Environmental Research 131 (2014) 165–173

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Environmental Research
journal homepage: www.elsevier.com/locate/envres

Reproductive biomarkers responses induced by xenoestrogens in the

characid fish Astyanax fasciatus inhabiting a South American reservoir:
An integrated field and laboratory approach
Paula S. Prado a, Ana Paula B. Pinheiro a, Nilo Bazzoli b, Elizete Rizzo a,n
a
Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, UFMG, Belo Horizonte, C.P. 486, 30161-970, Minas
Gerais, Brasil
b
Programa de Pós-Graduação em Zoologia de Vertebrados, Pontifícia Universidade Católica de Minas Gerais, PUC Minas, Belo Horizonte 30535-610, Minas
Gerais, Brasil

art ic l e i nf o a b s t r a c t

Article history: Field studies evaluating the effects of endocrine disruption chemicals (EDCs) on the fish reproduction are
Received 2 September 2013 scarce worldwide. The goal of this study was to assess hepatic levels of vitellogenin (Vtg), zona radiata
Received in revised form proteins (Zrp) and insulin-like growth factors (IGF-I and IGF-II), and relating them to reproductive
24 January 2014
endpoints in a wild fish population habiting a reservoir that receive domestic sewage, agricultural and
Accepted 7 March 2014
Available online 11 April 2014
industrial residues. Adult fish Astyanax fasciatus were sampled during the reproductive season in five
sites from the Furnas Reservoir, Grande River, and Paraguay–Paraná basin. As a control to field data, fish
Keywords: were experimentally exposed via dietary intake, to oestradiol benzoate (OB) for 7 days. Fish from site
Vitellogenin with little anthropogenic interference showed hepatic levels of Vtg, Zrp and IGF-I and IGF-II similar to
Zona radiata proteins
those from the non-treated experimental group. In sites located immediately downstream from the
IGF-I
municipal wastewater discharges, the water total oestrogen was 4120 ng/l, and male fish displayed
IGF-II
Over-ripening increased Vtg and Zrp and decreased IGF-I levels similar to OB treated fish. In females, levels of Vtg, Zrp,
Endocrine disruption IGF-I and IGF-II suggest an impairment of final oocyte maturation and spawning, as also detected by
frequency of over-ripening, follicular atresia and fecundity. At the sites that receive agricultural and
industrial residues, the water total oestrogen was o50 ng/l and females showed decreased Zrp and
increased IGF-II levels associated to reduced diameter of vitellogenic follicles, indicating an inhibition of
oocyte growth. Overall, the current study reports oestrogenic contamination impairing the reproduction
of a wild fish from a hydroeletric reservoir and, the data contribute to improving the current knowledge
on relationship between hepatic Vtg, Zrp and IGF-I and IGF-II, and reproductive endpoints in a teleost
fish. In addition, our data point out novel reproductive biomarkers (IGF-I, IGF-II and over-ripening) to
assessing xenoestrogenic contamination in freshwater ecosystems.
& 2014 Elsevier Inc. All rights reserved.

1. Introduction oestrogens in aquatic ecosystems since the consumption of oestrogens

Over recent decades, there is growing interest in evaluating the
adverse effects of endocrine disrupting chemicals (EDCs) in aquatic
environments. EDCs are exogenous compounds able to disrupt the
activity of the endocrine system responsible for homoeostasis, repro-
duction, and development of organisms (Diamanti-Kandarakis et al.,
2009). Among EDCs, xenoestrogens receive more attention due to
their ability to mimic natural oestrogens, binding to oestrogen
receptors, which have relatively low specificity (WHO/IPCS, 2002).
Municipal wastewater effluents are considered the main source of
n
Corresponding author. Fax: þ 55 31 34092771.
E-mail address: ictio@icb.ufmg.br (E. Rizzo).
and progestrogens in human medicine and animal farming has been
progressively increasing (Ying et al., 2002). Oestrogen concentrations
as low as 10–100 ng/l in water can adversely disrupt the endocrine
system, affecting the reproductive biology and the long-term sustain-
ability of fish populations (Hanselman et al., 2003; Kidd et al., 2007).
Understanding the effects of endocrine disrupting chemicals on
aquatic organisms is a challenge since they are continuously
exposed to increasing amounts of a mixture of contaminants in
their natural habitat (Allen and Moore, 2004). A range of repro-
ductive parameters has been reported as endpoints of xenoestro-
gen exposure in fish gonads, i.e. intersex, gonadal growth, oocyte
production, fertility and follicular atresia (Jobling et al., 2002;
Arukwe and Goksøyr, 2003; Robinson et al., 2003, Mills and
Chichester, 2005; Hutchinson et al., 2006; Tetreault et al., 2011).

http://dx.doi.org/10.1016/j.envres.2014.03.002
0013-9351/& 2014 Elsevier Inc. All rights reserved.
166 P.S. Prado et al. / Environmental Research 131 (2014) 165–173
In addition, cellular and molecular biomarkers are essential to
study the basic mechanisms of the action of EDCs (Denslow and
Sepúlveda, 2007, Arukwe and Røe 2008). Vitellogenin (Vtg) and
zona radiata proteins (Zrp) are typically synthesised in the liver of
females under the regulation of endogenous 17β-oestradiol (E2),
transported via the bloodstream to the ovary and taken up into
maturing oocytes (Arukwe and Goksøyr, 2003). Usually, Vtg and
Zrp concentrations are very low or absent in male and juvenile
fish, but can be induced upon xenoestrogen exposure, so they are
sensitive biomarkers of oestrogenic EDCs contamination in the
aquatic environment (Arukwe and Goksøyr, 2003; Genovese et al.,
2011, Truman and van den Hurk, 2012).
Recent studies have demonstrated that growth hormone (GH) –
insulin-like growth factor (IGF) – gonad interaction may be an
alternative axis in fish reproduction that acts in concert with the
hypothalamus – pituitary – gonad pathway (Huang and Weng, 2010).
In several teleosts, IGF-I as well as IGF-II may induce oocyte matura-
tion, each possibly having a species-specific role (Mukherjee et al.,
2006; Lokman et al., 2007; Weber et al., 2007; Reinecke, 2010b). In
addition, IGF-I can stimulate steroid biosynthesis during previtello-
genic oocyte growth (Campbell et al., 2006; Lokman et al., 2007). Both
in vivo and in vitro studies provide early evidences of an interaction
between EDCs and IGF-I (Reinecke, 2010a). In particular, E2 appears to
be able to down-regulate hepatic IGF-I expression at environmentally
relevant concentrations, but environmental oestrogen effects on the
IGF-II system still remains to be clarified (Shved et al., 2008; Reinecke,
2010b).
In South America, some native fish have been utilised as
bioindicator species for evaluating the environmental pollution
in field or experimental assays (Cazenave et al., 2009; Leonardi
et al., 2011; Fernandes et al., 2013). Among them, we highlight the
lambari Astyanax fasciatus from the Characidae family, a species
widely distributed in the La Plata River basin, abundant in several
Brazilian ecosystems, and that supports the fishing at the Furnas
Reservoir, Grande River, and Paraguay–Paraná basin. This small
characid species displays a non-migratory behaviour, omnivorus
feeding habit, prolonged reproductive season, multiple spawnings
through the year and first gonadal maturity at about 6.5 cm
standard length (Carvalho et al., 2009). Although some reports
have indicated this lambari as an appropriate species for ecotox-
icological studies in South America (Schulz and Martins-Júnior,
2001; Alberto et al., 2005; Carrasco-Letelier et al., 2006; Prado
et al., 2011), studies focused on the reproductive molecular
responses are missing.
Although the IGF system is involved in the fish reproductive
physiology, reports on IGF-I and IGF-II levels in environments
contaminated by EDC's are scarce. The goal of this study was to
assess the effects of xenoestrogenic contamination on hepatic
levels of Vtg, Zrp and IGF-I and IGF-II, and relate them to
the reproductive endpoints in A. fasciatus from the Furnas
Reservoir.
2. Material and methods
2.1. Study area
The Furnas Reservoir (Fig. 1) is a complex ecosystem with 22  109 m3 of water
and 1440 km2 of flooded total area, distributed in two major regions related to the
Grande River and Sapucaí River, each measuring about 250 km. This reservoir
situated on the Upper Paraná River, south-eastern Brazil, supplies water to a local
population of over 1 million people and receives more than 2000 t of waste solids
daily from the surrounding cities. Untreated municipal and industrial sewage and
pollution by pesticides has gradually reduced the water quality, requiring special
attention from Brazilian public institutions for conservation of fish fauna, which is
dominated by small and medium-sized species (Heleno, 2004; IGAM, 2008;
Sadauskas-Henrique et al., 2011).
2.2. Field collection
All procedures performed during fish collection followed the ethical principles
established by the Brazilian College of Animal Experimentation (COBEA) and the
study was approved by the Ethics Committee for Animal Experimentation (CETEA) of
the Federal University of Minas Gerais, Brazil. A total of 278 adult specimens of A.
fasciatus were caught during the reproductive season peak (November 2010 to
January 2011) at five sites: Barranco Alto, BA (211110 S; 451580 W); Fama, F (211240 S;
451500 W); Boa Esperança, BE (211040 S; 451330 W); Guapé, G (201450 S; 451550 W)
and Turvo, T (201400 S; 461130 W). The main environmental pollution sources in each
site are indicated in Fig. 1. Fish were sampled using 100 m of gillnets with a 3–4 cm
stretched mesh size deployed for about 14 h on the surface of the water and placed
30 m from the shoreline of the reservoir. The fish collection was supported by a team
of professional fishermen from the Furnas Hydrobiology and Hatchery Station.
2.3. Physic-chemical parameters and total oestrogen in the water
During the fish collections, temperature, pH, dissolved oxygen and conductivity
at 25 1C (specific conductance) were measured in each sampling site at 1 m depth
from the water's surface using an YSI 650 MDS multi-parameter instrument
(Table 1). Total phosphorus was determined in 1 l water samples processed at
the laboratory of Furnas Hydrobiology and Hatchery Station by the colorimetric
method (APHA, 1999).
Water samples (0.5 l from each site) were stored in plastic bottles and transported
in ice for the analysis of total oestrogen (oestrone, E1; 17β-oestradiol, E2 and oestriol,
E3) (Table 1) by applying the colorimetric test (ELISA kit Ecologiena, JapanEnviroChem-
icals, Ltd.). In the laboratory, the samples were filtered by glass fibre filters with 1 μm
pore size and, then 5 ml of methanol (MeOH) was added to elute the organic part of the
pellet. The test was performed on duplicate samples following the manufacturer's
recommendations. The competitive-ELISA coefficient of variation was up to 10% and the
quantitative analysis ranged from 0.05 to 3.0 mg/l. For all samples, duplicate measure-
ments were made at 450 nm using Versa Max Microplate Reader (Molecular
Devices, USA).
2.4. Biological indices and fecundity
Live specimens were kept in plastic containers with water from the reservoir
until dissection and samples collection. Total length (TL), body weight (BW), gonads
weight (GW) and liver weight (LW) were obtained for determining the gonadoso-
matic index (GSI ¼ 100 GW/BW), hepatosomatic index (HSI ¼100 LW/BW) and
Fulton condition factor (K1 ¼100 BW/TL3). To eliminate the influence of the gonads
and liver, K2 was also calculated (K2 ¼100 BW – (GWþ LW)/TL3).
For histology, a transversal section of the middle region of the left gonad was
obtained from each fish, then fixed in Bouin's fluid for 8–12 h and kept in 70%
ethanol for posterior processing in the laboratory. The right ovary from 20 fish/site
was fixed in 10% formalin for determining the fecundity in the laboratory. Liver
samples (n¼ 10 fish/sex) from each site were frozen in liquid nitrogen and
preserved at  80 1C for subsequent ELISA assays.
The batch fecundity was determined according to methodology established
previously (Otobo, 1978). Ovaries fixed in 10% formalin were washed with water and
dried on paper towel. Three samples of  100 mg were obtained from the cranial,
middle and tail ovarian regions. The vitellogenic oocytes were counted manually under
a stereomicroscopy for determining the batch fecundity (BF¼NO  GW/SW, where
NO¼ total number of vitellogenic oocytes in the sample, GW¼ gonads weight and
SW¼sample weight). The batch fecundity relative to TL was also calculated in order to
eliminate the interference of fish size (Arantes et al., 2010).
2.5. Experimental exposure to oestradiol benzoate (OB)
As control of the oestrogenic contamination in the Furnas Reservoir, a laboratory
study was conducted in December 2010. For this, adult specimens of A. fasciatus
were captured from the site Turvo (n¼75), then maintained for three months in
clean water of the fish culture tanks at the Furnas Hydrobiology and Hatchery
Station, as a recovery time. Following, they were transported to the laboratory,
separated at random into OB treated and non-treated groups at a density of 1.2 g of
live fish/l of water, and were acclimatised over three days in aquaria with
oxygenator, thermostat, mechanical, biological filters and a controlled photoperiod
(12 h light:12 h dark) and temperature (27–28 1C). During the experiment, the
treated group (positive control) was fed twice a day ad libitum with commercial
diet (Tetra Tropical granules, Tetra GmbH, Herrenteich, Germany) containing
Oestradiol Benzoate (OB, Tecnopec Brazil) dissolved in anhydrous alcohol, at a final
concentration of 20 μg/g OB. Fish from the non-treated group (negative control)
were fed with a commercial diet with ethanol only. After 7 days of the experiment,
the fish were killed by trasnversal section of spinal cord. For each specimen, the
following data were obtained: TL, BW, GW and LW for determining the GSI, HSI, K1
and K2. A transversal section of the middle region of the left gonad was obtained for
histology. Liver samples (n¼ 10/sex/group) were also frozen in liquid nitrogen and
 P.S. Prado et al. / Environmental Research 131 (2014) 165–173 167

Fig. 1. Main sources of pollution in the Furnas Reservoir, Grande River. Turvo (T), site located near the Furnas dam, that shows little anthropogenic interference; Barranco
Alto (BA) site which receives residues from agriculture; Fama (F) site which receives untreated industrial and domestic sewage from cities surrounding the Verde and Sapucaí
Rivers; Boa Esperança (BE) and Guapé (G) sites that receive all the untreated sewage from the urban areas.

Table 1
Water physic-chemical parameters and total oestrogen in five sites from the Furnas Reservoir, Grande River, and Paraguay–Paraná River basin.

Turvo (T) Barranco Alto (BA) Fama (F) Boa Esperança (BE) Guapé (G)

Temperature (1C) 23.95–26.14 23.17–27.08 22.51–25.68 24.26–26.06 24.29–26.97

Dissolved oxygen (mg/L) 7.63–8.00 6.43–7.91 4.75a–7.54 5.88–8.87 6.48–7.65
pH 7.20–7.33 5.87a–7.58 5.83a–7.38 6.95–7.97 7.24–7.41
Conductivity to 25 1C (mS/cm) 32.00–34.50 37.00–38.00 33.75–42.00 45.00–46.25 32.00–33.50
Total phosphorous (mg/L) 0.010–0.014 0.017–0.026 0.016–0.017 0.044a–0.062a 0.068a–0.180a
Total oestrogen (ng/L) o 50 o 50 o 50 130.50 121.00

Data of total oestrogen were obtained in January 2011.

a
Values of pH, dissolved oxygen and total phosphorous are different of Brazilian resolution CONAMA/357–2005 for freshwater Class II (www.mma.gov.br/port/conama/
res/res05/res35705.pdf).

preserved at  80 1C for subsequent analysis of a set of biomarkers such as those
assessed in the field study. To check the oestrogenic induction in fish, analyses of
total oestrogen were performed in 100 ml of liver homogenate using the colorimetric
test (ELISA kit, Ecologiena, JapanEnviroChemicals, Ltd.).
2.6. Vitellogenic follicles diameter, over-ripening, follicular atresia and intersex
For histology, gonad samples fixed in Bouin's fluid were gradually dehydrated in
ethanol, embedded in paraffin, sectioned at 5 mm thickness and then stained then three
sections were stained with haematoxylin and eosin (HE). In order to highlight the
distribution of cortical vesicles in vitellogenic follicles, some slides were stained with
Alcian Blue at pH 2.5. The longest diameter of the advanced vitellogenic follicles
(n¼ 250) was measured using 10 histological sections from each sampling site. Over-
ripening and follicular atresia frequencies (%) were calculated for total vitellogenic
follicles in each histological section and a mean was obtained by sampling site and
experimental group. Intersex gonads were recognised by the presence of isolated
perinucleolar oocytes inside the seminiferous tubules or grouped perinucleolar and
vitellogenic oocytes associated to the testicular tissue (Prado et al., 2011).
2.7. Biomarkers ELISA assays
Liver samples (n¼10/sex) from the experimentally exposed (positive and negative
controls) and field caught mature fish were submitted to indirect ELISA for Zrp, Vtg,
IGF-I and IGF-II. The frozen samples were homogenised in extraction buffer (50 mM
Tris–HCl pH 8.0 with 0.02% aprotinin and 1 mM phenylmethylsulfonyl fluoride) using
a Potter S homogeniser (Braun, Melsungen, Germany) at a 1:2 ratio of tissue weight:
buffer volume held in an ice-water cooled bath. Then, the extracts were sonicated
(3  , 10 s in each step) by an ultrasonic processor GEX 600 CE and centrifuged at
15,000g for 60 min at 4 1C. After pellet removal, the supernatant was stored in aliquots
at  80 1C until analysis. Total soluble protein contained in liver homogenates was
determined by the Bradford method using bovine serum albumin (BSA) as standard.
Duplicate samples of 100 μg/ml (Vtg and Zrp) and 500 μg/ml (IGF-I and II) were
incubated overnight in a 96-wells microplate (Nunc, Denmark), blocked with 2% BSA
and incubated again at 37 1C for 2 h with the primary antibodies:polyclonal-rabbit Zrp
and Vtg (salmon) at 1:500 (Biosense Laboratories AS, Norway), polyclonal-rabbit IGF-I
and IGF-II (human) at 1:1000 (Santa Cruz Biotechnology, Inc., USA). After washing
with PBS-Tween 0.05%, the plates were incubated with anti-rabbit IgG (1:2500, Sigma,
St. Louis, MO) conjugated with horseradish peroxidase for 2 h at 37 1C. Then they were
washed again and the reaction was revealed with 200 ml of 0.04% o-phenylenediamine
dihydrochloride (Sigma, St. Louis, MO, USA) in 0.05 M phosphate-citrate buffer
solution with 0.0025% hydrogen peroxide. The reaction was stopped by adding
100 ml of 5% H2SO4 to each well and the absorbance was measured at 495 nm using
a Versa Max Microplate Reader (Molecular Devices, USA). For validation of the ELISA
assays, dilution curves of liver homogenates of females and males were performed for
each protein analysed (Fig. S1). The cross-reactivity of the antibodies with the targeted
proteins of A. fasciatus was assessed by Western blot assays (Fig. S1) following
procedure established previously (Prado et al., 2011).
168 P.S. Prado et al. / Environmental Research 131 (2014) 165–173

2.8. Statistical analyses
Statistical analyses were carried out using GraphPad Instat software. Values
were expressed as means 7SEM, and results were considered significant at a 95%
confidence interval. A normality test followed by t-test was applied to evaluate the
biological and histological data from the experimental groups. One-way analyses of
variance (ANOVA) with Tukey's post-test were used to compare the biological and
histological data from field caught fish as well as biomarkers levels.
3. Results
3.1. Fish biological parameters
Field captured females were 12–14 cm TL and 22–39 g BW and,
males 11–13 cm TL and 14–26 g BW, with few significant differ-
ences among the sampling sites (Table 2). The highest values of
GSI were detected in females from T and males from BE. Overall,
HSI was significantly higher in females from BA and G and males
from BA, F and BE. The K1 values did not differ significantly among
females and they were significantly higher in males from BA
(Table 2). Excluding the gonads and liver weights, K2 values were
significantly higher in males from BA and females from BA and F.
Regarding the experimentally exposed fish, the treatment
with 20 mg/g OB for 7 days increased the hepatic total oestrogen by
about 70% in the females (Table 3). In the treated males,
115.8714.2 ng/100 ml of total oestrogen was detected in the liver
compared to less than 50 ng in non-treated males. For the biological
indices, higher GSI was observed in treated females and higher HSI in
treated males (Table 3). The K1 values were increased in males after
the OB treatment; however this effect was not observed when the
gonad and liver weights were excluded in K2. In females, K1 and K2
did not differ significantly between experimental groups (Table 3).

Table 2
Biological and reproductive parameters of A. fasciatus from the Furnas Reservoir, Grande River, and Paraguay–Paraná River basin.

Turvo (T) Barranco Alto (BA) Fama (F) Boa Esperança (BE) Guapé (G)

Females
N 31 32 32 48 49
Total length (cm) 13.88 7 0.16a 13.60 7 0.24a 13.90 70.18a 13.7070.18a 12.02 70.18b
Body weight (g) 36.22 7 1.26a 37.60 7 2.88a 38.93 71.77a 36.92 71.51a 22.6771.36b
Gonadosomatic index 15.007 1.37a 12.78 7 1.48b 10.75 71.08b 11.97 70.96b 11.13 71.13b
Hepatosomatic index 0.667 0.05a 0.83 7 0.09b 0.60 70.04a 0.60 70.03a 0.90 70.06b
Condition factor (K1) 1.357 0.02a 1.46 7 0.04a 1.44 70.04a 1.41 70.04a 1.26 70.03a
Condition factor (K2) 1.147 0.02a 1.32 7 0.03b 1.28 70.03bc 1.19 70.03ac 1.13 70.02a
Vitellogenic follicles Ø (mm) 552.58 7 3.32a 527.177 4.90b 549.26 73.02a 565.71 73.51c 573.23 73.73c
Over-ripening (%) 2.96 7 2.95a 12.93 7 8.81a 10.68 75.93a 35.5279.79b 33.37 76.82b
Follicular atresia (%) 0.707 0.24a 1.02 7 0.49a 0.82 70.30a 9.69 71.33b 4.76 71.91ab
Batch fecundity (103)* 36.82 7 1.87ac 41.767 4.83a 33.0373.28abc 26.3572.26bc 24.8672.62b
Batch fecundity/TL (103)* 2.707 0.15ac 2.96 7 0.28a 2.36 70.22abc 2.10 70.16bc 1.93 70.19b

Males
N 21 19 19 13 14
Total length (cm) 12.60 7 0.55a 12.03 7 0.41a 10.92 70.24ab 11.00 70.25ab 11.12 70.22ab
Body weight (g) 25.747 4.05a 24.68 7 2.39a 16.58 70.94b 16.3571.30b 14.7770.98b
Gonadosomatic index 1.88 7 0.34a 1.29 7 0.30a 1.27 70.30a 3.3370.35b 2.03 70.29a
Hepatosomatic index 0.43 7 0.04a 0.767 0.08b 0.58 70.05b 0.7070.07b 0.51 70.07ab
Condition factor (K1) 1.197 0.04a 1.38 7 0.01b 1.27 70.04ab 1.21 70.04ab 1.08 70.09a
Condition factor (K2) 1.147 0.04a 1.357 0.02b 1.25 70.04ab 0.95 70.02ab 1.22 70.03a

Values represent mean 7 SEM of data obtained during the peak of the reproductive season (November 2010 to January 2011). In a line, different letters indicate significant
differences among sites, ANOVA, p o 0.05.
n
n¼20 samples/site.

Table 3
Biometric data, biological indices, liver total oestrogen and reproductive parameters in A. fasciatus under experimental condition.

Females Males

Non-treated Treated Non-treated Treated

N 10 17 13 31
Total length (cm) 12.36 7 0.55a 12.487 0.82a 12.127 0.42a 10.78 7 0.11b
Body weight (g) 18.08 7 7 1.09a 17.067 0.99a 14.917 0.70a 12.717 0.43b
Gonadosomatic index 7.79 7 0.60a 9.167 0.78b 2.20 7 0.21a 2.117 0.13a
Hepatosomatic index 1.117 0.08a 1.067 0.10a 0.93 7 0.13a 1.32 7 0.10b
Condition factor (K1) 0.95 7 0.08a 1.017 0.09a 0.89 7 0.05a 1.02 7 0.01b
Condition factor (K2) 0.88 7 0.07a 0.917 0.08a 0.86 7 0.05a 0.99 7 0.01a
Liver total oestrogen (ng/100ml)* 159.20 7 8.94a 228.40 7 7.50b o 50 115.8 7 14.22
Vitellogenic follicles Ø (mm) 495.807 2.37a 509.50 7 2.57a
Over-ripening (%) nd 0.50 7 0.49
Follicular atresia (%) 9.647 2.41a 10.87 71.80a

Values represent mean7 SEM of data obtained during the peak of the reproductive season. In treated group, fish received commercial diet containing 20 mg/g of oestradiol
benzoate during 7 days. In non-treated group, fish received commercial diet containing ethanol only. In a line, different letters indicate significant differences between
groups, test T, p o 0.05. (nd) not detected.
n
n¼10 samples/group.
 P.S. Prado et al. / Environmental Research 131 (2014) 165–173 169

2 2
b Females

Zrp ELISA-absorbance (492nm)

Zrp ELISA-absorbance (492nm)

Males

b
ab b
1 1
a
a ab
ab
a a a
c a
c

0 0
Non-treated Treated T BA F BE G Non-treated Treated T BA F BE G
group group group group

1.5 1.5
Vtg ELISA-absorbance (492nm)

Vtg ELISA-absorbance (492nm)

b
b
b
1 b 1
b
ab
ab
ab ab
0.5 a 0.5 ab
ab
a a

0 0
Non-treated Treated T BA F BE G Non-treated Treated T BA F BE G
group group group group

Fig. 2. Indirect ELISA analysis showing hepatic levels of zona radiata proteins (Zrp) and vitellogenin (Vtg) in females (A, C) and males (B, D) of A. faciatus under experimental
and field conditions. Fish were captured at five sites from the Furnas Reservoir in January 2011: Turvo (T), Barranco Alto (BA), Fama (F), Boa Esperança (BE) and Guapé (G). In
experimental conditions, fish received commercial diet containing 20 mg/g oestradiol benzoate (treated group) and ethanol only (non-treated group) for 7 days. Data are
given as mean of ELISA absorbance values at 492 nm 7 standard error (n ¼10 sex/group). Statistically different values are marked with different letters, p o 0.05.

3.2. Hepatic levels of Zrp and Vtg and vitellogenic follicle diameter 3.4. Intersex, over-ripening, follicular atresia and fecundity

In females, ELISA assays revealed Zrp levels in sites BE and G In histological analyses, gonads with histological features of
statistically similar to OB treated group (positive control) while intersex represented 10% and 16% of total males fish caught in sites
those from site T were similar to the non-treated group (negative BE and G and they were not observed in T, BA and F. Ovarian
control) (Fig. 2A). Significantly lower Zrp levels were observed in sections exhibited perinucleolar and vitellogenic oocytes with a
females from sites BA and F. In males, changes in Zrp levels were central nucleus (Fig. 4A) or displaced toward the micropylar
not pronounced and, significantly increased levels were obtained region, a characteristic of the final oocyte maturation (Fig. 4B). In
in site G (Fig. 2B). Considering the analysis of hepatic Vtg, some sections, vitellogenic oocytes displayed features of over-
significantly higher levels were obtained in F, BE and G for females ripening i.e. increased diameter, thickened zona radiata, basophilic
and BE and G for males compared to the experimentally non- granules accumulated in the peripheric ooplasm, cortical vesicles
treated group (negative control) while no significant differences in clusters, yolk liquefaction, early formation of perivitelline space
were detected in fish from T and BA (Fig. 2C and D). while the follicular cells and theca remained unchanged (Fig. 4C
The mean diameter of vitellogenic follicles (Table 2) was and D). The percentage of over-ripening varied among females
significantly smaller in fish from BA (527.177 4.90 mm) and larger reaching up to 98% of vitellogenic oocytes in one female from site
in fish from BE (565.71 73.51 mm) and G (573.237 3.73 mm). No G. In the field assessment, the average percentage of over-ripening
significant variation in this parameter was detected in the experi- was statistically higher in BE (35.52 7 9.79) and G (33.37 76.82)
mental groups. compared to the other sampling sites (Table 2). In the experi-
mental assay, only one female of the treated group showed over-
3.3. Hepatic levels of IGF-I and IGF-II ripening features (Table 3).
Follicular atresia was below 5% in T, BA and F and G and a
In females and males, the experimental treatment with OB significant variation was detected in BE (Table 2). Elevated rates of
significantly reduced hepatic IGF-I levels and did not cause follicular atresia (10–50% of vitellogenic follicles) were detected in
significant changes for IGF-II (Fig. 3). In the field captured fish, some females from BE and G. In the laboratory assay, the follicular
significantly reduced levels of IGF-I were detected in sites BE and G atresia rates were about 10% in both groups, with no significant
for females and males, when compared to the non-treated group differences between treated and non-treated groups (Table 3).
and reduced levels were also detected in females from BA (Fig. 3A During follicular atresia, zona radiata splits and yolk liquefaction
and B). Levels of IGF-II were significantly elevated in females from were followed by hypertrophy of follicular cells to engulf the yolk
BA and F (Fig. 3C). In males, there was no significant difference of and zona radiata remnants, resulting in a progressive decrease in
IGF-II levels among sites and experimental groups (Fig. 3D). The the oocyte's diameter and theca thickening (Fig. 4G–I).
IGF-I and IGF-II levels observed in site T were statistically similar Batch fecundity of A. fasciatus in the Furnas reservoir ranged from
to the non-treated group for both genders. 12,347 to 68,046 eggs and 790 to 4447 eggs/cm TL, with significantly
170 P.S. Prado et al. / Environmental Research 131 (2014) 165–173

1 a 1
Females
IGF-I ELISA-absorbance (492nm)

IGF-I ELISA-absorbance (492nm)

Males
a a

a
b a
0.5 b 0.5
b
ab ab
b b
b b

0 0
Non-treated Treated T BA F BE G Non-treated Treated T BA F BE G
group group group group

0.8 0.8
IGF-II ELISA-absorbance (492nm)

IGF-II ELISA-absorbance (492nm)

b
b

ab ab
ab
0.4 0.4
a a a
a a a a
a
a

0 0
Non-treated Treated T BA F BE G Non-treated Treated T BA F BE G
group group group group

Fig. 3. Indirect ELISA analysis of hepatic levels of insulin-like growth factors (IGF-I) and (IGF-II) in females (A, C) and males (B, D) of A. faciatus under experimental and field
conditions. Fish were captured at five sites from the Furnas Reservoir in January 2011: Turvo (T), Barranco Alto (BA), Fama (F), Boa Esperança (BE) and Guapé (G). In
experimental conditions, fish received commercial diet containing 20 mg/g oestradiol benzoate (treated group) and ethanol only (non-treated group) for 7 days. Data are
given as mean of ELISA absorbance values at 492 nm 7 standard error (n¼ 10 sex/group). Statistically different values are marked with different letters, p o 0.05.

more low means in BE (26.3572.26) and G (24.8672.62), repre- in areas closest to the discharges (Williams et al., 1999; Marfil-
senting 37% and 41% reduction respectively (Table 2). Vega, et al., 2010) especially in semi-closed waters such as in the
Furnas Reservoir. In the sites T, BA and F the total oestrogen was
o50 ng/l, this may be because the municipal wastewater dis-
4. Discussion charges are far from these sampling sites. In contrast, input of
organic material, high conductivity and increased total phosphor-
Understanding the effects of endocrine disrupting chemicals on ous, resulting in water eutrophication, has been observed in BE
aquatic organisms is a challenge since they are continuously and G (Prado et al., 2011), which are highly contaminated sites
exposed to increasing amounts of a mixture of contaminants in with xenoestrogens.
their natural habitat. In this regard, the current study reports The dietary route is an effective method of hormonal treatment
oestrogenic contamination in the Furnas Reservoir and the repro- for fish as confirmed by increased levels of total oestrogen in
ductive effects on the feral fish A. fasciatus by applying an treated males of the present study and it is a technique often used
integrated approach based on field and experimental assessment in aquaculture (Angus et al., 2001; Cabas et al., 2011). Moreover,
of multiple biomarkers of endocrine disruption. Besides the 20 μg/g of OB for 7 days was sufficient to increase the hepatic
analysis of Zrp and Vtg, the data contribute to improving the concentration of total oestrogen in females and males and induce
current knowledge on the relationship between hepatic IGF-I and hepatic expression of Zrp and Vtg in males of A. fasciatus, hence it
IGF-II and reproductive endpoints in teleost fish. was used as a positive control for biomarkers analyses data. The
Few studies on xenoestrogens effects have been reported in induction of hepatic Zrp and Vtg in males may have resulted in
closed or semi-enclosed waters such as lakes (Jin et al., 2013). In increased HSI and also influenced the K values. In mosquitofish,
the present study, the ELISA assays revealed the presence of 130.5 dietary intake of synthetic oestrogen 17α-ethynyl oestradiol (EE2)
and 121.0 ng/l of total oestrogen in water from sites BE and G, at 10 mg/g for 7 days stimulated the Vtg production in adult males
respectively, then confirming the high oestrogenic contamination (Tolar et al., 2001) while exposure to 800 ng/l OB for 3 weeks
from municipal wastewater discharges in these sites. Oestrogens increased the Zrp levels in medaka and testis–ova was also
have been measured in sewage effluents at concentrations of 0.5– observed (Hirakawa et al., 2012). Handling and transport evokes
62 ng/l in Europe (Kuch and Ballschmitter, 2001, Johnson et al., a variety of responses in fish that will possibly cause physiological
2005; Hinteman et al., 2006), 176 ng/l in Canada (Atkinson et al., adaptation affecting growth rates, feeding and behaviour (Barton,
2012) reaching 1150 ng/l in Korea (Sim et al., 2011). Concentrations 2002). In this study, fish from experimental groups maintained in
from 600 to 5000 ng/l of oestrone and from 6.8 to 6000 of 17β captivity for recovery (90 days) presented higher rates of follicular
oestradiol were detected in water from the Rico stream, Jabotica- atresia (  10%) than field captured fish, except for BE. In general,
bal city and in the Atibaia River, Campinas city, in São Paulo State, when fish are under culture conditions, failures occur in the
south-eastern Brazil (Lopes, 2010). After discharge in the super- gonadal maturation with increased follicular atresia and hormonal
ficial waters, the total concentrations of oestrogen decrease by induction is necessary to stimulate spawning (Micale et al., 1999).
about 75% in 72 h under oxygenated conditions, however, the Levels of Vtg and Zrp in males are highly specific biomarkers of
continuous discharge of sewage effluents maintain the high levels exposure to oestrogens (Sumpter and Jobling, 1995; Lee et al.,
 P.S. Prado et al. / Environmental Research 131 (2014) 165–173 171

Fig. 4. Histological sections of mature ovaries in A. fasciatus (A, B). (A) Vitellogenic follicles (VF) with central nucleus (N) and perinucleolar follicles (PN); (B) vitellogenic
follicle with nucleus displaced toward the micropyle (arrow); (C) ovary showing numerous over-ripe oocytes (OR); (D) detail of over-ripe oocyte with basophilic material
concentrated in the peripheral ooplasm (arrows), thickened zona radiata (ZR), yolk liquefaction (Y) and cortical vesicles in clusters (CV); (E) cortical vesicles (CV) stained with
Alcian Blue pH 2.5 in a healthy vitellogenic follicle; (F) peripheral ooplasm stained with Alcian Blue pH 2.5 in an over-ripe oocyte with perivitelline space in formation (PV);
(G) early follicular atresia with yolk liquefaction (Y); (H) advanced follicular atresia with hypertrophied follicular cells (FC); and (I) final follicular atresia with zona radiata
remnants (ZR) and thickening of the theca (T). Bar scale: 193.0 mm (A), 20.6 mm (B), 244.3 mm (C), 77.3 mm (D), 41.3 mm (E), 31.4 mm (F), 117.9 mm (G), 108.5 mm (H), and
206.3 mm (I).

2002a, 2002b; Truman and van den Hurk, 2012; Leonardi et al.,
2011; Genovese et al., 2011). The elevated levels of these proteins
detected in the current study in male fish from BE and G were
similar to those detected in the OB treated group associated to
intersex records support the effects of oestrogenic contamination
in fish from the Furnas Reservoir. Females from BE and G also
showed increased hepatic levels of Zrp and Vtg, enlarged diameter
of vitellogenic follicles and high frequency of over-ripening in the
ovaries. These changes suggest a continuous ovarian growth
stimulated by xenoestrogens, leading to a delay in the steroido-
genic shift that triggers final oocyte maturation and spawning
(Johnson et al., 2008). This spawning delay may generate over-
ripening which is characterised as aging of oocytes in the ovary or
in the coelomic cavity and is associated with a decrease in
fecundity and egg viability (Flett et al., 1996; Lahnsteiner, 2000).
In a field study, over-ripening was reported in fish from a
xenoestrogen polluted site from Lake Erie, USA and associated
with poor fertilisation and low survival at hatching (Flett et al.,
1996).
Additionally, decreased IGF-I levels as detected in this study
may be impairing the final oocyte maturation in the females from
sites BE and G. In mummichog (Fundulus heteroclitus), the stimu-
lating effect of IGF-I on oocyte maturation was even more rapid
and effective than 17α, 20β-dihydroxy-4-pregnen-3-one (DHP),
the major maturation-inducing steroid hormone, that is synthe-
sised by the interaction of the theca and granulosa cell layer
(Negatu et al., 1998). in vitro, treatment of post-vitellogenic
follicles with IGF-I increased concentration of DHP, regulating
the final oocyte maturation (Mukherjee et al., 2006). Also, IGF-I
can induce oocyte maturational competence and resumption of
meiosis (germinal vesicle breakdown, GVBD) by a steroid-
independent pathway in some fish species (Weber et al., 2007).
Low frequency of follicular atresia (o10%) was observed in the
field study and significant differences were detected only in site
BE. Thus, over-ripening was a suitable biomarker for assessing
xenoestrogenic contamination in A. fasciatus, while follicular
atresia did not reflect the environmental impact in the Furnas
reservoir. In contrast, increased rate of follicular atresia has been
observed under natural and experimental conditions when fish are
exposed to EDCs (Janz et al., 2007; Kidd et al., 2007; Kaptaner and
Ünal, 2010). As IGF-I act as cell survival factor (Reinecke, 2010b)
the decreased levels of IGF-I as detected in BE and G in the present
study could activate the apoptosis pathways to lead to follicular
atresia, thus reducing the fecundity. Although few studies evalu-
ated xenoestrogen effects on fish fecundity in field conditions,
experimental data showed that exposure to environmental
172 P.S. Prado et al. / Environmental Research 131 (2014) 165–173
relevant concentration (5 ng/L) of EE2 in the zebrafish (Danio rerio)
caused a 56% reduction in fecundity and complete failure in
fertilisation (Nash et al., 2004). Similarly, in medaka Oryzias latipes,
rates of fertilisation and hatching were significantly reduced in
groups exposed to 60 and 120 ng/L of EE2 (Hashimoto et al., 2009).
In the sites BA and F, the concentrations of total oestrogen were
below of the detection limit and induction of Vtg and Zrp in males
was not observed. Higher frequency of resting (stage 1) and
reduced diameter of vitellogenic follicles (Prado et al., 2011,
present study) associated to impaired Zrp levels in females as
observed in current study suggest a possible inhibition of ovarian
development. This could be caused by contamination from agri-
cultural and industrial wastes that changed the physiochemical
water parameters i.e. pH and dissolved oxygen rates. Moreover,
elevated concentrations of aluminium, iron and zinc as well as
organochlorine (aldrin/dieldrin, endosulfan, heptachlor/hepta-
chlor epoxide and metolachlor) as detected in these sites
(Fernandes et al., 2013), could disrupt the endocrine system of
A. fasciatus.
In ovaries, IGF-II seems to promote proliferation of follicular
cells and oocyte growth and maturation in red seabream (Kagawa
et al., 1994), mummichog (Negatu et al., 1998), and short-finned
eel (Lokman et al., 2007). In this sense, increased hepatic levels of
IGF-II observed in females from BA and F in the present study,
indicate a possible physiological response in an attempt to recover
ovary maturation. in vitro studies in rainbow trout ovaries showed
no significant change in the IGF-I mRNA levels during acquisition
of oocyte maturational competence (OMC), however, IGF-II mRNA
levels were significantly higher in females exhibiting high OMC
(Bobe et al., 2003). Also, in mummichog, IGF-II induced GVBD, a
marker for resumption of meiosis (Negatu et al., 1998). In the
present study, the lower levels of IGF-II in females from BE and G
when compared to BA and F can be related to the over-ripening
process. Thus, our data indicate the IGF system as a potential
target of EDC's in fish, particularly in females.
Fish from site T showed reproductive and biomarkers responses
similar to those from the non-treated experimental group (nega-
tive control), indicating a low risk of oestrogenic contamination.
This site has little anthropogenic interference, is surrounded by
dense vegetation and far from industrial or urban pollution
sources and it has been considered a reference site in the Furnas
Reservoir (Prado et al., 2011, Fernandes et al., 2013).
In summary, our results indicate that xenoestrogenic contam-
ination in the Furnas Hydroelectric Reservoir induced changes in
hepatic levels of Zrp, Vtg, IGF-I and IGF-II which may be related to
the reproductive impairment observed in A. fasciatus. Besides Zrp,
Vtg and intersex, the novel reproductive biomarkers used in the
current study, i.e. IGF-I, IGF-II and over-ripening, were appropriated
to assessing xenoestrogenic contamination in the Furnas Reservoir
and are indicated to monitoring programs in South America.
Acknowledgments
We thank Furnas Centrais Elétricas S/A, especially Mr. Dirceu
Marzulo Ribeiro for supporting the fish collections; Dr. Patrícia
M. Martinelli and collaborators for ELISA assistance and also Dr.
Steve Latham for review the English language. The work was
supported by grants from Brazilian Research Foundations: CNPq,
FAPEMIG and CAPES.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.envres.2014.03.002.
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