Ecotoxicol. Environ.
Saf. 147, 688–698.
Castro, Í.B., Iannacone, J., Santos, S., Fillmann, G., 2018. TBT is still a matter of concern
in Peru. Chemosphere 205, 253–259.
Chiavarini, S., Massanisso, P., Nicolai, P., Morabito, R., 2003. Butyltins concentration
levels and imposex occurrence in snails from the Sicilian coasts (Italy). Chemosphere
50, 311–319.
Crise, A., Kaberi, H., Ruiz, J., Zatsepin, A., Arashkevich, E., Giani, M., Karageorgis, A.P.,
Prieto, L., Pantazi, M., Gonzalez-Fernandez, D., Ribera d’Alcalà, M., Tornero, V.,
Vassilopoulou, V., Durrieu de Madron, X., Guieu, C., Puig, P., Zenetos, A., Andral, B.,
Angel, D., Altukhov, D., Ayata, S.D., Aktan, Y., Balcıoğlu, E., Benedetti, F.,
Bouchoucha, M., Buia, M.C., Cadiou, J.F., Canals, M., Chakroun, M., Christou, E.,
Christidis, M.G., Civitarese, G., Coatu, V., Corsini-Foka, M., Cozzi, S., Deidun, A.,
Dell’Aquila, A., Dogrammatzi, A., Dumitrache, C., Edelist, D., Ettahiri, O., Fonda-
Umani, S., Gana, S., Galgani, F., Gasparini, S., Giannakourou, A., Gomoiu, M.T.,
Gubanova, A., Gücü, A.C., Gürses, Ö., Hanke, G., Hatzianestis, I., Herut, B., Hone, R.,
Huertas, E., Irisson, J.O., İşinibilir, M., Jimenez, J.A., Kalogirou, S., Kapiris, K.,
Karamfilov, V., Kavadas, S., Keskin, Ç., Kideyş, A.E., Kocak, M., Kondylatos, G.,
Kontogiannis, C., Kosyan, R., Koubbi, P., Kušpilić, G., La Ferla, R., Langone, L.,
Laroche, S., Lazar, L., Lefkaditou, E., Lemeshko, I.E., Machias, A., Malej, A.,
Mazzocchi, M.G., Medinets, V., Mihalopoulos, N., Miserocchi, S., Moncheva, S.,
Mukhanov, V., Oaie, G., Oros, A., Öztürk, A.A., Öztürk, B., Panayotova, M.,
Prospathopoulos, A., Radu, G., Raykov, V., Reglero, P., Reygondeau, G., Rougeron,
N., Salihoglu, B., Sanchez-Vidal, A., Sannino, G., Santinelli, C., Secrieru, D., Shapiro,
G., Simboura, N., Shiganova, T., Sprovieri, M., Stefanova, K., Streftaris, N., Tirelli, V.,
Tom, M., Topaloğlu, B., Topçu, N.E., Tsagarakis, K., Tsangaris, C., Tserpes, G., Tuğrul,
S., Uysal, Z., Vasile, D., Violaki, K., Xu, J., Yüksek, A., Papathanassiou, E., 2015. A
MSFD complementary approach for the assessment of pressures, knowledge and data
gaps in Southern European Seas: the PERSEUS experience. Mar. Pollut. Bull. 95 (1),
28–39.
da Costa, M.B., Zamprogno, G.C., Otegui, M.B.P., de Morais, L.C., Pedruzzi, F.C., Dalbem,
G.B., da Silva, D.A., Resende, A.C.B., 2017. A temporal and spatial monitoring of
organotin pollution in a harborside region of Brazil by imposex and ecological quality
ratio using Leucozonia nassa. Environmental Monitoring and Assessment 189, 650.
EC, 2008. Directive 2008/105/EC of the European Parliament and of the Council of16
December 2008 on environmental quality standards in the field of waterpolicy,
amending and subsequently repealing Council Directives 82/176/EEC,83/513/EEC,
84/156/EEC, 84/491/EEC, 86/280/EEC and amending Directive2000/60/EC of the
European Parliament and of the Council. European Union.
Erdelez, A., Furdek Turk, M., Štambuk, A., Župan, I., Peharda, M., 2017. Ecological
quality status of the Adriatic coastal waters evaluated by the organotin pollution
biomonitoring. Mar. Pollut. Bull. 123, 313–323.
Galante-Oliveira, S., Langston, W.J., Burt, G.R., Pereira, M.E., Barroso, C.M., 2006.
Imposex and organotin body burden in the dog-whelk Nucella lapillus L. along the
Portuguese coast. Appl. Organomet. Chem. 20, 1–4.
Galante-Oliveira, S., Oliveira, I., Santos, J.A., Pereira, M.D.L., Pacheco, M., Barroso, C.M.,
2010. Factors affecting RPSI in imposex monitoring studies using Nucella lapillus (L.)
as bioindicator. J. Environ. Monit. 12, 1055–1063.
Galante-Oliveira, S., Oliveira, I., Ferreirra, N., Santos, J.A., Pacheco, M., Barroso, C.M.,
2011. Nucella lapillus L. imposex levels after legislation prohibiting TBT antifoulants:
temporal trends from 2003 to 2008 along the Portuguese coast. J. Environ. Monit. 13
(2), 304–312.
Gibbs, P.E., Bebianno, M.J., Coelho, M.R., 1997. Evidence of the differential sensitivity of
neogastropods to tributyltin (TBT) pollution, with notes on a species (Columbella
rustica) lacking the imposex response. Environ. Technol. 18 (12), 1219–1224.
Gibbs, P.E., Bryan, G.W., 1996. TBT-induced imposex in neogastropod snails: masculi-
nization to Mass Extinction Tributyltin: case study of an environmental contaminant.
Cambridge University Press.
Gibbs, P.E., Bryan, G.W., Pascoe, P.L., Burt, G.R., 1987. The use of the dog-whelk, Nucella
lapillus, as an indicator of tributyltin (TBT) contamination. J. Marine Biol. Assoc. U.K.
67 (3), 507–523.
Hagger, J.A., Jones, M.B., Lowe, D., Leonard, D.R.P., Owen, R., Galloway, T.S., 2008.
Application of biomarkers for improving risk assessments of chemicals under the
Water Framework Directive: a case study. Mar. Pollut. Bull. 56 (6), 1111–1118.
Horiguchi, T., Shiraishi, H., Shimizu, M., Yamazaki, S., Morita, M., 1995. Imposex in
973
Ecological Indicators 93 (2018) 966–974
Japanese gastropods (Neogastropoda and Mesogastropoda): effects of tributyltin and
triphenyltin from antifouling paints. Mar. Pollut. Bull. 31 (4–12), 402–405.
Lahbib, Y., Abidli, S., González, P.R., Alonso, J.I.G., Menif, N.T.-E., 2011. Potential of
Nassarius nitidus for monitoring organotin pollution in the lagoon of Bizerta (northern
Tunisia). J. Environ. Sci. 23 (9), 1551–1557.
Lahbib, Y., Abidli, S., Menif, N.T.E., 2018. Organotin pollution along the tunisian coasts:
status of imposex before and after the ratification of the anti-fouling systems (AFS)
convention. Springer International Publishing, Cham, pp. 1669–1671.
Langston, W.J., Pope, N.D., Davey, M., Langston, K.M., O’Hara, S.C.M., Gibbs, P.E.,
Pascoe, P.L., 2015. Recovery from TBT pollution in English Channel environments: a
problem solved? Mar. Pollut. Bull. 95 (2), 551–564.
Laranjeiro, F., Pérez, S., Navarro, P., Carrero, J.A., Beiras, R., 2015a. The usefulness of a
sediment bioassay with the gastropod Nassarius reticulatus in tributyltin monitoring
programs. Chemosphere 139, 550–557.
Laranjeiro, F., Sánchez-Marín, P., Galante-Oliveira, S., Barroso, C., 2015b. Tributyltin
pollution biomonitoring under the Water Framework Directive: proposal of a multi-
species tool to assess the ecological quality status of EU water bodies. Ecol. Ind. 57,
525–535.
Laranjeiro, F., Beiras, R., Barroso, C., 2017. The use of the biomarker imposex to assess
the environmental status of aquatic ecosystems regarding tributyltin pollution. Ecol.
Ind.
Laranjeiro, F., Sánchez-Marín, P., Oliveira, I.B., Galante-Oliveira, S., Barroso, C., 2018.
Fifteen years of imposex and tributyltin pollution monitoring along the Portuguese
coast. Environ. Pollut. 232, 411–421.
Law, R., Hanke, G., Angelidis, M., Batty, J., Bignert, A., Dachs, J., Davies, I., Denga, Y.,
Duffek, A., Herut, B., Hylland, K., Leonards, P., Lepom, P., Mehtonen, J., Piha Henna,
E., Roose, P., Tronczynski, J., Velikova V., Vethaak D., 2010. Marine Strategy
Framework Directive. Task Group 8 Report. Contaminants and Pollution Effects.
Lyons, B.P., Thain, J.E., Stentiford, G.D., Hylland, K., Davies, I.M., Vethaak, A.D., 2010.
Using biological effects tools to define Good Environmental Status under the
European Union Marine Strategy Framework Directive. Mar. Pollut. Bull. 60 (10),
1647–1651.
Martinez-Haro, M., Beiras, R., Bellas, J., Capela, R., Coelho, J.P., Lopes, I., Moreira-
Santos, M., Reis-Henriques, A.M., Ribeiro, R., Santos, M.M., Marques, J.C., 2015. A
review on the ecological quality status assessment in aquatic systems using com-
munity based indicators and ecotoxicological tools: what might be the added value of
their combination? Ecol. Ind. 48, 8–16.
Matthiessen, P., Gibbs, P.E., 1998. Critical appraisal of the evidence for tributyltin-
mediated endocrine disruption in mollusks. Environ. Toxicol. Chem. 17 (1), 37–43.
Mattos, Y., Stotz, W.B., Romero, M.S., Bravo, M., Fillmann, G., Castro, Í.B., 2017. Butyltin
contamination in Northern Chilean coast: is there a potential risk for consumers? Sci.
Total Environ. 595, 209–217.
Micu, S., Comanescu G., Kelemen B., 2009. “The imposex status in Rapana venosa po-
pulation from Agigea littoral.” Analele stiintifice ale Universitătii Al. I. Cuza” Iasi, s.
Biologie animală: 133–137.
Oehlmann, J., Schulte-Oehlmann, U., Tillmann, M., Markert, B., 2000. Effects of endo-
crine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part
I: bisphenol A and octylphenol as xeno-estrogens. Ecotoxicology 9, 383–397.
Oliveira, I. B., Galante-Oliveira, S., Sousa, A. C. A. ,Takahashi, S., Tanabe, S., Thomas, K.,
Suter, M. J.-F., Barroso C.M., 2012. Imposex and organotin (OT) levels in Nucella
lapillus along the Portuguese coast: a re-survey in 2011. SETAC Europe 22nd Annual
Meeting, Berlin.
OSPAR, 2004. Provisional JAMP Assessment Criteria for TBT – Specific Biological Effects.
London.
OSPAR, 2012. Finding common ground – towards regional coherence in implementing
the Marine Strategy Framework Directive in the North-East Atlantic region through
the work of the OSPAR Commission: 34.
Rato, M., Ferreira, N., Santos, J., Barroso, C.M., 2009. Temporal evolution of imposex in
Nassarius reticulatus (L.) along the Portuguese coast: the efficacy of EC regulation
782/2003. J. Environ. Monit. 11, 100–107.
Rial, D., Bellas, J., Ruiz, J.M., 2018. Critical analysis of the relationship between imposex
and butyltin body burden in Nassarius reticulatus and Nucella lapillus. Environ. Pollut.
237, 523–530.
Ruiz, J.M., Carro, B., Albaina, N., Couceiro, L., Míguez, A., Quintela, M., Barreiro, R.,
2017. Bi-species imposex monitoring in Galicia (NW Spain) shows contrasting
achievement of the OSPAR Ecological Quality Objective for TBT. Mar. Pollut. Bull.
114, 715–723.
Rilov, G., Gasith, A., Evans, S.M., Benayahu, Y., 2000. Unregulated use of TBT-based
antifouling paints in Israel (eastern Mediterranean): high contamination and imposex
levels in two species of marine gastropods. Mar. Ecol. Prog. Ser. 192, 229–238.
Rodríguez, J.G., Borja, Á., Franco, J., García Alonso, J.I., Garmendia, J.M., Muxika, I.,
Sariego, C., Valencia, V., 2009. Imposex and butyltin body burden in Nassarius nitidus
(Jeffreys, 1867), in coastal waters within the Basque Country (northern Spain). Sci.
Total Environ. 407 (14), 4333–4339.
Santos, M.M., Vieira, N., Santos, A.M., 2000. Imposex in the Dogwhelk Nucella lapillus (L.)
along the Portuguese Coast. Mar. Pollut. Bull. 40, 643–646.
Santos, M.M., Ten Hallers-Tjables, C.C., Santos, A.M., Vieira, N., 2002. Imposex in Nucella
lapillus, a bioindicator for TBT contamination: re-survey along the Portuguese coast to
monitor the effectiveness of EU regulation. J. Sea Res. 48, 217–223.
Santos, M.M., Reis-Henriques, M.A., Guillot, R., Lima, D., Franco-Duarte, R., Mendes, I.,
Queirós, S., Castro, L.F.C., 2008. Anti-androgenic effects of sewage treatment plant
effluents in the prosobranch gastropod Nucella lapillus. Compar. Biochem. Physiol.,
Part C 148, 87–93.
Sarradin, P.M., Lapaquellerie, Y., Astruc, M., Latouche, C., Astruc, M., 1995. Long term
behaviour and degradation kinetics of tributyltin in a marina sediment. Sci. Total
Environ. 170, 59–70.
F. Laranjeiro et al.
Solé, M., Morcillo, Y., Porte, C., 1998. Imposex in the commercial snail Bolinus brandaris
in the northwestern Mediterranean. Environ. Pollut. 99 (2), 241–246.
Sousa, A., Laranjeiro, F., Takahashi, S., Tanabe, S., Barroso, C.M., 2009. Imposex and
organotin prevalence in a European post-legislative scenario: temporal trends from
2003 to 2008. Chemosphere 77 (4), 566–573.
Sousa, A., Mendo, S., Barroso, C.M., 2005. Imposex and organotin contamination in
Nassarius reticulatus (L.) along the Portuguese coast. Appl. Organomet. Chem. 19,
315–323.
Stroben, E., Oehlmann, J., Fioroni, P., 1992a. The morphological expression of imposex in
Hinia reticulata (Gastropoda: Buccinidae): a potential indicator of tributyltin pollu-
tion. Mar. Biol. 113, 625–636.
Stroben, E., Oehlmann, J., Fioroni, P., 1992b. Hinia reticulata and Nucella lapillus.
Comparison of two gastropod tributyltin bioindicators. Mar. Biol. 114, 289–296.
Terlizzi, A., Geraci, S., Gibbs, P.E., 1999. Tributyltin (TBT)-induced imposex in the
neogastropod Hexaplex trunculus in Italian waters: morphological aspects and eco-
logical implications. Ital. J. Zool. 66, 141–146.
Titley-O'Neal, C.P., Munkittrick, K.R., MacDonald, B.A., 2011. The effects of organotin on
female gastropods. J. Environ. Monit. 13 (9), 2360–2388.
UNEP/MAP, 2015. Main elements of the Integrated Monitoring and Assessment
Programme related to Ecological Objectives 5, 9. 10. United Nations Environment
Programme – Mediterranean Action Plan.
974
Ecological Indicators 93 (2018) 966–974
UNEP/MAP, 2016. Draft Integrated Monitoring and Assessment Guidance. United Nations
Environment Programme – Mediterranean Action Plan.
Vethaak, A.D., Davies, I.M., Thain, J.E., Gubbins, M.J., Martínez-Gómez, C., Robinson,
C.D., Moffat, C.F., Burgeot, T., Maes, T., Wosniok, W., Giltrap, M., Lang, T., Hylland,
K., 2017. Integrated indicator framework and methodology for monitoring and as-
sessment of hazardous substances and their effects in the marine environment.
Marine Environ. Res. 124, 11–20.
van Hoof, L., Hendriksen, A., Bloomfield, H.J., 2014. Sometimes you cannot make it on
your own; drivers and scenarios for regional cooperation in implementing the EU
Marine Strategy Framework Directive. Marine Policy 50 (Part B), 339–346.
Wernersson, A.-S., Carere, M., Maggi, C., Tusil, P., Soldan, P., James, A., Sanchez, W.,
Dulio, V., Broeg, K., Reifferscheid, G., Buchinger, S., Maas, H., Van Der Grinten, E.,
O’Toole, S., Ausili, A., Manfra, L., Marziali, L., Polesello, S., Lacchetti, I., Mancini, L.,
Lilja, K., Linderoth, M., Lundeberg, T., Fjällborg, B., Porsbring, T., Larsson, D.,
Bengtsson-Palme, J., Förlin, L., Kienle, C., Kunz, P., Vermeirssen, E., Werner, I.,
Robinson, C., Lyons, B., Katsiadaki, I., Whalley, C., den Haan, K., Messiaen, M.,
Clayton, H., Lettieri, T., Carvalho, R., Gawlik, B., Hollert, H., Di Paolo, C., Brack, W.,
Kammann, U., Kase, R., 2015. The European technical report on aquatic effect-based
monitoring tools under the water framework directive. Environ. Sci. Eur. 27 (1),
1–11.