The unpredictable effects of mixtures of androgenic and estrogenic chemicals on fish early life.
ABSTRACT Estuarine shallow areas and coastal lagoons are known to receive and concentrate multiple inputs, either from land, rivers or coastal areas, being intensively impacted by chemical contamination, namely endocrine disrupting chemicals (EDCs). Despite the ubiquitous coexistence of several classes of EDCs in most of these aquatic ecosystems, there is still limited information regarding their combined effects. Furthermore, given the immediate implications for population dynamics, the available laboratory studies almost invariably focus on very specific life history stages, such as embryonic development or reproduction, thus creating a gap on our knowledge of what happens in between. During this 'intermediate phase', the newborn larvae and juveniles face numerous challenges whose outcome may impair reproduction or even survival. The black-striped pipefish, Syngnathus abaster, member of the Syngnathidae family (comprising pipefish, seahorses and seadragons), usually breeds in coastal areas such as estuaries, where its newborns are immediately exposed to EDCs. Given the ongoing decline of pipefish populations, together with the observed shrinkage and fragmentation of seagrass meadows, known to be impacted by EDCs, a first reasonable question to address is if pipefish newborns respond to environmentally relevant concentrations of ubiquitous EDCs, either single or in combination. Hence, a seven days exposure experiment to the estrogenic chemical ethinylestradiol (EE(2)) and the androgenic chemical tributyltin (TBT), single and in binary mixtures, was conducted. Selected behavioural (e.g. predator avoidance) and developmental variables (e.g. growth) were monitored in pipefish juveniles after EDCs insult. The obtained results indicate that EE(2), TBT, or their combined exposure, do impact pipefish early life. However, the pattern of results emerging from the measured variables clearly indicates that mixtures significantly modulate newborn responses in distinct ways when compared to individual chemical's exposure. These findings further demonstrate the importance of addressing the issue of chemical mixtures of pollutants acting through dissimilar mode of action. Independently of all the observed response variations, an ultimate conclusion seems certain: EE(2) and TBT, single or in combination, induce disruption patterns able to imbalance pipefish survival. Since these (as well as other) contaminants are present in estuarine areas, profound implications in population structure could be expected, ranging from a decrease in recruitment to a disruption of sexual selection. Inexorably, these stressors simultaneously operate in already declining populations.
The unpredictable effects of mixtures of androgenic and estrogenic chemicals
on fish early life
M.P. Sárriaa, M.M. Santosa, M.A. Reis-Henriquesa, N.M. Vieiraa,b, N.M. Monteiroc,d,⁎
aCIMAR/CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 177, 4050-123 Porto, Portugal
bDepartamento de Biologia, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal
cCIBIO, Research Centre in Biodiversity and Genetic Resources, Campus Agrário de Vairão, R. Padre Armando Quintas, 4485-661 Vairão, Portugal
dCEBIMED, Faculdade de Ciências da Saúde da Universidade Fernando Pessoa, Rua Carlos da Maia, 296, 4200-150 Porto, Portugal
a b s t r a c t a r t i c l ei n f o
Received 1 September 2010
Accepted 8 November 2010
Available online 27 November 2010
Early life history
Estuarine shallow areas and coastal lagoons are known to receive and concentrate multiple inputs, either from
land, rivers or coastal areas, being intensively impacted by chemical contamination, namely endocrine
disrupting chemicals (EDCs). Despite the ubiquitous coexistence of several classes of EDCs in most of these
aquatic ecosystems, there is still limited information regarding their combined effects. Furthermore, given the
immediate implications for population dynamics, the available laboratory studies almost invariably focus on
very specific life history stages, such as embryonic development or reproduction, thus creating a gap on our
knowledge of what happens in between. During this ‘intermediate phase’, the newborn larvae and juveniles
face numerous challenges whose outcome may impair reproduction or even survival.
The black-striped pipefish, Syngnathus abaster, member of the Syngnathidae family (comprising pipefish,
seahorses and seadragons), usually breeds in coastal areas such as estuaries, where its newborns are
immediately exposed to EDCs. Given the ongoing decline of pipefish populations, together with the observed
shrinkage and fragmentation of seagrass meadows, known to be impacted by EDCs, a first reasonable question
to address is if pipefish newborns respond to environmentally relevant concentrations of ubiquitous EDCs,
either single or in combination. Hence, a seven days exposure experiment to the estrogenic chemical
ethinylestradiol (EE2) and the androgenic chemical tributyltin (TBT), single and in binary mixtures, was
conducted. Selected behavioural (e.g. predator avoidance) and developmental variables (e.g. growth) were
monitored in pipefish juveniles after EDCs insult. The obtained results indicate that EE2, TBT, or their
combined exposure, do impact pipefish early life. However, the pattern of results emerging from the
measured variables clearly indicates that mixtures significantly modulate newborn responses in distinct ways
when compared to individual chemical's exposure. These findings further demonstrate the importance of
addressing the issue of chemical mixtures of pollutants acting through dissimilar mode of action.
Independently of all the observed response variations, an ultimate conclusion seems certain: EE2and TBT,
single or in combination, induce disruption patterns able to imbalance pipefish survival. Since these (as well
as other) contaminants are present in estuarine areas, profound implications in population structure could be
expected, ranging from a decrease in recruitment to a disruption of sexual selection. Inexorably, these
stressors simultaneously operate in already declining populations.
© 2010 Elsevier Ltd. All rights reserved.
Endocrine disrupting chemicals (EDCs), a group of exogenous and
endogenous compounds able to interfere with hormone-controlled
physiological processes, are increasingly widespread in the environ-
ment. The adverse effects of EDCs upon the fauna and flora, together
with the persistence in the natural ecosystems of several classes of
EDCs (Trussell, 2001), fully justified the establishment of a growing
public concern (COM(1999)706; WHO, 2002; EPA(2009)EDSP),
which was rapidly translated into a research increase in endocrine
disruption mediated by contaminant exposure (e.g. crustaceans:
Rodríguez et al., 2007; gastropods: Ellis and Pattisina, 1990;
amphibians and reptiles: Crain and Guillette, 1998; Kloas, 2002;
fish: Fent et al., 1998; Thibaut and Porte, 2004; Mills and Chichester,
2005; Rempel and Schlenk, 2008; Ankley et al., 2009; birds and
mammals: Ottinger and Saal, 2002; humans: Murray et al., 2001;
Waring and Harris, 2005; Caserta et al., 2008). From all the impacted
ecosystems, the aquatic environments are amongst the most
thoroughly studied, given the increasing concentration of chemicals
either from point and non-point sources (Sumpter, 2005; Matthiessen
et al., 2006).
Environment International 37 (2011) 418–424
⁎ Corresponding author. Tel.: +35 1252660411; fax: +35 1252661780.
E-mail address: firstname.lastname@example.org (N.M. Monteiro).
0160-4120/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/envint
The best-documented example of endocrine disruption in wildlife
is still the masculinisation of neogastropod females (imposex) caused
by the antifoulingagenttributyltin (TBT) (Santoset al., 2005). The TBT
masculinisation effect has also been recognized in fish (Mcallister and
Kime, 2003; Shimasaki et al., 2003; Santos et al., 2006). In fact, TBT is
now known to affect several animal taxa (Moore et al., 1991; Fisher et
al., 1999; Jensen et al., 2004; Zhang et al., 2008; Guo et al., 2010;
Revathi and Munuswamy, 2010), an already anticipated outcome
given the persistent nature of this xenobiotic together with its wide
distribution, being easily detected both in freshwater, estuarine and
coastal ecosystems (Fent, 1996). Besides TBT, other contaminant
classes have also been thoroughly studied, with a particular incidence
on estrogenic chemicals (Jaser et al., 2003; Vosges et al., 2008;
Gyllenhammar et al., 2009; Soares et al., 2009; Saaristo et al., 2010). In
natural ecosystems, EDCs occur in complex mixtures such as
estrogenic, androgenic, anti-estrogenic or anti-androgenic. Hence,
ecotoxicological evaluations of a single contaminant may not reflect
the multitude of antagonistic or synergistic stimuli that wildlife
species surely faces (Lopes et al., 2005). Recent studies have clearly
demonstrated the importance of addressing the issue of mixture
effects of EDCs if we want to improve risk assessment of this class of
chemicals. Today, it is well established that mixtures of estrogenic
chemicals(ECs),acting througha similarmode ofaction, operate in an
additive manner. In fact, mixtures of ECs elicited biological responses
for concentrations that were known to be ineffective when the
selected compounds were administered alone (Silva et al., 2002; Brian
et al., 2005; Correia et al., 2007). In contrast to the somewhat
predictable effects of mixtures of ECs, the information available on
mixture effects of EDCs that act through dissimilar modes of action
seems to be a rather unexplored ground (Spurgeon et al., 2010).
Interestingly, despite the ubiquitous coexistence of both estrogenic
and androgenic contaminants in aquatic ecosystems, only a few
published studies have addressed their combined effects in fish
(Santos et al., 2006; Micael et al., 2007; Greco et al., 2007; Sun et al.,
While the consequences of EDCs exposure, single or in combina-
tion, have been mainly addressed either during the embryonic
development or reproductive phase, given the immediate implica-
tions for population dynamics, attention must be devoted also to the
time period that lies in between. During this phase, the newborn
larvae, or juvenile, face numerous challenges such as feeding or
escaping predation. In fact, since predation is the main cause of early
mortality (Cushing, 1974), any disturbance on the basic escape
mechanisms, including startle responses or locomotion performance,
potentially impact population structure more than imbalances
occurring during reproduction (Houde, 1987). All of the above-cited
behaviours can be severely impacted by EDCs (Little et al., 1990;
Carlson et al., 1998; Zhou and Weis, 1998; McGee et al., 2009).
However, data regarding effects on fish larvae movement and
development under EDC exposure are still scarce, and even more so
when xenobiotics are administered in mixtures, since the resulting
interactions are vastly unknown.
Estuarine shallow areas and coastal lagoons are amongst the most
productive aquatic habitats (Kneib, 1997). These particular environ-
ments perform a relevant ecological function, serving as spawning
and nursery areas for numerous fish species (Franco et al., 2006).
Interestingly, these areas are also intensively impacted by chemical
contamination (Ridgway and Shimmield, 2002; Pojana et al., 2007).
Specifically, Zostera sp. seagrass meadows have been recurrently
reported as impacted by EDC exposure (Francois et al., 1989;
Chesworth et al., 2004). This marine angiosperm, as well as related
aquatic vegetation, has declined extensively worldwide (Martin et al.,
2010), probably due to the ongoing anthropogenic pressure, which is
causing increased fragmentation and degradation of estuarine
habitats. This habitat loss is especially critical for some syngnathid
species, already considered globally threatened given the declining
and Vincent, 2005), it remains to be addressed if EDC impacts not only
the estuarine primary production but also the syngnathid populations.
The family Syngnathidae (seahorses, seadragons and pipefishes)
presents unique reproductive characteristics, namely male pregnancy
which occurs in a specialized brooding area, the marsupium (Monteiro
et al., 2005). Since some species, such as black-striped pipefish,
Syngnathus abaster Risso (1826), migrate to estuarine areas during the
breeding season, it can predicted that these populations, comprising
both reproductively mature individuals as well as their newborns, may
be at risk. Even though there is virtually no information on endocrine
disruption effects on syngnathids, this family, and especially S. abaster,
can be viewed as an interesting model for the assessment of ecosystem
health due to several factors: i) it is a species with a considerable life
span, ii) it migrates to estuarine areas in order to breed, iii) male
pregnancy and larvae release from the marsupium occurs within the
estuarine zone, and iv) the first stages of development, from larvae to
ready to migrate juveniles, occurs in the estuary, especially within
seagrass meadows.If we take into consideration that thepopulationsof
S. abaster are also in decline (Pombo et al., 2002), a first reasonable
question to address is if pipefish, especially newborns, are vulnerable to
environmentally relevant concentrations of EDCs. Furthermore, since
most of the current research is mainly focused on the action of EDCs on
reproduction, it seems important to evaluate non-reproductive param-
eters, such as growth and activity patterns, that can act as rapid-
measurable variablesable toreflect theeffectsof EDCs ata muchearlier
stage of development. Any maladaptive alteration on larvae/juvenile
morphology or behaviour can be translated into increased mortality
rates,whichcan greatly contribute to the observedpopulationdeclines.
Thus, in the present study, special attention was devoted to the larvae
response to the presence of a potential predator (the mosquitofish,
Gambusia affinis), given that predation modulates early mortality,
influencing fish larvae density that reach adulthood.
Finally, considering that newborn syngnathids immediately
contact with TBT and the synthetic EC ethinylestradiol (EE2), two
molecules known to sometimes elicit antagonic responses in marine
organisms, it is important to clarify not only the hazard posed by each
chemical independently, but also recognize the interactions created
under mixture exposures. Furthermore, by simultaneously monitor-
ing several distinct variables, either behavioural or morphological, it
will be possible to determine if EDC mixtures globally induce any type
of “predictable” alteration patterns in newborn pipefish.
2. Materials and methods
17-α-ethinylestradiol [C20H24O2] (EE2, purity≥98%, Sigma-
Aldrich); tributyltin chloride [C12H27ClSn] (TBTCl, purity 97%,
Sigma-Aldrich); acetone (pro-analysi, purity≥99.8%, Merck,
2.2. Biological model
Syngnathus abaster (Pisces; Syngnathidae) is a small brown-green
benthonic euryhaline pipefish with a restricted distribution that
includes the Mediterranean, Black Sea and the Atlantic coast of
southwest Europe up to southern Biscay (Dawson, 1986) (Fig. 1). This
pipefish occurs either in coastal areas or in brackish and fresh waters
habitats (Cakic et al., 2002), between 0.5 and 5 m, within a
temperature range of 8 to 24 °C (Dawson, 1986). Males exhibit a
brood pouch located ventrally on the tail (Urophori; Herald, 1959)—
that consists of two skin folds that contact medially with their free
edges. Females, usually larger than males deposit the oocytes in this
specialized incubating area (marsupium), where the male fertilizes
M.P. Sárria et al. / Environment International 37 (2011) 418–424
them. Newborn pipefish abandon the marsupium, exhibiting a
uniform dark colouration that is similar to that of the adults, and no
further care is provided (Silva et al., 2006).
2.3. Normalization of prime conditions
88 pregnant males were captured, during the breeding season, at
Ria de Aveiro, Portugal (40° 39′ N; 8° 39′ W) and initially maintained
in a 150 L aquarium. Since parental quality or spawning investment
may influence larvae morphology and behaviour (see Silva et al.,
2009), special care was taken to ensure that the selected newborns
were randomly selected from the hatchlings of a pool of 20 different
males(all ofwhichwithinthesamestageofpregnancy,as determined
by visual inspection). Gambusia affinis (Poeciliidae) adult females
were also collected at Aveiro, using a hand net. The mosquitofish were
maintained in separate aquaria, but in similar conditions as those
described for S. abaster. Since the role of the mosquitofish was to be
used as a potential predator (visual stimulus), the individuals were
kept fasting, in order to avoid a complete lack of interest in S. abaster
2.4. EDCs exposure
Five days after exiting the marsupium, 180 newborns were
randomly selected from a pool of approximately 1000 individuals.
Juveniles, in groups of 10, were transferred to 5 L aquaria and exposed
to EE2 (nominal concentrations: 3 and 9 ng/L), TBTCl (nominal
concentrations: 10 and 50 ngSn/L) as well as all possible combina-
tions, during seven days. Acetone was used as organic solvent
(0.000114%). Thus, a total of 9 treatments, including control, were
replicated twice and maintained in closely controlled physicochem-
ical conditions (25‰ artificial salt water; 14:10 h photoperiod; 21±
1 °C). 75% water renovation was conducted every other day.
Newborns were fed daily with Brachionus plicatilis and 48 h Artemia
2.5. Video recording and collected data
At the end of the exposure (day 7), the juveniles were randomly
assigned to two 30 L aquaria. These aquaria were physically divided
into two asymmetrical sections. A larger section (about two thirds)
contained the pipefish juveniles while the smaller section contained
the potential predator(G. affinis). The glass division that separatedthe
two sections allowed the juveniles to see the mosquitofish, but
prevented any chemical communication, meaning that the pipefish
were not receiving any chemical stimulus from the G. affinis females
that, in turn, were not subject to any kind of EDC exposure. The
stimulus fish were initially kept from sight by an opaque blind.
Given that newly born S. abaster tend to adopt a benthic
distribution (Silva et al., 2006) while avoiding potential predators, it
could be expected that newborns would tend to occupy the quadrant
of the aquaria located near the bottom and further away from the
glass division. Thus, this was the area selected for the registry of the
selected variables: (1) number of movement bursts and (2) time
spent on this more secluded area. Ten minutes after juvenile
transference, the visual barrier was slowly removed and the juvenile
S. abaster behaviour was digitally video-recorded (Sony DCR-SR36)
for ten minutes.
Preliminary observations showed that juveniles individually
placed in one aquarium tended to adopt abnormal behaviours
(ranging from complete immobility to random patterns of locomo-
tion). Thus, it was decided to mimic the conditions observed in the
wild, where juveniles are present in groups, by placing a group of five
individuals together, while only registering the behaviour of three.
The response pattern obtained seemed to be consistent across
treatments (see Results section), thus ruling out the effects of a
hypothetical pseudo-replication, where the behaviour of one larvae
would strongly influence the response patterns of the others.
At the end of the behavioural experiments, all 180 juveniles were
photographed and measured (LT) to the nearest millimetre. Measure-
mentsof the eye,pupil anddorsalfin (see Fig.2a)were obtainedusing
UTHSCSA IMAGE TOOL (v3.00). These variables were selected given
their potential implications in vital processes such as swimming
performance (predator escape or prey capture) or vision (predator
and prey recognition).
2.6. Statistical analysis
All assumptions were met prior to data analysis. ANOVA model
(two factors: EE2and TBT, each with three levels: zero, lower and
higher EDC concentration) was conducted to determine if pipefish
growth was affected by the selected contaminants, using body length
as the dependent variable. To avoid biases associated with covariates,
two ANCOVAs were conducted to determine the influence of the EDCs
Fig. 1. Geographical distribution of Syngnathus abaster (as presented by Dawson, 1986).
M.P. Sárria et al. / Environment International 37 (2011) 418–424
on the pupil surface and dorsal fin length (eye surface and body
length, respectively, were used as co-variables). The two behavioural
variables considered (number of movement bursts and time spent on
the secluded area) were used as dependent variables in a MANOVA
design [two factors: EE2and TBT (each with three levels: zero, lower
and higher EDC concentration)], to test for differences between
treatments. Post hoc comparisons were conducted using Student–
Newman–Keuls (SNK). A P value of 0.05 was used for significance
testing. Analyses were performed in STATISTICA (v7).
3.1. Larvae mortality
Given the team's past experience in rearing S. abaster juveniles in
aquaria, the juvenile mortality during the experimental period was
considered reasonably negligible (7 out of 180 juveniles, scattered
among 6 aquaria out of 18).
3.2. EDCs exposure and morphological development
in juvenile growth, a significant interaction was observed between
the selected EDCs (Table 1A). Even though the obtained pattern is of
difficult interpretation, a general trend suggests that while EE2seems
to depress growth, TBT produces the opposite results. Nevertheless, it
can also be perceived that growth is diminished when juveniles are
(SNK post-hoc test, data not shown), suggesting that TBT effects in
juvenile growth are counteracted when in presence of the highest EE2
concentrations (Fig. 2b).
When considering the effects of the selected EDCs on the surface of
the fish pupil, when adjusted for global eye surface (see Fig. 2c), the
ANCOVA results show a significant interaction between the consid-
ered factors (Table 1B). An increase in EE2concentration is translated
Fig. 2. The effects of EDCs exposure on Syngnathus abaster morphological development: a) a juvenile and a detail of the head showing the selected variables; b) effects of TBT and EE2,
single or in combination, on body length; c) pupil surface; and d) dorsal fin length.
Syngnathus abaster morphometric analysis. (A) ANOVA results on the body length of
juveniles exposed to EE2(nominal concentrations: 0, 3 and 9 ng/L) and TBT (nominal
concentrations: 0, 10 and 50 ngSn/L), as well as all possible combinations. (B) ANCOVA
results on the pupil surface of juveniles exposed to EE2and TBT, as well as all possible
combinations (eye surface used as co-variable). (C) ANCOVA results on the dorsal fin
length of juveniles exposed to EE2and TBT, as well as all possible combinations (body
length used as co-variable).
Source of variationDF
Body length (LT)
M.P. Sárria et al. / Environment International 37 (2011) 418–424
into larger pupil surfaces. When TBT is administered alone, a similar
pattern is also visible. Nevertheless, when both contaminants are
combined, an opposite trend is observed with a decreasing pupil size.
The ANCOVA results on the effects of EDCs in dorsal fin length
(adjusting for fish size), showed no significant interaction (Table 1C).
Both considered factors, EE2and TBT, produced a similar (parallel)
pattern of results, with the tested concentrations increasing the
length of the dorsal fin (see Fig. 2d). SNK showed that both tested TBT
concentrations were significantly different from the control. Although
a similar pattern was visible for EE2(see Table 1C, where EE2showed
significant effects), thepost-hoc testwas notable to detect differences
between the concentration levels.
3.3. EDCs exposure and juvenile behaviour
When simultaneously considering the number of movement
bursts and time spent on the aquarium's most secluded area, the
MANOVA showed a significant interaction between the tested
contaminants. Overall, similarly to what was observed for the pupil
surface, EE2seems to increase both the number of bursts and time
spent on the protected area (see Fig. 3). The highest EE2concentration
produced a significantly higher number of movement bursts (SNK,
data not shown). TBT alone favours an increase in time spent in the
surveyed area, but not in the amount of bursts. When in combination,
it seems that TBT is able to depress EE2effects on the number of bursts
in the protected area, whereas EE2at 9 ng/L significantly decreased
the time spent in the protected area at the highest TBT exposure level.
Far from being considered a model organism for ecological risk
assessment in aquatic environments, the present study has provided
evidence that pipefish are sensitive to EDCs, even when exposed for a
short time period during juvenile stages. This early life response is
especially interesting if the long life span of this species is taken into
account, meaning that imbalances in fish physiology or behaviour can
be detected very early on, without the need of waiting for sexual
maturity(where the majorityof the endpointstraditionally addressed
in endocrine disruption studies are concentrated). Variables related to
basic survival mechanisms, such as predator recognition and
avoidance, or phenotypic alterations, which ultimately relate to
population structure and maintainability (if juveniles do not reach
adulthood, no offspring will be produced), do have the potential to
become valid early warning signals. In fact, recent investigations,
using juvenile zebrafish (Danio rerio), unveiled specific behavioural
fingerprints elicited by small neuroactive molecules (Kokel et al.,
2010;Rihel et al., 2010). These results demonstrate the power of high-
throughput locomotor responses in the discovery and characteriza-
tion of psychotropic drugs. Relatively simple behavioral tests may,
thus, realistically help estimate toxic effects that would only become
apparent, when using more orthodox approaches, after longer time
periods (Little et al., 1990). Theserapid tests can act as reliableproxies
for the determination of toxic effects even in species not usually
considered for ecological risk assessment.
Fish larvae development is strictly dependent on a coordinated
regulation of energy metabolism. Since lipids are viewed as the most
energetic cost-effective biomolecules, any chemical able to disrupt
lipid homeostasis, such as organotin compounds (Grun et al., 2006),
can be viewed as having the potential to interfere with pipefish
development. Alterations in size can have profound implications in
pipefish reproductive dynamics since size does matter when it comes
to selecting a partner. Both male and female S. abaster prefer larger
partners (Silva et al., 2008, 2010).
As the generality of syngnathids, S. abaster mating choices are
highly dependent on visual communication (Silva et al., 2007).
Therefore, it can be predicted that any disruption on the sensory
machinery responsible for picking up visual cues (e.g. size, coloration
or behaviour) can severely impact sexual signaling. Recently, Sundin
et al. (2010) stated that an environmental change, such as water
turbidity, hampered the strength of mate choice in the sex role-
reversed species Syngnathus typhle, due to vision impairment. Males
of the broad-nosed pipefish did not seem to compensate for a
reduction in visibility by using alternative sensory cues, such as
olfaction, neither did females compensate by increasing their
courtship activity. Even considering that there is limited information
regarding the effects of toxicants on eye function and visual
processing in fish species (Weis et al., 1987; Fent, 1992; Bentivegna
and Piatkowski, 1998), the obtained results show that, when exposed
to the mixture of TBT plus EE2, pipefish juveniles developed smaller
pupils and became more lethargic. Unlike most vertebrates, the iris of
most teleosts does not present muscle cells and, thus, is unable to
dilate and contract. The pupil diameter generally remains unchanged,
even in response to a variation in ambient illumination (Douglas et al.,
2002). Guthrie and Banks (unpublished data) demonstrated that
several hours of light or dark adaptation produce no measurable
change in the diameter of the pupil of the perch's eye. However, under
the effect of chemicals, a reduction of the pupil's surface was observed
within two hours. Even though it remains to be demonstrated that an
alteration in pupil diameter is directly translated into poorer eyesight,
diminished the amount of time spent in the most secluded area. This
‘kamikaze’ behaviour (spending time near a starving mosquitofish)
will surely decrease the chances of reaching sexual maturity, where
visual communication is also crucial for reproduction.
Fig. 3. The effects of EDCs exposure on Syngnathus abaster behaviour: a) effects of TBT
and EE2, single or in combination, in the number of movement bursts and b) time spent
on the aquarium's most secluded area.
M.P. Sárria et al. / Environment International 37 (2011) 418–424
The study of mixtures of EDCs in the environment is a major
emerging issue in ecotoxicology and environmental risk assessment.
Whereas most ecotoxicological studies have typically focused on
single chemical exposures, the contact with complex mixtures,
outside the laboratory, is the rule, not the exception (Hotchkiss et
al., 2008). While the concept of concentration additive effects seems
to accurately predict the toxicity of chemicals witha commonmode of
action, such as ECs, the established approaches have a limited use to
predict the effects of mixtures of other classes of chemicals or EDCs
acting through a dissimilar mode of action (Vijver et al., 2010;
Spurgeon et al., 2010). In the present study the results from the
measured variables clearly indicate that mixtures of TBT and EE2
significantly modulate larvae responses in an unpredictable way
when compared to single chemical exposure. Hence, the effects of the
combined exposure of TBT and EE2are difficult to forecast, given that
different patterns emerged when different variables and concentra-
tionswere considered. Nevertheless, althoughlargelyunpredictable, a
common denominator can be withdrawn from all the experiments:
mixtures of TBT and EE2 have the potential to severely impact
endpoints known to affect pipefish offspring survival.
All the presented results indicate that EE2 and TBT influence
morphological and behavioral development during pipefish early life.
Nevertheless, the combined effects of the selected contaminants are
far from producing a predictable outcome. Some of the measured
variables showed clear interactions between the tested compounds.
This unpredictability stresses the importance of considering mixture
effects in environmental risk assessment of EDCs. Independently of all
the observed response variation, an ultimate conclusion seems
certain: EE2 and TBT, single or in combination, induce disruption
patterns able to imbalance pipefish early survival. Since these (as well
as other) contaminants are indeed present in estuarine areas,
profound implications in population structure could be expected
ranging from a decrease in recruitment to a disruption of sexual
selection, all simultaneously operating in already declining
The Portuguese Foundation for Science and Technology is
acknowledged for financial support under the research project
POCI/MAR/60895/2004 and M.P.S. doctoral grant (SFRH/BD/31041/
2006). The authors are grateful to the technical support of Carlos Rosa,
Hugo Santos and Ricardo Lacerda (CIIMAR-BOGA).
Ankley GT, Bencic DC, Breen MS, Collette TW, Conolly RB, Denslow ND, et al. Endocrine
disrupting chemicals in fish: developing exposure indicators and predictive models
of effects based on mechanism of action. Aquat Toxicol 2009;92(3):168–78.
Bentivegna CS, Piatkowski T. Effects of tributyltin on medaka (Oryzias latipes) embryos
at different stages of development. Aquat Toxicol 1998;44:117–28.
Brian JV, Harris CA, Scholze M, Backhaus T, Booy P, Lamoree M, et al. Accurate prediction
of the response of freshwater fish to a mixture of estrogenic chemicals. Environ
Health Perspect 2005;113(6):721–8.
Cakic P, Lenhardt M, Mickovic D, Sekulic N, Budakov LJ. Biometric analysis of Syngnathus
abaster populations. J Fish Biol 2002;60:1562–9.
Carlson RW, Bradbury SP, Drummond RA, Hammermeister DE. Neurological effects on
startle response and escape from predation by medaka exposed to organic
chemicals. Aquat Toxicol 1998;43:51–68.
Caserta D, Maranghi L, Mantovani A, Marci R, Maranghi F, Moscarini M. Impact of
endocrine disruptor chemicals in gynaecology. Hum Reprod 2008;14(1):59–72.
Chesworth JC, Donkin ME, Brown MT. The interactive effects of the antifouling
herbicides Irgarol 1051 and Diuron on the seagrass Zostera marina (L.). Aquat
COM(1999)706. Communication from the Commission to the Council and the European
Parliament. Community Strategy for Endocrine Disrupters.
Correia AD, Freitas S, Scholze M, Gonçalves JF, Booj P, Lamoree MH, et al. Mixtures of
estrogenic chemicals enhance vitellogenic response in sea bass. Environ Health
Crain DA, Guillette Jr LJ. Reptiles as models of contaminant-induced endocrine
disruption. Anim Reprod Sci 1998;53(1–4):77–86.
Cushing DH. The Possible Density-Dependence of Larval Mortality and Adult Mortality
in Fishes. In: Blaxter JH, editor. The Early Life History of Fish. Cambridge: Cambridge
University Press; 1974. p. 103–11.
Dawson CE. In: Whitehead PJP, Bauchot ML, Hureau JC, Nielsen J, Tortonese E, editors.
Syngnathidae in fishes of the north-eastern Atlantic and the Mediterranean. Paris:
Unesco; 1986. p. 628–39.
Douglas RH, Collin SP, Corrigan J. The eyes of suckermouth armoured catfish
(Loricariidae, subfamily Hypostomus): pupil response, lenticular longitudinal
spherical aberration and retinal topography. J Exp Biol 2002;205:3425–33.
Ellis DV, Pattisina LA. Widespread neogastropod imposex:a biological indicator of global
TBT contamination. Mar Pollut Bull 1990;21:248–53.
EPA(2009)EDSP. Environmental Protection Agency. Endocrine Disruptor Screening
Program: Policies and Procedures for Initial Screening.
Fent K. Embryotoxic effects of tributyltin on the minnow Phoxinus phoxinus. Environ
Fent K. Ecotoxicology of organotin compounds. Crit Rev Toxicol 1996;26:1-117.
Fent K, Woodin BR, Stegeman JJ. Effects of triphenyltin and other organotins on hepatic
monooxygenase system in fish. Comp Biochem Physiol C Pharmacol Toxicol
Fisher WS, Oliver LM, Walker WW, Manning CS, Lytle TF. Decreased resistance of
eastern oysters (Crassostrea virginica) to a protozoan pathogen (Perkinsus marinus)
aftersublethal exposure to tributyltin oxide. Mar Environ Res 1999;47(2):185–201.
Franco A, Franzoi P, Malavasi S, Riccato F, Torricelli P, Mainardi D. Use of shallow water
habitats by fish assemblages in a mediterranean coastal lagoon. Estuar Coast Shelf
Francois R, Short FT, Weber JH. Accumulation and persistence of tributyltin in eelgrass
(Zostera marina L.) tissue. Environ Sci Technol 1989;23(2):191–6.
Greco L, Capri E, Rustad T. Biochemical responses in Salmo salar muscle following
exposure to ethynylestradiol and tributyltin. Chemosphere 2007;68:564–71.
Grun F, Watanabe H, Zamanian Z, Maeda L, Arima K, Cubacha R, et al. Endocrine-
disrupting organotin compounds are potent inducers of adipogenesis in verte-
brates. Mol Endocrinol 2006;20:2141–55.
Guo S, Qian L, Shi H, Barry T, Cao Q, Liu J. Effects of tributyltin (TBT) on Xenopus tropicalis
embryos at environmentally relevant concentrations. Chemosphere 2010;79(5):
Gyllenhammar I, Holm L, Eklund R, Berg C. Reproductive toxicity in Xenopus tropicalis
after developmental exposure to environmental concentrations of ethynylestra-
diol. Aquat Toxicol 2009;91(2):171–8.
Herald ES. From pipefish to seahorse — a study of phylogenetic relationships. Proc
Californian Acad Sci 1959;29:465–73.
Hotchkiss AK, Rider CV, Blystone CR, Wilson VS, Hartig PC, Ankley GT, et al. Fifteen years
after “wingspread”—environmental endocrine disrupters and human and wildlife
Houde ED. Fish early life dynamics and recruitment variability. Am Fish Soc Symp
IUCN. In: Baillie JEM, Hiltor-Taylor C, Stuait SN, editors. 2004 IUCN Red List of
Threatened Species, Global Species Assessment. Gland: IUCN; 2004.
Jaser W, Severin GF, Jütting U, Jüttner I, Schramm KW, Kettrup A. Effects of 17α-
ethinylestradiol on the reproduction of the cladoceran species Ceriodaphnia
reticulata and Sida crystalline. Environ Int 2003;28(7):633–8.
Jensen HF, Holmer M, Dahllöf I. Effects of tributyltin (TBT) on the seagrass Ruppia
maritime. Mar Pollut Bull 2004;49(7–8):564–73.
Kendrick AJ, Hyndes GA. Patterns in the abundance and size-distribution of syngnathid
fishes among habitats in a seagrass-dominated marine environment. Estuar Coast
Shelf Sci 2003;57(4):631–40.
Kloas W. Amphibians as a model for the study of endocrine disruptors. Int Rev Cytol
Kneib RT. The role of tidal marshes in the ecology of estuarine nekton. Oceanogr Mar
Biol Annu Rev 1997;35:163–220.
Kokel D, Bryan J, Laggner C, White R, Cheung CYJ, Mateus R, et al. Rapid behavior-based
identification of neuroactive small molecules in the zebrafish. Nat Chem Biol
Little EE, Archeski RD, Flerov BA, Kozlovskaya VI. Behavioral indicators of sublethal
toxicity in rainbow trout. Arch Environ Contam Toxicol 1990;19:380–5.
Lopes J, Dias J, Cardoso A, Silva C. The water quality of the Ria de Aveiro lagoon,
Portugal: from the observations to the implementation of a numerical model. Mar
Environ Res 2005;60(5):594–628.
Martin-Smith KM, Vincent ACJ. Seahorse declines in the Derwent estuary, Tasmania in
the absence of fishing pressure. Biol Conserv 2005;123(4):533–45.
Martin P, Sébastien D, Gilles T, Isabelle A, Montaudouin X, Emery E, et al. Long-term
evolution (1988–2008) of Zostera spp. meadows in Arcachon Bay (Bay of Biscay).
Estuar Coast Shelf Sci 2010;87(2):357–66.
Matthiessen P, Arnold D, Johnson AC, Pepper TJ, Pottinger TG, Pulman KGT.
Contamination of headwater streams in the United Kingdom by oestrogenic
hormones from livestock farms. Sci Total Environ 2006;367:616–30.
Mcallister BG, Kime DE. Early life exposure to environmental levels of the aromatase
inhibitor tributyltin cause masculinisation and irreversible sperm damage in
zebrafish (Danio rerio). Aquat Toxicol 2003;65:309–16.
McGee MR, Julius ML, Vajda AM, Norris DO, Barber LB, Schoenfuss HL. Predator
avoidance performance of larval fathead minnows (Pimephales promelas) following
short-term exposure to estrogen mixtures. Aquat Toxicol 2009;91(4):355–61.
M.P. Sárria et al. / Environment International 37 (2011) 418–424
Micael J, Reis-Henriques MA, Carvalho AP, Santos MM. Genotoxic effects of binary
mixtures of xenoandrogens (tributyltin, triphenyltin) and a xenoestrogen
(ethinylestradiol) in a partial life-cycle test with zebrafish (Danio rerio). Environ
Mills LJ, Chichester C. Review of evidence: are endocrine-disrupting chemicals in the
aquatic environment impacting fish populations? Sci Total Environ 2005;343(1–3):
Monteiro NM, Almada VC, Vieira MN. Implications of different brood pouch structures
in syngnathid reproduction. J Mar Biol Assoc UK 2005;85:1235–41.
Moore DW, Dillon TM, Suedel BC. Chronic toxicity of tributyltin to the marine
polychaete worm, Neanthes arenaceodentata. Aquat Toxicol 1991;21(3-4):181–98.
Murray TJ, Lea RG, Abramovich DR, Haites NE, Fowler PA. Endocrine disrupting
chemicals: effects on human male reproductive health. Early Pregnancy 2001;5(2):
Ottinger MA, Saal FS. Impact of environmental endocrine disruptors on sexual
differentiation in birds and mammals hormones. Brain Behav 2002:325–83.
Pojana G, Gomiero A, Jonkers N, Marcomini A. Natural and synthetic endocrine
disrupting compounds (EDCs) in water, sediment and biota of a coastal lagoon.
Environ Int 2007;33(7):929–36.
Pombo L, Elliott M, Rebelo JE. Changes in the fish fauna of the Ria de Aveiro estuarine
lagoon (Portugal) during the twentieth century. J Fish Biol 2002;61(Supplement
Rempel MA, Schlenk D. Effects of environmental estrogens and anti-androgens on
endocrine function, gene regulation, and health in fish. Int Rev Cell Mol Biol
Revathi P, Munuswamy N. Effect of tributyltin on the early embryonic development in
the freshwater prawn Macrobrachium rosenbergii (De Man). Chemosphere 2010;79
Ridgway J, Shimmield G. Estuaries as repositories of historical contamination and their
impact on shelf seas. Estuar Coast Shelf Sci 2002;55(6):903–28.
Rihel J, Prober DA, Arvanites A, Lam K, Zimmerman S, Jang S, et al. Zebrafish behavioral
profiling links drugs to biological targets and rest/wake regulation. Science
Rodríguez EM, Medesani DA, Fingerman M. Endocrine disruption in crustaceans due to
pollutants: a review. Comp Biochem Physiol A Mol Integr Physiol 2007;146(4):
Saaristo M, Craft JA, Lehtonen KK, Lindström K. Exposure to 17α-ethinylestradiol
impairs courtship and aggressive behaviour of male sand gobies (Pomatoschistus
minutus). Chemosphere 2010;79(5):541–6.
Santos MM, Castro LFC, Vieira MN, Micael J, Morabito R, Massanisso P, et al. New
insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus.
Comp Biochem Physiol 2005;141:101–9.
Santos MM, Micael J, Carvalho AP, Morabito R, Booy P, Massanisso P, et al. Estrogens
counteract the masculinizing effect of tributyltin in zebrafish. Comp Biochem
Physiol C Toxicol Pharmacol 2006;142(1–2):151–5.
Shimasaki Y, Kitano T, Oshima Y, Inoue S, Imada N, Honjo T. Tributyltin cause
masculinization in fish. Environ Toxicol Chem 2003;22(1):141–4.
Silva E, Rajapakse N, Kortenkamp A. Something from “nothing”—eight weak estrogenic
chemicals combined at concentrations below NOECs produce significant mixture
effects. Environ Sci Technol 2002;36(8):1751–6.
Silva K, Monteiro NM, Almada VC, Vieira MN. Early life history of Syngnathus abaster. J
Fish Biol 2006;68(1):80–6.
Silva K, Vieira MN, Almada VC, Monteiro MN. The effect of temperature on mate
preferences and female-female interactions in Syngnathus abaster. Anim Behav
Silva K, Vieira MN, Almada VC, Monteiro NM. Can the limited marsupium space be a
limiting factor for Syngnathus abaster females? Insights from a population with size
assortative mating. J Anim Ecol 2008;77:390–4.
Silva K, Almada VC, Vieira MN, Monteiro NM. Female reproductive tactics in a sex-role
reversed pipefish: scanning for male quality and number. Behav Ecol 2009;20(4):
Silva K, Vieira MN, Almada VC, Monteiro NM. Reversing sex role reversal: compete only
when you must. Anim Behav 2010;79:885–93.
Soares J, Coimbra AM, Reis-Henriques MA, Monteiro NM, Vieira MN, Oliveira JMA, et al.
Disruption of zebrafish (Danio rerio) embryonic development after full life-cycle
parental exposure to low levels of ethinylestradiol. Aquat Toxicol 2009;95(4):
Spurgeon D, Jones O, Dorne JL, Svendsen C, Swain S, Sturzenbaum S. Systems toxicology
approaches for understanding the joint effects of environmental chemical
mixtures. Sci Total Environ 2010;408(18):3725–34.
Sumpter JP. Endocrine disrupters in the aquatic environment: an overview. Acta
Hydroch Hydrob 2005;33(1):9-16.
Sundin J, Berglund A, Rosenqvist G. Turbidity hampers mate choice in a pipefish.
Sun L, Zha J, Wang Z. Effects of binary mixtures of estrogen and antiestrogens on
Japanese medaka (Oryzias latipes). Aquat Toxicol 2009;93(1):83–9.
Thibaut R, Porte C. Effects of endocrine disrupters on sex steroid synthesis and
metabolism pathways in fish. J Steroid Biochem Mol Biol 2004;92(5):485–94.
Trussell RR. Endocrine disruptors and the water industry. J Am Water Works Assn
Vijver MG, Peijnenburg WJ, De Snoo GR. Toxicological mixture models are based on
inadequate assumptions. Environ Sci Technol 2010;44(13):4841–2.
Vosges M, Braguer JC, Combarnous Y. Long-term exposure of male rats to low-dose
ethinylestradiol (EE2) in drinking water: effects on ponderal growth and on litter
size of their progeny. Reprod Toxicol 2008;25(2):161–8.
Waring RH, Harris RM. Endocrine disrupters: a human risk? Mol Cell Endocrinol
Weis JS, Weis P, Wang F. Developmental effects of tributyltin on the fiddler crab (Uca
pugilator) and the killifish (Fundulus heteroclitus). Oceans '87: the Ocean, an
International Workplace, International Organotin Symposium, 4; 1987. p. 1456–60.
World Health Organization (WHO). IPCS Global Assessment of the State-of-the-Science
of Endocrine Disruptors. International Programme on Chemical Safety, WHO/PCS/
Zhang J, Zuo Z, Chen R, Chen Y, Wang C. Tributyltin exposure causes brain damage in
Sebastiscus marmoratus. Chemosphere 2008;73(3):337–43.
Zhou T, Weis JS. Swimming behavior and predator avoidance in three populations of
Fundulus heteroclitus larvae after embryonic and/or larval exposure to methylmer-
cury. Aquat Toxicol 1998;43:131–48.
M.P. Sárria et al. / Environment International 37 (2011) 418–424