Ecotoxicology and Environmental Safety 74 (2011) 1879–1887

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

0147-65

doi:10.1

n Corr

E-m

journal homepage: www.elsevier.com/locate/ecoenv

Reproductive disruption in lambari Astyanax fasciatus froma Southeastern Brazilian reservoir

Paula S. Prado a, Cassia C. Souza a, Nilo Bazzoli b, Elizete Rizzo a,n

a Departamento de Morfologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, UFMG, Belo Horizonte, C.P. 486, 30161-970 Minas Gerais, Brazilb Programa de Pos-Graduac- ~ao em Zoologia de Vertebrados, Pontifıcia Universidade Catolica de Minas Gerais, PUC Minas, Belo Horizonte 30535-610, Minas Gerais, Brazil

a r t i c l e i n f o

Article history:

Received 25 September 2010

Received in revised form

3 June 2011

Accepted 21 July 2011Available online 9 August 2011

Keywords:

Astyanax fasciatus

Furnas Reservoir

Endocrine disruption

Intersex

Zona radiata proteins

13/$ - see front matter & 2011 Elsevier Inc. A

016/j.ecoenv.2011.07.017

esponding author. Fax: þ55 31 34092771.

ail address: ictio@icb.ufmg.br (E. Rizzo).

a b s t r a c t

The aim of this study was to assess the health indicators and reproductive endpoints in a wild

population of lambari Astyanax fasciatus, a small characid fish widely distributed in South America. A

range of biomarkers, from the molecular to population levels, was evaluated in adult fish sampled from

five sites from the Furnas Reservoir, Grande River. At the sites that receive untreated agriculture and

industrial residues, fish showed reduced body size and delayed gonadal maturation. Fish from the sites

located immediately downstream from the municipal wastewater discharges exhibited feminisation,

intersex and evidence of contamination by xenoestrogens. Elevated levels of zona radiata proteins were

found by Western blot in the livers of male fish from three contaminated sites. Overall, this study

provides the first evidence of endocrine disruption in a wild fish population inhabiting the Furnas

Reservoir, and suggests water contamination by oestrogenic compounds and agricultural residues with

a possible risk to human health and wildlife.

& 2011 Elsevier Inc. All rights reserved.

1. Introduction

It is well established that a wide range of structurally diversechemicals of anthropogenic origin share the ability to disturb thephysiology and endocrinology of organisms (Goksøyr, 2006).These hormonally active pollutants known as endocrine disrupterchemicals (EDCs) affect the health of both humans and wildlife.Among EDCs, chemicals that have oestrogen-like properties arereferred as xenoestrogens (Denslow and Sepulveda, 2007).Although domestic sewage is widely recognised as one of themajor discharge sources of xenoestrogens, the impact of sewageeffluents has rarely been investigated in South America.

The Furnas hydroelectric power plant was built on the GrandeRiver, Upper Parana River basin, Southeastern Brazil, in the mid-60s. It is complex reservoir with 22 billion m3 of water and twomajor sub-regions that correspond to the Grande and SapucaiRivers, each one measuring about 250 km. The Furnas Reservoirfloods a total area of 1440 km2 and supplies water to a localpopulation of over 1 million people. Besides receiving more than2000 tonnes of waste solids daily from the surrounding cities, thetourism industry has developed in marginal areas of the reservoirin recent years. Untreated sewage and pollution by pesticides hasgradually reduced the water quality requiring special attention

ll rights reserved.

from public institutions in Brazil (Santos-Neto and Siqueira, 2005;IGAM, 2008). Proliferation of cyanobacteria and fish contaminatedwith toxin microcystin has been reported recently in tilapia,representing a risk to human health (Deblois et al., 2008).

Aquatic organisms are targets of EDCs and fish studies canprovide a basis for effective cross-species extrapolation ofresponses (Arukwe and Goksøyr, 2003; Ankley et al., 2009). EDCscan disrupt different levels of biological organisation from themolecular to the ecosystem (Denslow and Sepulveda, 2007).Recent investigations indicate the importance of consideringmultiple endpoints at different biological levels for accurate andeffective assessment of the integrated effects of EDCs (Ankleyet al., 2009).

The lambari do rabo vermelho Astyanax fasciatus (Cuvier, 1819),characiform fish from the family Characidae, is widely distributedin the La Plata River basin and is abundant in several Brazilianecosystems. Besides its importance as a forage fish, it also supportsfishing in the Furnas Reservoir becoming an important proteinsource for riverine populations (Carvalho et al., 2009). Due to itswide distribution in both clean and polluted waters, small size,omnivorous feeding habit and a reproductive strategy with multi-ple spawning peaks during the year, this lambari has beenindicated as a sentinel species for environmental investigationsand exposure assays (Schulz and Martins-Junior, 2001; Albertoet al., 2005; Carrasco-Letelier et al., 2006).

In fishes, two critical time windows share enhanced sensitivityto EDCs: gonad differentiation during early life-stages, and the

Table 1Localisation and type of impact in five sites from the Furnas Reservoir, Grande

River.

Sampling sites Impact

Turvo (T) 201400S;

461130W

Reference site with little anthropogenic

interference, located about 30 km from Capitolio

city, the largest city near the site, with 8.000

inhabitants and without industrial activity

Barranco Alto (BA)

211110S; 451580W

Agricultural area that receives residues from the

coffee, potatoes and corn crops cultures, mainly

nitrogen, phosphate and potassium (NPK) fertilizers,

organochlorine (endosulfan, DDT, DDE),

organophosphate (malathion) and carbamate

(furadan) insecticides and glyphosate (roundup) and

atrazine herbicides

Fama (F) 211240S;

451500W

Urban area that receives untreated industrial and

domestic sewage from the cities surrounding the

Verde and Sapucaı Rivers. The major sources of

pollution are the industries of electro-electronics,

information technology, metallurgy, plasticizers and

processors of grains

Boa Esperanc-a (BE)

211040S; 451330W

Urban area downstream of untreated sewage

discharges (approximately 91 tonnes of waste solids

P.S. Prado et al. / Ecotoxicology and Environmental Safety 74 (2011) 1879–18871880

reproductive period in adults. During early development, fishesare susceptible to exogenously administered sex steroids causingsex reversal, which results in irreversible changes in tissuedifferentiation, altering the sex and in the long-term, impactingthe sustainability of wild populations (Kidd et al., 2007). In adults,expression of vitellogenin (Vtg) and zona radiata proteins (Zrp) inmale fish are key biomarkers of xenoestrogen contamination withhigh mechanistic relevance. Usually, these proteins are synthe-sised in the liver of females under stimulation by ovarianestradiol, whereas their induction in males and juveniles maybe triggered by exposure to EDCs (Arukwe and Goksøyr, 2003).The major molecular constituents of zona radiata in teleostspecies are 3–4 proteins with molecular masses between 47 and129 kDa (Modig et al., 2007). Some studies have shown that theexpression of Zrp precedes that of Vtg, suggesting a highersensitivity to oestrogenic induction (Arukwe and Røe, 2008).The detection of high concentrations of Vtg or Zrp in wild maleshas been considered an early warning sign of environmentalpollution by oestrogenic EDCs (Maradonna and Carnevali, 2007).Additionally, body size, gonadal development and conditionfactor are frequently used as indicators of overall health inexperimental and field studies as they are biomarkers of highecological relevance (Ankley et al., 2009).

Due to the lack of studies dealing with the effects of environ-mental pollution on South American neotropical ichthyofauna, thepurpose of this study was to evaluate health indicators, reproduc-tive parameters and endocrine disruption biomarkers in A. fasciatus

inhabiting contaminated sites in the Furnas Reservoir.

daily) outfall from the Boa Esperanc-a city with

39.000 inhabitants

Guape (G) 201450S;

451550W

Urban area downstream of untreated sewage

discharges (more than 6 tonnes of waste solids

daily) outfall from the Guape city with 14.000

inhabitants

Data obtained from IGAM (2008), Tanure (2003), IBGE (2004) and EMBRAPA

(2011).

2. Material and methods

2.1. Fish sampling and water parameters

A total of 1265 adult specimens of A. fasciatus were caught quarterly at five sites

in the Furnas Reservoir, Grande River (Fig. 1), thus totalling eight fish collections

Fig. 1. Localisation of sampling sites in the Furnas Reservoir in Minas Gerais, Southeast

(BE); Guape (G). The highlighted area in black on the map of Minas Gerais state indica

performed during 2006 and 2007. The environmental pollution sources at the

sampling sites, i.e. Barranco Alto (BA), Fama (F), Boa Esperanc-a (BE) and Guape (G),

are described in Table 1. A site surrounded by dense vegetation and that is far from

industrial or urban pollution sources was chosen as the reference (Turvo, T). In each

sampling site, fish were captured using 100 m of gillnets with a 3–4 cm stretched

mesh size deployed for about 14 h on the surface of water and placed at a distance

of 30 m from the shoreline of the reservoir. The collections were supported by a

ern Brazil. Reference sites: Turvo (T); Barranco Alto (BA); Fama (F); Boa Esperanc-a

tes the Furnas Reservoir area.

P.S. Prado et al. / Ecotoxicology and Environmental Safety 74 (2011) 1879–1887 1881

team of professional fishermen from Furnas Hydrobiology and Hatchery Station.

The research was approved by the Committee of Ethics and Animal Experimenta-

tion (CETEA, 2011) from the Federal University of Minas Gerais.During the fish collections, temperature, pH, dissolved oxygen and conductiv-

ity at 25 1C (specific conductance) were measured at each sampling site at a depth

of 1 m from the water’s surface using the YSI 650 MDS multi-parameter instru-

ment. Water transparency was evaluated using Secchi disc depth. Suspended solid

residues were determined by the Furnas Hydrobiology and Hatchery Station’s

laboratory for samples of 1 L of water that were filtered using a vacuum pump and

application of the gravimetric method.

2.2. Gonad maturation and morphometry

Specimens captured alive were kept in plastic containers with water from the

reservoir until dissection. A transversal section of the middle region of the left

gonad was obtained from each fish. This was then fixed in Bouin’s fluid for 8–12 h

and kept in 70% ethanol for histological processing. The gonad samples were then

gradually dehydrated in ethanol, embedded in paraffin, sectioned at 5 mm

thickness and then stained with haematoxylin–eosin (HE). The following gonad

maturation stages were established based on their macroscopic and microscopic

features: (1) resting, (2/3) advanced maturation/ripe, (4A) partially spent and (4B)

totally spent (Table 2). The sex ratio (females/males) was assessed for each site

and sampling period, following gender validation by histology.

Morphometric analyses of gonadal maturation were conducted on histological

sections of gonads in stage 3 (n¼10 to 15/site), sampled during the reproductive

period’s peak (January 2007). For females, proportions (%) of perinucleolar, vitello-

genic and atretic follicles were determined in ovarian sections (n¼15/site), and the

diameter of vitellogenic follicles (n¼250/site) was measured for each site. For males,

the diameter of seminiferous tubules (n¼150/site) and amount of spermatogenic

cysts per field (n¼75/site) were determined for each site, under 400� magnification.

2.3. Health indicators

After excision of the left gonad, all specimens were preserved in 10% formalin

solution and kept in 70% ethanol for 60 days at room temperature (25 1C) for

biometrical analyses. In the laboratory, total length (TL), as well as body weight

(BW), right gonad weight (GW�2), liver weight (LW) and coelomic fat weight

(CFW) were measured for each fish. A deviation of about þ5.5% in weight and

�2.5% in length has been noted using this methodology when compared to

measurements of live specimens (Anderson and Neumann, 1996; Carvalho et al.,

2009). The following health and reproductive parameters were calculated as

previously established (Le Cren, 1951; Magalh~aes et al., 2004; Carvalho et al.,

2009): gonadosomatic index (GSI¼100 GW�2/BW), hepatosomatic index

(HSI¼100 LW/BW), coelomic fat index (CFI¼100 CFW/BW) and Fulton condition

factor (K¼100 BW/TL3).

Table 2Macroscopic and microscopic features of gonadal maturation stages of Astyanax

fasciatus form Furnas Reservoir.

Females Males

Resting (1) Translucent and thin ovaries

surrounded by fat. Presence of

initial and advanced

perinucleolar follicles and

oogonia nests

Slender and transparent testis

with reduced volume and

surrounded by fat.

Seminiferous tubules with

spermatogonia only and

occluded lumen

Maturation/

ripe (2/3)

Consistent yellowish ovaries,

increased volume, occupying

gradually most of the coelomic

cavity with follicles visible to

the naked eye. Presence of

perinucleolar, previtellogenic

and vitellogenic follicles

Milky white testis, increased

volume, occupying gradually

large part of the coelomic

cavity. Seminiferous tubules

with cysts of spermatogenic

cells and lumen full of

spermatozoa embedded in

acidophilic secretion

Partially

spent

(4A)

Flaccid ovaries with

haemorrhagic areas and few

follicles visible. Presence of

perinucleolar, previtellogenic,

vitellogenic and postovulatory

follicles

Flaccid testis with

haemorrhagic and transparent

areas. Few cysts of

spermatogenic cells with

tubular lumen partially empty

Totally

spent

(4B)

Very flaccid and haemorrhagic

ovaries, without follicles visible.

Presence of perinucleolar,

postovulatory and atretic

follicles

Testis with reduced volume

and transparent aspect.

Seminiferous tubules

containing spermatogonia and

empty tubular lumen with

residual spermatozoa only

2.4. Zona radiata (eggshell) proteins

In January 2007, we also collected fresh liver samples from 5 males from each

sampling site and 5 females from Turvo (reference site) for analyses of the zona

radiata proteins (Zrp) by Western blotting. In the field, the samples were frozen in

liquid nitrogen and preserved at �80 1C for assays. In the laboratory, the samples

were homogenised in extraction buffer (50 mM Tris–HCl pH 8.0, 0.02% aprotinin

and 1 mM phenylmethylsulfonyl fluoride) using a Potter S homogeniser (Braun,

Melsungen, Germany) held in an ice-water cooled bath. Then, the extracts were

sonicated (3�10 s) in an ultrasonic processor GEX 600 CE and centrifuged at

15000g for 60 min at 4 1C. After pellet removal, the supernatant was stored in

aliquots at –80 1C until analysis. Total soluble protein was determined by the

Lowry method using bovine serum albumin (BSA) as standard. To assess the

method’s sensitivity, aliquots containing 400, 200, 100, 50 and 25 mg proteins of

female liver samples from Turvo were submitted to 7.5% sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS-PAGE). The 100 mg aliquots of proteins

provided sufficient material to clearly observe the three immunoreactive bands

corresponding to Zrp-a (�70 KDa), Zrp-b (�50 KDa) and Zrp-g (�35 KDa)

proteins, therefore this concentration was chosen for the further analysis. Aliquots

containing 100 mg of proteins were loaded into each lane and submitted to 7.5%

(SDS-PAGE). Following this, the separated proteins were transferred to nitrocellu-

lose membranes, blocked with 2% casein and incubated with rabbit anti-salmon

Zrp primary antibody (1:1000, Biosense AS., Norway) for 1 h. After rinsing, the

membranes were incubated with goat anti-rabbit secondary antibody (1:2500,

Sigma, St. Louis, Mo.) conjugated with peroxidase for 2 h and finally the bands

were revealed using 4-chloro-1-naphthol and 3.30-diaminobenzidine (DAB). For

quantification, the protein assay was performed in triplicate and the density of the

bands on immunoblots was estimated by scanning densitometry using Scion

Image Software. The specificity of the primary antibody was assessed by

immunohistochemistry. Briefly, samples of ovaries fixed in methanol/dimethyl

sulfoxide were embedded in paraplast, sectioned, immunostained with rabbit

anti-salmon Zrp primary (1:200), incubated with goat anti-rabbit secondary

antibody (1:2000) and revealed with DAB.

2.5. Statistical analyses

Statistical analyses were carried out using GraphPad Instat software. Values

were expressed as means 7SEM, and results were considered significant at 95%

confidence interval. One-way analysis of variance (ANOVA) followed by Tukey’s

test was used to compare the water quality parameters. The frequency distribu-

tion of gonad maturation stages and density of Zrp bands in Western blot were

analysed by ANOVA with Dunnett’s multiple comparison post test. Nonparametric

Kruskal–Wallis followed by Dunn’s test was performed to evaluate the biological

indices. To detect deviation from the (2 females/1 male) expected sex ratio Fisher’s

exact test was performed for sampling periods.

3. Results

3.1. Water quality and health indicators

Mean values of temperature, pH and dissolved oxygen did notvary significantly among sites (Table 3, Fig. S1). Minimum valuesof temperature were obtained in July 2006/2007 (dry season) andmaximum values in January 2007 (rainy season) (Fig. 2). Tem-peratures above 30 1C in January 2006 and pH below 6.0 inJanuary 2007 were recorded at site F. The mean depth of theSecchi disc was significantly lower in the impacted sites com-pared to reference site (T) and it was not scored at the site BE dueto high water contamination. Dissolved oxygen values below5.0 mg/L were recorded during January 2007 at all impacted sites.Conductivity at 25 1C (specific conductance) and suspended solidresidues were significantly highest at site BE (po0.001).

Considering the biometric data and biological indices, thehighest means for TL and BW were recorded (po0.001) at thereference site (T). Fish from impacted sites BA and F hadsignificantly reduced TL and BW, when compared to other sites(Table 4). Higher values of GSI were observed in females fromsites T, BE and G, and males from sites BA, BE and G (Table 4).Comparing whole sampling, HSI for females and males weresignificantly higher in all impacted sites. Fish from BE showedthe highest CFI while K showed few variations among sites overthe sampling periods (Table 4, Fig. S2).

Table 3Water quality parameters at five sites from the Furnas Reservoir during 2006 and 2007.

Turvo Barranco Alto Fama Boa Esperanc-a Guape

Temperature (1C) 24.2770.94a (21–27) 24.8471.31a (20–30) 24.8571.51a (19–33) 25.4771.19a (20–27) 24.9370.97a (21–29)

pH 7.4970.23a (6.3–8.1) 7.5770.31a (6.0–8.5) 7.4770.33a (5.8–8.6) 7.8370.24a (6.7–8.6) 7.4470.25a (6.3–8.5)

Dissolved oxygen (mg/L) 7.1470.39a (5.7–8.5) 6.8070.47a (4.2–8.5) 7.1170.60a (3.7–9.1) 7.2170.65a (4.3–10.8) 6.4170.35a (4.3–7.6)

Conductivity to 25 1C (mS/cm) 38.1071.31a (35–45) 40.0071.87a (32–50) 39.2871.56a (31–46) 62.1475.45b (49–95) 36.4371.07a (32–42)

Secchi disc depth (m) 4.0770.30a (3.3–5.3) 1.8670.27b (0.8–3.3) 2.0870.47b (0.4–4.0) nd 2.4070.20b (1.6–3.1)

Total suspended solids (mg/L) 0.9270.59a (0.4–1.9) 2.8572.87a (1.3–9.3) 3.3571.97a (0.8–12.2) 8.1771.91b (5.4–15.8) 1.9770.33a (1.5–3.1)

Volatile solids (mg/L) 0.8070.32a (0.2–1.7) 1.5570.61a (0.5–3.3) 1.8270.53a (0.5–3.0) 5.7070.93b (3.7–7.6) 0.9270.31a (0.3–1.7)

Fixed solids (mg/L) 0.4270.20a (0.1–1.0) 1.3070.75a (0.3–3.5) 1.5370.63a (0.3–3.3) 2.4770.26b (1.2–3.3) 1.1570.19a (0.6–1.5)

Values represent mean7SEM of eight samplings. Minimum and maximum values included in parenthesis. In a line, different letters indicate significant differences among

sites, po0.05. (nd) not determined.

Fig. 2. Cyclical variations of temperature (A) and seasonal distribution of the

gonadosomatic indices (GSI) for females (A) and males (B) of Astyanax fasciatus

caught in the Furnas Reservoir during 2006 and 2007. Sampling sites: reference

site, Turvo (T); Barranco Alto (BA); Fama (F); Boa Esperanc-a (BE); Guape (G).

Values represent mean7SEM. Asterisks indicate significant difference compared

to T, po0.05.

P.S. Prado et al. / Ecotoxicology and Environmental Safety 74 (2011) 1879–18871882

3.2. Gonadal maturation

The GSI distribution over the sampling periods showed anevident reproductive seasonality in females and followed by thegonad maturation cycle at the reference site (T), with GSI peaks inJanuary when temperature was more elevated (Fig. 2). Similarly,the lowest values of GSI occurred in July coinciding with theperiod of lowest temperature. In contrast, alterations to thispattern were recorded at the impacted sites, especially at site F,where low GSI means were observed, even in periods of hightemperature. Conversely to females, variations of GSI in male fish

were less pronounced at all sites, with the highest meansobtained in BE and G during January 2006 and 2007.

In all sampling sites, the frequency (%) of gonadal maturationstages, determined macro- and microscopically, showed thatfemales and males in stages 2/3 (maturation/ripe) and 4A (par-tially spent) were predominant in most periods, except in F(Fig. 3). Resting females were significantly more abundant in siteF and resting males were not sampled at sites BE and G. Intersexgonads were found in BE (25% January 2006 and 29% January2007) and G (25% January 2007) from fish classified macroscopi-cally as males during the reproductive peak (Fig. 3). No intersexfish were founded at sites T, BA and F.

The morphometric analyses of gonadal maturation in stage3 showed a distribution close to 40% of perinucleolar follicles and60% of vitellogenic follicles in females from site T (Table 5).Perinucleolar follicles predominated in females from sites BA(60%) and F (64%). Follicular atresia was low in all sampling sites,varying from 3% to 6%, with no significant differences among sites.The diameter of vitellogenic follicles was lower in fish from sitesBA, F and BE compared to site T. In males, the mean diameter ofthe seminiferous tubules was significantly lower in site F andhigher in site G. Cysts of spermatogonia were less frequent in fishfrom all impacted sites while spermatocytes were more numerousin fish from site F and spermatids were abundant in sites BE and G.

3.3. Sex ratio, intersex and zona radiata proteins (Zrp)

Considering the whole sampling from each site, the sex ratio wassignificantly similar to the expected (2 females/1 male) in the sites T(2.3/1, Fisher’s test, p¼0.5556) and F (1.8/1, p¼0.6596). An accen-tuated prevalence of females to males was detected in sites BE (4/1,p¼0.0069) and G (6.8/1, po0.0001). Unlike, the sex ratio (1.3/1,p¼0.0245) was significantly different to the expected in BA. Thesedeviations from the expected sex ratio were confirmed analysing thebinomial distribution of data for each sampling period (Fig. S3).

At the maturation stage, normal ovarian tissue showed peri-nucleolar and vitellogenic follicles (Fig. 4A) and the testiculartissue had seminiferous tubules with germ cell cysts in differentdevelopmental phases (Fig. 4B). Intersex fish showed ovarianfollicles organised in two different patterns: (1) focal arrange-ment with large patches of perinucleolar and vitellogenic folliclesassociated with testicular tissue in distinct foci (Fig. 4C); (2)multifocal arrangement with single or small clusters of perinu-cleolar follicles within seminiferous tubules (Fig. 4D). The focalpattern (Fig. 4E and F) was predominant in the intersex gonads,and vitellogenic follicles occupied a considerable space in thehistological sections, disrupting the testicular structure. Atreticfollicles were also found in the intersex gonads, but postovulatoryfollicles were not detected.

In Western blotting, liver samples of females exhibited threeimmunoreactive bands corresponding to Zrp-a (�70 KDa), Zrp-b

Table 4Biological indices and sex ratio in A. fasciatus captured at five sites from the Furnas Reservoir during 2006 and 2007.

Turvo Barranco Alto Fama Boa Esperanc-a Guape

Females N 223 110 185 136 245

TL (cm) 13.8670.11a 11.6670.14b 11.9670.13b 12.9270.09c 13.1470.10c

BW (g) 34.6070.89a 20.3970.89b 22.2770.77b 29.1770.75c 29.8370.73c

GSI 6.7470.39a 4.4570.46b 4.4370.47b 7.3670.46a 7.0370.40a

HSI 0.5370.01a 1.0070.06b 0.8770.03b 0.9470.09bc 0.7170.02c

CFI 0.6670.07a 0.5570.08a 0.7670.07a 1.8170.20b 0.9370.09ab

K 1.2470.01a 1.2170.02a 1.2270.01a 1.3270.01b 1.2570.01a

Males /intersex N 100 83 101 43 39

TL (cm) 13.1470.18a 10.7170.10b 10.4270.10b 11.5570.14c 12.0570.20ac

BW (g) 27.7470.89a 15.6570.89b 13.3370.77c 19.4370.75a 21.4670.73a

GSI 1.1170.13a 1.5570.09b 0.8970.08a 2.4970.19c 1.5870.18b

HSI 0.4670.02a 0.8870.03b 0.8370.04bc 0.6570.04c 0.6070.03ac

CFI 1.8070.14a 1.3670.14ab 1.0770.10b 2.1270.28a 1.5970.28ab

K 1.1870.01a 1.2570.02b 1.1470.02a 1.2370.02ab 1.1870.03ab

Values represent mean7SEM. In a line, different letters indicate significant differences among sites, po0.05. (TL) total length; (BW) body weight; (GSI) gonadosomatic

index; (HSI) hepatosomatic index; (CFI) coelomic fat index; (K) condition factor.

P.S. Prado et al. / Ecotoxicology and Environmental Safety 74 (2011) 1879–1887 1883

(�50 KDa) and Zrp-g (�35 KDa) monomers. In male fish, themonomers Zrp-b and Zrp-g were detected in fish from sites F andG, while only Zrp-g occurred in site BE. In quantifying the bandsin immunoblotting, Zrp expression was significantly higher infemale fish from T (reference site) and male fish from sites F, BEand G, when compared to male fish from T and BA (Fig. 5A). Inovaries, the immunostaining of Zrp proteins was observed in thezona radiata (eggshell) of vitellogenic follicles, confirming thespecificity of the antibody (Fig. 5B).

4. Discussion

The current study examined multiple endpoints, ranging fromthe molecular to population levels, for an accurate evaluation ofthe health and reproductive indicators of A. fasciatus in the FurnasReservoir. This highly integrated assessment supports the hypoth-esis of water contamination in BA, F, BE and G and also the use ofthis feral characid as a useful bioindicator of environmentalimpact in the Furnas Reservoir.

According to the major biological responses, the sampling sitesinvestigated in the present study were grouped according to threedifferent patterns: (1) T (reference site), where the fish showedthe best health and reproductive status; (2) BA and F, where fishhad reduced body size and delayed gonadal maturation; (3) BEand G, where feminisation and intersex were detected. In thereference site (T), the physical–chemical parameters of the water(temperature, pH and dissolved oxygen) were within the normalranges for reproductive success of neotropical fish species (LoweMcConnel, 1987). In contrast, dissolved oxygen below 5.0 andlower water transparency were detected in BA, F, BE and G, sitesthat receive residues from industrial and domestic sewage as wellas agriculture. High temperatures around 30 1C occurred at sites F,BE and G where the domestic sewage is the main source ofpollution. Changes in the water quality were detected mainly inJanuary, the reproductive peak, and may have influenced thehealth and reproductive endpoints of A. fasciatus. Due to therecent increase in tourism, Furnas Reservoir, requires specialattention by the public institutions for conservation of the wildfish populations, such as A. fasciatus, which is well adapted tolentic environment.

The health parameters of fish have been used to evaluate thebiological responses to complex mixtures of contaminants and tounderstand the mechanism of interactive chemical effects on

biological systems, principally in field studies (Filby et al.,2007). In the present study, the higher HSI values observed inall impacted sites, as compared to T, could be associated withliver disorders that need to be further investigated in A. fasciatus

in the Furnas Reservoir. Diverse hepatic alterations such as lipidaccumulation, hypertrophy for removal of toxicants or an alteredsynthesis of vitellogenin, Zrp and other proteins inducible by EDC,as well as oxidative stress, have been reported in teleosts exposedto organic compounds and heavy metals (Fishelson, 2006; vanDyk et al., 2007). These results indicate a need to evaluate theendocrine disruptor residues and heavy metals in the FurnasReservoir.

In the sites BA and F, female and male fish had reduced bodysize when compared to fish from sites T, BE and G, suggesting agrowth deficiency. In these sites, the smallest fish reachinggonadal maturation was 8.8 cm, a larger size than the length offirst sexual maturation (close to 6.0 cm) reported for Astyanax

(Carvalho et al., 2009). Moreover, analyses of frequency ofgonadal maturation stages and GSI suggest a delayed gonadalmaturation in sites BA and F. This suggestion is supported bymorphometric analyses, where a smaller diameter of vitellogenicfollicles and a prevalence of perinucleolar follicles were obtainedat both sites, as well as the smaller diameter of seminiferoustubules were recorded in fish from site F. Also for females, GSI infish from site F did not follow the cyclical variations of tempera-ture, exhibiting means below 2 in periods of high temperatures(above 25 1C). Several factors can influence the size and gonadalmaturation of fish, including food availability, genetic profile,abundance of predators and environmental status (Bagenal andTesch, 1978). Impaired development and reproduction has beenreported in several fish exposed to EDCs, such as in the mosquito-fish Gambusia sp. subject to paper mill effluent in the Fenhollo-way River, Florida, USA (Orlando et al., 2007), and in pollutedwetlands in Western Australia (Game et al., 2006). Under experi-mental conditions, Shved et al. (2008) observed long-lastingconsequences to growth and sexual development and also altera-tions to mRNA levels for insulin-like growth factor (IGF-I) andgrowth hormone (GH) in tilapia Oreochromis niloticus, which wereexposed to 17a-ethinylestradiol. Despite the evidences, addi-tional studies are needed to clarify the relationship betweenpollution sources and impacts on health and reproductiondetected in fish from sites BA and F.

In the sites, which receive untreated domestic sewage, theorganic material load reduced the water quality in a way that

Fig. 3. Seasonal distribution of the relative frequencies (%) of the gonad maturation stages for females and males of Astyanax fasciatus caught at five sites from the Furnas

Reservoir during 2006 and 2007. Gonad maturation stages: 1¼resting, 2/3¼maturation/ripe, 4A¼partially spawned/spent and 4B¼totally spawned/spent. Asterisks

indicate significant difference, po0.05.

Table 5Morphometry of the gonadal maturation in A. fasciatus captured at five sites from the Furnas Reservoir during 2006 and 2007.

Turvo Barranco Alto Fama Boa Esperanc-a Guape

Females Perinucleolar follicles (%) 39.2273.21 60.4779.31n 64.0177.82n 40.2972.01 32.4472.25

Vitellogenic follicles (%) 60.6873.21 39.5379.31n 35.9877.82n 59.7172.01 67.5672.25

Vittelogenic follicles diameter (mm) 589.9073.54 567.2073.62n 560.8074.25n 561.4073.44n 587.8073.57

Males Spermatogonia cysts (%) 27.3673.40 17.5772.50n 18.4172.18n 15.9371.85n 12.5071.85n

Spermatocytes cysts (%) 58.3674.06 65.6472.09 70.4972.74n 62.1472.07 65.5071.58

Spermatids cysts (%) 14.2872.71 16.7971.21 11.1071.21 21.9371.24n 22.0071.51n

Seminiferous tubule diameter (mm) 177.8673.51 175.4873.48 151.9473.07n 173.6473.46 190.4773.12n

Values represent mean7SEM. In a line, asterisks indicate significant differences from the reference site (Turvo), po0.05.

P.S. Prado et al. / Ecotoxicology and Environmental Safety 74 (2011) 1879–18871884

Fig. 4. Gonadal tissue sections of Astyanax fasciatus sampled in Furnas Reservoir. (A) Ovarian tissue in maturation (stage 2) with perinucleolar (PF) and vitellogenic follicles

(VF). (B) Testicular tissue in maturation with seminiferous tubules containing spermatogenic cysts (SC) and spermatozoa (Z). (C) Intersex gonad with focal arrangement of

the ovarian tissue (OV) into the testis (TE). (D) Intersex gonad with multifocal arrangement of perinucleolar follicles into the testis. (E) and (F) Focal intersex gonad

showing both perinucleolar and vitellogenic follicles (E) and spermatozoa among vitellogenic follicles (F). Scale bars: 150 mm (A, B, C, E), 50 mm (D), 25 mm (F).

P.S. Prado et al. / Ecotoxicology and Environmental Safety 74 (2011) 1879–1887 1885

significantly increased the conductivity at 25 1C (specific conduc-tance), especially in site BE, where around 70% of organic material(volatile solids) were recorded. In general, organically pollutedenvironments show high productivity levels and the few species,which survive in these environments may become abundant, suchas A. fasciatus (Alberto et al., 2005). Considering this, the highestCFI and K averages observed in the current study for fish from siteBE may be reflecting the abundant nutrients associated withsewage discharges. Similar results were reported for domestic-sewage-polluted sites in the Sinos and Camamducaia Rivers,Brazil (Schulz and Martins-Junior, 2001; Alberto et al., 2005).According to Filby et al. (2007), xenoestrogens from humanwastes are associated with solid residues in domestic sewageand cause adverse effects on reproduction and development offeral fish, therefore constituting a pervasive ecological problem. Inthe sites BE and G, higher GSI averages and elevated frequenciesof maturation/ripe females suggest a continuous ovarian growthpossibly stimulated by xenoestrogens from human wastes, thusleading to a delay in the steroidogenic shift that triggers finaloocyte maturation and spawning, as has also been reported inother species (Johnson et al., 2008).

Sex ratio (2 females/1 male) has been frequently detected infield studies, mainly in Astyanax species (Carvalho et al., 2009;Mazzoni et al., 2005) and could be related to the selectivity of

gillnets used during the samplings such as detected in sites T andF. However, we also detected females consistently more numer-ous than males in sites BE and G. Additionally, we found in thesesites a high proportion of intersex gonads, which is considered asabnormal in gonochoristic species such as A. fasciatus. Althoughthe genotype of intersex fish was not determined, the macro andmicroscopic appearance of the gonads, with prevalence of testi-cular tissue showing mature sperm, indicate that A. fasciatus

intersex specimens detected in present study were males. Inter-sex and fish feminisation are conditions that could be caused byexposure to oestrogenic and/or anti-androgenic substances dur-ing early life, thus they have been widely used to assess environ-mental pollution (Arukwe and Goksøyr, 2003; Mills andChichester, 2005; Jobling et al., 2006). Intersex gonads werefound in several fish species captured in EDC polluted rivers inNorth America, England as well as the Mediterranean Sea (Fossiet al., 2004; Hinck et al., 2009; Jobling et al., 2009). In the roach, aproportion of about 20% of intersex was found in medium-risksites with oestrogenic contamination in English rivers (Joblinget al., 2006). Compared to the intersex scores determined for theroach, the predominance of focal pattern detected in the currentstudy suggests an elevated risk of oestrogenic contamination insites BE and G. Intersex fish and a skewed sex ratio may impactthe development and reproduction of offspring (Kidd et al., 2007);

Fig. 5. Image analyses of immunoblot assays in liver (A) and immunohistochem-

ical localisation in vitellogenic follicle (B) for zona radiata proteins (Zrp) in

Astyanax fasciatus from the Furnas Reservoir. Values represent mean7SEM. (Tf)

females from Turvo, (Tm) males from Turvo, (Bam) males from Barranco Alto, (Fm)

males from Fama, (BEm) males from Boa Esperanc-a and (Gm) males from Guape.

Zona radiata (ZR). Scale bar: 12 mm. Asterisks indicate significant difference

among samples, po0.05.

P.S. Prado et al. / Ecotoxicology and Environmental Safety 74 (2011) 1879–18871886

thus the populations of A. fasciatus may be affected in the longterm in the Furnas Reservoir.

The effects of xenoestrogen contamination in the FurnasReservoir were also assessed at molecular level through analysesof zona radiata proteins (Zrp). Analysing the reproductive end-points at population and organism levels, we expected to find Zrpexpression in males from BE and G, but significant expression wasalso detected in male fish from F. This result should be an earlysign of xenoestrogen contamination in site F. The differentialexpression of Zrp-g or Zrp-b and Zrp-g proteins observed in thecurrent study may be caused by the wide variety of contaminantswith different properties in sampling sites. In this context, onlythe Zrp-b protein expression was detected in the plasma ofAtlantic salmon Salmo salar treated with a single chemical, butthe three Zrp monomers were detected when different chemicalswere combined with nonyphenol (Arukwe et al., 2000). Inductionof Zrp was also reported in the liver and plasma of Xiphias gladius

L. taken from EDC contaminated areas in the Mediterranean Sea(Fossi et al., 2004). Due to a rapid and sensitive response, Zrpexpression is able to predict changes at higher levels of biologicalorganisation (Arukwe and Røe, 2008). The molecular detection ofZrp in male fish from sites F, BE and G, combined with thefeminisation detected by intersex and deviation of the sex ratio inBE and G, provides the first evidence of endocrine disruption inpopulations of A. fasciatus and suggests xenoestrogen contamina-tion in the Furnas Reservoir.

5. Conclusion

Overall, our results indicate that adverse effects on the healthand reproductive status detected in A. fasciatus may be caused by

exposure to residues from agriculture as well as untreatedindustrial and domestic sewage released into the Furnas Reser-voir. Feminisation and intersex fish suggest a risk of oestrogeniccontamination in the reservoir. Urgent planning for treatment ofthe industrial and domestic sewage before final disposal into thereservoir and implementation of EDCs monitoring programs arerecommended to avoid the long-term detrimental effects on thepopulations of native fishes inhabiting the Furnas Reservoir. Wealso propose additional field and experimental studies to inves-tigate the mechanisms and timing of sexual differentiation in A.

fasciatus and to better understand the responses to effluents andpollutants.

Acknowledgments

We thank Furnas Centrais Eletricas S.A, especially Mr. DirceuMarzulo Ribeiro for support the fish collections; Dr. Carlos D.Chavez Olortegui and collaborators for imunoblotting assistanceand also Dr. Steve Latham for review the English language. Thework was supported by grants from Brazilian Research Founda-tions: CNPq, FAPEMIG and CAPES.

Appendix A. Supporting information

Supplementary data associated with this article can be foundin the online version at doi:10.1016/j.ecoenv.2011.07.017.

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