Seasonal and Spatial Distribution of Several Endocrine-Disrupting Compounds in the Douro River...

Seasonal and Spatial Distribution of SeveralEndocrine-Disrupting Compounds in the DouroRiver Estuary, Portugal

Claudia Ribeiro Æ Maria Elizabeth Tiritan ÆEduardo Rocha Æ Maria Joao Rocha

Received: 10 August 2007 / Accepted: 22 February 2008 / Published online: 27 March 2008

� Springer Science+Business Media, LLC 2008

Abstract Recent studies in the Douro River estuary show

signs of pollution in the area and of fish endocrine disruption.

However, the chemical nature of the local contamination has

not been fully investigated nor have studies checking for the

simultaneous presence of endocrine-disrupting chemicals

(EDCs), either of animal (estrone, E1; estradiol, E2),

pharmaceutical (17a-ethynylestradiol, EE2), vegetal

(daidzein, DAID; genistein, GEN; biochanin A, BIO-A), or

industrial (bisphenol A, BPA; 4–octylphenol, 4-OP; 4-no-

nylphenol, 4-NP) origins. Thus, the main objective of this

study was to examine the presence of these EDCs in estuarine

water samples collected, in every season of the year, at nine

sampling stations along the estuarine gradient. All samples

were processed by two-step solid-phase extraction (Oasis

HLB followed by silica) prior to high-performance liquid

chromatography with diode-array detection (HPLC-DAD)

and gas chromatography–mass spectrometry (GC-MS)

analyses. The current data showed that E1 and EE2, all

phytoestrogens, and BPA were identified and measured in

this estuary. In contrast, 4-OP was only detected by GC-MS

and E2 and 4-NP were not found. Additionally, E1 (up to

112.9 ng/L) and EE2 (up to 101.9 ng/L) were both measured

in biologically hazardous amounts in winter. In the year

sampled, the phytoestrogens suggested a possible seasonal

pattern of fluctuation. Both DAID (up to 888.4 ng/L) and

GEN (183.6 ng/L) were maximal in early summer, whereas

BIO-A (up to 191.4 ng/L) reached its highest concentrations

in winter. BPA (up to 10.7 lg/L) also attained highest

levels in winter. In December 2005, it is hypothesized that

E1, EE2, and BPA concentrations were atypically high due

to current drought conditions. Almost all assayed EDCs

existed in all seasons and, therefore, might have contributed

to endocrine disruption of aquatic animals, previously

documented by the high rate of ovotestis in fish caught in this

estuary.

Introduction

The Douro River flows along 900 km, from its source near

Duruelo de la Sierra (Spain) to its estuary at Porto

(Portugal). Due to its dimensions and huge watershed

(*98, 000 km2), this river passes throughout many differ-

ent regions that include densely inhabited areas, industrial

poles, and large agricultural fields, which, as a whole,

contribute to its pollution. This river has 51 large dams that

affect the drainage rates of the river to the Atlantic Ocean

(Vieira and Bordalo 2000). One dam, the Crestuma-Lever,

was built inside the innate estuary of the Douro River,

controlling the last 21.6 km of the river course and forming

a basin where the accumulation of pollutants is inevitable.

Thus, in addition to chemicals from upstream sources,

C. Ribeiro � M. E. Tiritan � M. J. Rocha (&)

Department of Pharmaceutical Sciences, Superior Institute of

Health Sciences (ISCS-N), Rua Central de Gandra, 1317,

4585-116 Gandra, PRD, Portugal

e-mail: [email protected]

C. Ribeiro � E. Rocha � M. J. Rocha

Interdisciplinary Centre for Marine and Environmental Research

(CIIMAR), CIMAR Associate Laboratory, University of Porto

(UPorto), Porto, Portugal

C. Ribeiro � M. E. Tiritan

Centre of Studies of Organic Chemistry, Phytochemistry and

Pharmacology of Oporto University (CEQOFFUP), Porto,

Portugal

C. Ribeiro � E. Rocha

Institute of Biomedical Sciences Abel Salazar (ICBAS),

University of Porto (UPorto), Porto, Portugal

123

Arch Environ Contam Toxicol (2009) 56:1–11

DOI 10.1007/s00244-008-9158-x

contaminants leached from the untreated sewages and sew-

age treatment plants (SWTPs) located in the margins of this

highly industrialized and densely inhabited estuary (Vieira

and Bordalo 2000). In fact, recent studies involving the

measurement of pollutants such as insecticides (Ferreira

et al. 2002), inorganic metals (Mucha et al. 2004; Ramalhosa

et al. 2005), polycyclic aromatic hydrocarbon (PAHs)

(Ferreira et al. 2006), and bisphenol A (BPA) (Almeida et al.

2007) establish this estuary as extremely polluted. Estrogens

or estrogen mimics (phytoestrogens, alkylphenols, etc.),

which can contribute to diverse fish disorders (Mills and

Chichester 2005), were apparently never investigated in this

area. Those compounds, labeled herein as endocrine-dis-

rupting chemicals (EDCs), include several classes of natural

or pharmaceutical estrogens (17b-estradiol, estrone, and

ethynylestradiol), phytoestrogens (daidzein, genistein, and

biochanin A), and industrial pollutants (BPA, 4-octylphenol,

and 4-nonylphenol). Despite their different origins and dis-

tinct physical chemical properties, all of these EDCs could

cause endocrine disruption in fish (Kiparissis et al. 2003;

Mills and Chichester 2005). In fact, in vivo and in vitro

experiments have demonstrated that estrogens, either of

natural or pharmaceutical origins, and industrial pollutants

can exhibit additive or synergic toxic effects that are

responsible for the appearance of ovotestis in aquatic ani-

mals (Mills and Chichester 2005). Thus, because urban,

industrial, and agricultural pollution clearly exists in the

Douro River estuary and because studies in local fish, such as

the grey mullet (Mugil cephalus) (Ferreira et al. 2002),

suggest the presence of endocrine disruption in this area, the

main objectives of this study were to (1) identify and quantify

EDCs in Douro estuarine water, targeting natural and

pharmaceutical estrogens, phytoestrogens, BPA, and alkyl-

phenols, (2) determine possible seasonal variations for the

studied compounds, and (3) establish which compounds

were present in hazardous concentrations.

Materials and Methods

Sampling Area

The Douro River estuary is about 22 km long and is

located on the west coast of Portugal (41�080 N, 8�400 W).

Its average depth is about 8 m, with a natural semidiurnal

tidal range of 2–3 m (Bordalo and Vieira 2005). For this

study, nine sampling stations (S1 to S9) were selected from

the river outlet, near the Atlantic Ocean, to the Crestuma-

Lever dam (Fig. 1). Sampling stations S1, S3, S6, and S9

were located on the north bank of the river at the Porto city

margin, whereas S2, S4, S5, S7, and S8 were located at the

opposite side, bordering the other highly industrialized and

densely inhabited district, the Gaia city.

Sampling Collection

At the peak of winter (December 2005), spring (March

2006), summer (July 2006), and autumn (October 2006),

2 L of estuarine water samples were systematically col-

lected at a depth of 1 m, using a peristaltic sampler pump

(Global Water, Model WS300), along the estuarine gradi-

ent. Here, sampling occurred at both high and low tides of

the Douro River estuary and mean water temperatures

ranged from *7.0�C (winter) to 25.0�C (summer). Other

physicochemical parameters such as pH, salinity (%), and

conductivity are shown in Table 1. During sampling, all

bottles were rinsed two or three times before the collection

of the water samples, which were immediately filtrated, to

eliminate particulate matter and other suspended solids,

through a 47-mm GF/C glass fiber filter, acquired from

Millipore (Ireland). After this procedure, each filter was

washed several times with small amounts of CH3OH that

were added to the latter filtrate. Finally, all samples were

acidified with H2SO4 to pH 2 to prevent biodegradation and

kept at *5�C during transport to the laboratory.

Chemicals

Estrone (E1), 17b-estradiol (E2), ethynylestradiol (EE2),

daidzein (DAID), genistein (GEN), biochanin A (BIO-A),

bisphenol A (BPA), 4-octylphenol (4-OP), and the deriv-

atizing reagent N-methyl-N-trimethylsilyltrifluoroacetamide

(MSTFA) were purchased from Sigma-Aldrich (Steinhein,

Germany), whereas 4-nonylphenol (4-NP) was purchased

from Riedel-de-Haen (Seelze-Hannover, Germany). Stock

solutions of individual standards were prepared by dissolv-

ing known amounts of each compound in CH3OH:CH3CN

Fig. 1 Map of the Douro River estuary showing the locations of the

nine sampling areas (S1 to S9) selected for this study, the main

industrial poles, and sewage treatment plants of this area

2 Arch Environ Contam Toxicol (2009) 56:1–11

123

(50:50, v/v) high-performance liquid chromatography

(HPLC) grade, acquired from Sigma-Aldrich (Steinhein,

Germany), to obtain final concentrations of 500 mg/L. All

other solvents were analytical grade from Sigma-Aldrich

(Steinhein, Germany). Ultrapure water was supplied by a

Milli-Q water system.

Sample Preparation

All water samples, previously filtered and acidified, were

preconcentrated by a solid extraction phase (SPE) proce-

dure using a 500-mg Oasis HLB Cartridge (polymer of

N-vinylpyrrolidone and divinylbenzene) purchased from

Waters Corporation (Milford, MA, USA). Prior to use, this

cartridge was sequentially washed with 25 mL of

CH2Cl2:CH3OH (50:50, v/v), 12 mL of CH3OH, and

25 mL of ultrapure Milli-Q water (Ribeiro et al. 2007).

Samples were applied to cartridges under vacuum to obtain

a constant flow rate of 5–7 mL/min. Cartridges were

washed with 25 mL of ultrapure Milli-Q water, followed

by 1 mL of CH3OH and eluates discarded. Cartridges were

dried kept under vacuum aspiration for 30 min and eluted

with 20 mL of CH2Cl2:CH3OH (50:50, v/v). The dark and

sticky eluate was further ‘‘purified’’ on a prewashed 1-g

Sep-Pak silica cartridge (CH2Cl2:CH3OH 50:50, v/v; from

Waters Corporation, Milford, MA, USA). Seven milliliters

of the same solvent composition were used to elute samples

into a round-bottomed tube, which was taken to dryness

under N2 in a thermostatic bath at 40�C. Samples were

resuspended in 200 lL of CH3OH:CH3CN (50:50, v/v).

Twenty microliters of each sample were injected in tripli-

cate into the high-performance liquid chromatography with

diode-array detection (HPLC-DAD) system for quantita-

tive analysis (Ribeiro et al. 2007) and evaporated to

dryness and derivatizated with 50 lL of MSTFA before

injection in the gas chromatography–mass spectrometry

Table 1 Ranges of the physicochemical parameters measured in each of the sampling sites (S1 to S9) of the Douro River estuary

Parameters Sampling station Winter December 2005 Spring March 2006 Summer July 2006 Autumn October 2006

Low tide High tide Low tide High tide Low tide High tide Low tide High tide

pH S1 7.5 7.6 7.3 7.5 8.3 7.9 7.9 8.2

S2 7.9 7.9 7.4 7.5 7.9 7.7 7.8 8.1

S3 7.5 7.7 7.3 7.7 8.3 7.3 8.0 8.0

S4 7.7 7.8 7.7 7.6 8.2 7.9 8.0 8.2

S5 7.7 7.4 7.6 7.5 8.0 7.8 7.8 8.1

S6 7.8 7.8 7.5 7.3 7.9 7.9 8.0 8.1

S7 7.5 7.4 7.5 7.6 7.8 8.0 7.9 8.0

S8 7.5 7.4 7.6 7.4 7.6 7.9 7.8 7.8

S9 7.5 7.5 7.7 7.5 7.8 7.7 7.9 8.0

Salinity (%) S1 4.0 12.7 0.0 0.7 6.1 8.9 20.6 31.1

S2 5.8 6.0 0.0 0.0 7.1 8.5 10.0 30.3

S3 1.2 7.9 0.0 0.0 5.9 11.1 15.4 31.0

S4 8.1 10.9 0.0 0.0 6.1 11.1 13.5 31.4

S5 10.4 11.9 0.0 0.0 4.7 6.2 7.9 23.7

S6 2.0 2.3 0.0 0.0 1.8 1.9 3.4 14.5

S7 0.0 0.0 0.0 0.0 2.2 3.1 7.4 12.1

S8 0.0 0.0 0.0 0.0 0.0 0.0 1.3 1.5

S9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Conductivity (mS/cm) S1 7.3 21.2 0.8 1.1 10.8 15.3 33.0 47.7

S2 10.2 10.6 0.2 0.3 10.4 18.8 17.0 46.7

S3 1.9 13.7 0.2 0.8 12.5 14.6 25.3 47.6

S4 13.9 18.4 0.2 0.9 10.8 18.7 22.4 48.2

S5 17.7 19.9 0.2 0.2 8.5 11.0 13.7 37.4

S6 3.1 3.5 0.2 0.2 2.8 2.9 6.3 24.0

S7 0.2 0.2 0.2 0.2 4.2 5.7 7.4 12.1

S8 0.1 0.1 0.1 0.2 0.4 0.4 2.0 2.3

S9 0.1 0.3 0.2 0.2 0.6 0.6 0.5 0.9

Arch Environ Contam Toxicol (2009) 56:1–11 3

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(GC-MS) equipment. Recovery assays, for the SPE steps,

demonstrated that all compounds had recoveries higher

than 69%, except 4-NP (53%) (Table 2).

Instrumental and Main Methodological Characteristics

The HPLC system consisted of a LiChroCART C18

reversed-phase analytical column 250 9 4-mm-inner

diameter, 5-lm particle size (Merck, Darmstadt, Germany)

and a Merck Hitachi HPLC apparatus, equipped with the

LaChrom diode array detector L-7455. Data acquisition was

performed by a HPLC System Manager HSM D-7000,

Version 3.0 (Merck-Hitachi). All analyses in this system

followed a previously validated method developed for the

present estuarine water samples (Ribeiro et al. 2007).

Briefly, the method was as follows: mobile-phase compo-

sition CH3CN:H2O (25:75, v/v) acidified with CF3CO2H,

pH 2, 1 mL/min with organic solvent increasing linearly, at

room temperature, as indicated in Table 3. After each

chromatographic run, the amount of CH3CN increased up to

100% and was maintained isocratically for 5 min before a

new injection. During the application of this method, sev-

eral quality control parameters were performed taking in

consideration the International Conference on Harmoniza-

tion (1996) rules; that is, the robustness of the method was

constantly checked. For that, the instrumental precision and

accuracy were monitored regularly by the measurement of

the peak areas of injections containing both standard mix-

ture and fortified matrix (relative standard deviation

(RSD) \ 1%). Furthermore, the maintenance of the reten-

tion times among samples and standards was always

verified as each analyzed sample was injected both indi-

vidually (n = 2) and spiked with all standards (n = 1).

Finally, only the areas of peaks showing a purity test higher

than 99% (this value was calculated automatically by the

HPLC-DAD software) were considered for the quantitative

analysis, which was based on calibration curves created for

this propose using a Douro River estuarine matrix spiked

with standards of all EDCs (Table 2).

The presence of compounds of interest in the samples

was confirmed by GC-MS on derivatized samples. The GC

system consisted in a Varian CP 3800 apparatus equipped

with a VF-5ms-type capillary column (30 m 9 0.25 mm

inner diameter; df: 0.25 lm) connected to an ion trap MS

(Varian Saturn 2200). The GC-MS analytical procedure

used for the current estuarine water samples was based in

several published protocols that were adjusted to the cur-

rent samples (Ballesteros et al. 2006; Lee et al. 2004;

Shareef et al. 2006; Silveira 2007). As the majority of the

EDCs analyzed (Fig. 2) contain several hydroxyl groups,

MSTFA was the preferred derivatization reagent used to

prepare the trimethylsilyl (TMS) derivatives (Lee et al.

2004; Shareef et al. 2006). Data acquisition was obtained

using the selected ion monitoring (SIM) mode. The iden-

tification of each chromatographic peak was achieved by

comparing the retention times with coinjected standards

and matching the characteristic ions of standards and

samples (Table 4).

Results

Data are displayed in Figs. 3 and 4 and in Tables 1, 2, and

4–7. In Table 5, it is shown that E1 and EE2 were found in

Table 2 Chromatographic data and calibration results obtained by the standard addition technique

Chemicals

(EDCs)

Wavelength

(nm)

Retention

times

(tM, min)

Linear

dynamic

range (lg/mL)

Intercept Slope R2 Recovery

(%)

LOD

(ng/L)

LOQ

(ng/L)

DAID 246 8.10 0.40–20.0 5,172.3 57,460 0.998 104 10.0 31.7

GEN 260 10.60 0.40–20.0 9,622.9 52,857 0.999 100 3.2 9.8

BPA 278 12.60 0.90–135.0 1,723.5 7,817.9 0.998 99 8.0 24.5

E2 280 13.60 0.80–20.0 1,236.3 3,530.1 0.999 87 7.0 21.3

EE2 280 14.40 1.00–10.0 –152.7 4,056.8 0.999 108 18.0 54.5

E1 280 15.20 0.80–10.0 1,497.3 3,398.0 0.992 116 15.0 44.0

BIO-A 260 15.70 0.12–10.0 –403.9 36,004 0.998 92 12.4 37.5

4-OP 278 25.90 2.00–140.0 30.1 5,340.9 0.997 69 3.8 12.0

4-NP 278 27.80 2.00–140.0 -53.6 4,598.0 0.999 53 7.0 21.8

LOD = limit of detection; LOQ = limit of quantificaion

Table 3 Organic gradient for the chromatographic separation of nine

EDCs by HPLC-DAD

Time (min) Organic solvent (%)

0 25

5 40

14 55

17 57

30 90

36 100


123

the Douro River estuarine waters. These chemicals, quan-

tified only in winter (Fig. 3A), showed their highest levels

at the sampling point S4 (up to 112.9 ng/L in low tide) for

E1 and at the sampling station S5 (up to 101.9 ng/L in high

tide) for EE2. Later, during all other analyzed seasons, E1

and EE2 became undetectable by HPLC-DAD (Table 2),

whereas their presence were confirmed by GC-MS, as

demonstrated by the retention time and fragmentation

patterns characteristic of both E1 and EE2 shown in

Table 4 and Fig. 4A and 4B. In contrast, the presence of E2

was not detected on any occasion either by HPLC-DAD or

GC-MS. The measurement, by HPLC-DAD, of both E1

and EE2 in winter can be related with the absence of

pluviosity and low temperatures registered in this period.

This observation is also supported by the values of salinity

and conductivity found in winter (Table 1).

Table 6 shows the concentrations of all phytoestrogens

measured. Only BIO-A was quantifiable in all seasons

(Fig. 3A–3C). Therefore, in every season of the year, the

highest levels of BIO-A were measured at the following

sampling stations: S1 (up to 112.1 ng/L in low tide) in

winter, S7 (up to 191.4 ng/mL in low tide) in spring, S3 (up

Fig. 2 Chemical structure of all

EDCs investigated in the Douro

River estuary

Table 4 Ions and fragment ratios used for identification of the nine proposed analytes by GC-MS

EDCs Main

sourceaMass weight

(g/mol)

Retention

times (min)

Ions selected in

standard mixtures (m/z)

(% relative abundance)

4-OP Industrial and municipal effluents 206 12.0 278 (30), 179 (100)

4-NP Industrial and municipal effluents 220 13.1 292 (34), 179 (100)

BPA Industrial effluents 228 15.7 372 (6), 357 (100)

E1 Urban and municipal effluents 270 19.7 342 (100), 257 (45)

E2 Urban and municipal effluents 272 19.9 416 (100), 285 (75)

EE2 Municipal effluents 296 21.4 425 (100), 285 (40)

BIO-A Agricultural runoff 284 22.4 428 (8), 413 (100)

DAID Agricultural runoff 254 23.4 398 (100), 383 (85)

GEN Agricultural runoff 270 23.7 471 (1), 399 (100)

a Data from Lintelmann et al. (2003) and Lagana et al. (2004)


123

to 23.1 ng/L in high tide) in summer, and at S3 (87.3 ng/L

in low tide) in autumn. In contrast, the levels of DAID and

GEN were very low or undetectable by HPLC-DAD

(Table 2) in winter, spring, and autumn but showed note-

worthy concentrations in summer (Table 6). Thus, in July

(Fig. 3C), the highest levels of DAID were measured at the

sampling station S9 (up to 888.4 ng/L in high tide) and

those of GEN at the sampling station S5 (up to 197.4 ng/L

in high tide). Here, it is important to stress that the identity

of all phytoestrogens referred in this study were all con-

firmed by GC-MS, as is shown in Fig. 4C–4E. Thus, the

retention times and fragmentation patterns characteristic of

DAID, GEN, and BIO-A (Table 4) were found in all ana-

lyzed samples.

In Table 7, it is demonstrated that BPA was the main

industrial pollutant measured in our study, as 4-OP was

under the detection limits of the HPLC-DAD method and

4-NP was never detected. Therefore, BPA was measured in

all seasons at the sampling points S1 and S3 and S5 and S7

located near the industrial poles and sewage treatment

plants of the Porto and the Gaia city, respectively (Fig. 1).

Mostly in summer and autumn, BPA levels became, in

several sampling stations, under the detection limits of the

current HPLC-DAD method (Table 2). Nonetheless, the

current GC-MS analysis demonstrated that BPA and 4-OP

were always present in this estuary, whereas 4-NP was not.

In Table 4 and Fig. 4F and 4G are shown the fragmentation

peaks characteristic of the BPA and 4-OP.

Discussion

The data of this study are the first of their kind advanced

for the estuary of the Spanish–Portuguese Douro River.

Fig. 3 Chromatograms of several the samples: (A) S5, low tide,

winter (December 2005), (B) S1, low tide, spring (March 2006), (C)

S9, high tide, summer (July 2006), and (D) S4, low tide, autumn

(October 2006) spiked with the standard mixture containing 0.10 lg/L

of DAID and GEN, 0.23 lg/L of BPA, 0.20 lg/L of E2 and E1,

0.25 lg/L of EE2, 0.03 lg/L of BIO-A, and 0.5 lg/L of 4-OP and

4-NP


123

Our study showed that the water contained varying

amounts of natural estrogens and of xenoestrogens, which,

in accordance with recent bibliographic data, at times

reached deleterious amounts of EDCs (Mills and Chich-

ester 2005). Estrogens of natural and pharmaceutical origin

found in winter samples were similar to those reported in

Japanese wastewaters (Komori et al. 2004). Such values

(Table 5), commonly accepted as hazardous for the aquatic

fauna (Metcalfe et al. 2001; Segner et al. 2003), were

extremely high when compared with other European

estuaries (Belfroid et al. 1999; Noppe et al. 2007; Vethaak

et al. 2005). Nonetheless, we believe that the estrogenic

load might be atypical because in 2005 there occurred the

most severe regional drought in the last 60 years in this

region, and on such occasions, the drainage rates of the

river to the Atlantic Ocean are very low, ranging from 0 to

13,000 m3/s (Bordalo et al. 2006; Vieira and Bordalo

2000). This hypothesis is also supported by the data

obtained later for all other sampling seasons, when the

levels of both E1 and EE2 became undetectable by HPLC-

DAD (Table 2). It is important to stress that the precipi-

tation levels in this area returned to normal later

Fig. 4 MS spectra of all

detected compounds in the

Douro River estuary. The

arrows point to the peaks (m/z)

used for the identification of E1

(A), EE2 (B), DAID (C), GEN

(D), BIO-A (E), BPA (F), and

4-OP (G)


123

Table 5 Concentrations of E1 and EE2 in each of the sampling sites (S1 to S9) of the Douro River estuary

Estrogens (ng/L) Sampling station Winter December 2005 Spring March 2006 Summer July 2006 Autumn October 2006


E1 S1–S3 \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a

S4 112.9 \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a

S5 99.8 \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a

S6–S7 \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a

S8 103.9 \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a

S9 \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a \15.0a

EE2 S1 \18.0a 56.0 \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a

S2–S3 \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a

S4 97.7 \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a

S5 \18.0a 101.9 \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a

S6 83.1 62.3 \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a

S7 \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a

S8–S9 \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a \18.0a

a Less than detection limit

Table 6 Concentrations of DAID, GEN, and BIO-A in each of the sampling sites (S1 to S9) of the Douro River estuary

Phytoestrogens (ng/L) Sampling station Winter December 2005 Spring March 2006 Summer July 2006 Autumn October 2006


DAID S1 \10.0a \10.0a \10.0a \10.0a 231.3 495.9 \10.0a \10.0a

S2 \10.0a \10.0a \10.0a \10.0a 597.4 205.3 \10.0a \10.0a

S3–S4 \10.0a \10.0a \10.0a \10.0a \10.0a \10.0a \10.0a \10.0a

S5 25.9 \10.0a \10.0a \10.0a 245.1 603.5 \10.0a \10.0a

S6 \10.0a 14.6 \10.0a \10.0a \10.0a \10.0a \10.0a \10.0a

S7 \10.0a \10.0a \10.0a \10.0a \10.0a \10.0a \10.0a \10.0a

S8 \10.0a \10.0a \10.0a \10.0a 202.1 292.5 \10.0a \10.0a

S9 \10.0a \10.0a \10.0a \10.0a 145.3 888.4 \10.0a \10.0a

GEN S1 \3.2a \3.2a \3.2a \3.2a \3.2a 72.4 \3.2a \3.2a

S2 \3.2a \3.2a \3.2a \3.2a 183.6 83.9 \3.2a \3.2a

S3 \3.2a \3.2a \3.2a \3.2a \3.2a 177.3 \3.2a \3.2a

S4 \3.2a \3.2a \3.2a \3.2a \3.2a \3.2a \3.2a \3.2a

S5 \3.2a \3.2a \3.2a \3.2a \3.2a 197.4 \3.2a \3.2a

S6–S7 \3.2a \3.2a \3.2a \3.2a \3.2a \3.2a \3.2a \3.2a

S8 \3.2a \3.2a \3.2a \3.2a 48.4 90.5 \3.2a \3.2a

S9 \3.2a \3.2a \3.2a \3.2a 29.1 122.8 \3.2a \3.2a

BIO-A S1 112.1 57.1 134.4 109.6 \12.4a 18.7 61.4 17.1

S2 \12.4a \12.4a 124.2 50.9 \12.4a \12.4a \12.4a \12.4a

S3 \12.4a 76.1 108.8 107.9 \12.4a 23.1 87.3 \12.4a

S4 94.0 \12.4a 93.5 95.9 \12.4a \12.4a 43.2 \12.4a

S5 78.0 75.1 49.6 72.0 \12.4a \12.4a 28.9 \12.4a

S6 \12.4a \12.4a 46.8 104.0 \12.4a \12.4a 38.9 37.1

S7 52.2 63.1 191.4 82.1 \12.4a \12.4a 56.2 31.2

S8 38.7 41.2 90.1 52.5 \12.4a \12.4a 24.1 17.9

S9 56.2 79.3 60.6 122.6 \12.4a \12.4a 33.3 \12.4a



123

(Cararmelo and Orgaz 2007), causing a visible increase of

the caudal of this estuary in spring and a significant

decrease in the levels of the salinity and conductivity

during this period (Table 1). These parameters, closely

related with pluviosity (Ellis et al. 1977), indicate an

obvious dilution effect of all local pollutants. However,

because we demonstrated, by GC-MS, the continuous

presence of E1 and EE2 in all seasons, their potential

deleterious effects cannot be ruled out, as both estrogens

might produce endocrine disruption and reproductive dis-

turbances even in very low nanograms per liter ranges

(Metcalfe et al. 2001; Segner et al. 2003). These results

are in accordance with recent studies that classified the

Douro estuary as the second most polluted of Portugal

(Vasconcelos et al. 2007), with documented intersex in

local estuarine fish (Ferreira et al. 2002). Thus, the present

data confirm the need of regular local monitoring studies

for evaluating natural and pharmaceutical estrogens, as it is

well known that these chemicals induce vitellogenesis,

intersex, and feminization in male fish and affects human

reproduction (Waring and Harris 2005). In contrast, E2 was

never detected in these estuarine waters, possibly due to

rapid degradation of E2 to E1 and/or because humans

excrete more E1 than E2 in their urine (Johnson et al.

2000).

Phytoestrogens are about 10-2- to 10-3-fold less potent

than natural and pharmaceutical estrogens (Benassayag

et al. 2002; Gutendorf and Westendorf 2001). Nonetheless,

because they are still able to induce endocrine disruption in

fish (Kiparissis et al. 2003) and DAID, GEN, and BIO-A

were never measured in the Douro River, this study reports

the levels of these compounds in this estuary. Thus, in

winter, spring, and autumn, occasions when both agricul-

tural activities are at the resting stage, both DAID and GEN,

which are among the most important components of fruits,

cereals, and vegetables (Liggins et al. 2000a, b, 2002), were

undetectable or showed very low levels (Table 6). In con-

trast, the concentrations of BIO-A were highest in winter

and early spring, which is consistent with the seasonal

variation of the indigenous flora of this estuary. In fact, this

estuary not only contains considerable amounts of red clo-

ver (Trifolium pratense L., Fabaceae) and papilonaceous

plants (Papilionaceae) but also holds many species of

grasses that are rich sources of BIO-A at that time of the

year (Booth et al. 2006), which, by leaching, can easily

reach the estuarine waters. Later, in early summer, this

depiction reversed and, although the concentrations of both

DAID and GEN were particularly high, those of BIO-A

become minimal. These data are consistent with both the

vast vineyards and orchards (mainly of apple, orange, and

almond) located in both margins of this river. In autumn, the

seasonal cycle of the studied phytoestrogens seemed to be

completed with the rise of BIO-A concentrations and the

remarkable decrease of both DAID and GEN.

Because the concentrations of all measured phytoestro-

gens were below the hazardous levels described in the

in vitro experiments conducted by Kiparissis et al. (2003)

and because the last study demonstrated that only con-

centrations above 1000 lg/L GEN produce gonadal

intersex in the Japanese medaka (Oryzias latipes), our

current data suggest that, individually, all measured phy-

toestrogens might not exert any adverse effect in the local

fauna. However, further biological studies are needed in

this estuary to confirm if the total load of all these com-

pounds have potential for synergistic activity, either with

other phytoestrogens or other estrogenic chemicals able to

produce endocrine disruption in aquatic species.

As for industrial pollution, the Douro estuary is partic-

ularly affected by discharges of debris from plating, soap,

textile, and tannery industries located near its margins or in

its main tributaries (Fig. 1). Extremely high levels of BPA

(Table 7) were found in winter, possibly due to the drought

Table 7 Concentrations of BPA measured in each sampling sites (S1 to S9) of the Douro River estuary

Industrial pollutants (lg/L) Sampling station Winter December 2005 Spring March 2006 Summer July 2006 Autumn October 2006


BPA S1 10.7 5.5 6.6 7.0 1.0 4.2 1.7 \0.08a

S2 \0.08a \0.08a 6.3 2.5 \0.08a 1.3 \0.08a \0.08a

S3 0.25 9.0 5.2 5.5 0.7 3.1 2.5 \0.08a

S4 \0.08a \0.08a 4.8 5.0 \0.08a \0.08a 1.1 \0.08a

S5 9.9 7.8 1.9 4.6 \0.08a 1.6 0.9 \0.08a

S6 \0.08a \0.08a 0.1 3.9 \0.08a \0.08a 0.9 \0.08a

S7 5.5 7.6 0.3 4.8 0.7 2.3 0.7 \0.08a

S8 4.3 4.7 5.1 0.7 0.7 2.1 \0.08a \0.08a

S9 6.2 7.6 3.3 4.7 \0.08a 1.7 \0.08a \0.08a



123

conditions of December 2005. In fact, later the levels of

BPA decreased, becoming similar to those reported in other

polluted rivers either in Portugal (Azevedo et al. 2001;

Quiros et al. 2005), Spain (Cespedes et al. 2005), or The

Netherlands (Vethaak et al. 2005). The permanent presence

of BPA in the sampling points S1 and S3, and S5 and S7,

located near highly urbanized and industrial poles (Fig. 1),

confirm the impact of the human activities in both margins

of this estuary. Relative to the possible hazardous effects of

BPA, the concentration in the Douro estuary were lower

than those reported for endocrine disruption in several

studies (i.e., lower than 160 lg/L) (Mills and Chichester

2005; Staples et al. 2002). Nonetheless, we think that fur-

ther studies should be conducted with water from this

estuary in order to investigate the possible ‘‘cocktail

effect’’ (i.e., total estrogenic effect) promoted by the mix-

ture of all EDCs detected and/or quantified in this area.

Finally, no typical gradient and influence of tides were

found along this estuary for the assayed EDCs. These

observations were probably due to the artificial hydrody-

namics of this estuary, a result of the Crestuma-Lever dam

as suggested by Bordalo et al. (2006) in studies concerning

the water quality of this estuary. However, the combination

of drought and diminished discharges of the dam, like those

registered in December 2005, warns that in the Douro

River estuary, moderate or low concentrations of EDCs

might suddenly become extremely high and then be

potentially risky to wildlife.

Conclusions

Our data (1) showed the presence of natural and pharma-

ceutical estrogens, phytoestrogens, and selected industrial

pollutants in the Douro River estuarine waters, (2) dem-

onstrated the occurrence of hazardous amounts of E1 and

EE2 in December 2005 as well as their continuous pres-

ence in this estuary, (3) reported, for the first time, the

environmental concentrations of DAID, GEN, and BIO-A

in the Douro estuary, (4) suggested a possible seasonal

fluctuation trend for the studied phytoestrogens, (5)

revealed that BPA was a significant pollutant of this estu-

ary, whereas 4-OP and 4-NP were not (6) supported the

need for further monitoring studies targeting estrogenic

chemicals, and (7) warned that biologic studies are needed

to evaluate possible endocrine disruptive ‘‘cocktail effects’’

of the EDCs in the local fauna.

Acknowledgments This study was financially supported by the

Fundacao para a Ciencia e Tecnologia (FCT; PhD Grant SFRH/BD/

18231/2004/SLU, attributed to Claudia Ribeiro) and by the Cooper-

ativa de Ensino Superior, Politecnico e Universitario, CRL (CESPU)/

ISCS-N (Research Projects 1F/13/2005/CESPU and 2F/02/2006/

CESPU).

References

Almeida C, Serodio P, Florencio MH, Nogueira JMF (2007) New

strategies to screen for endocrine-disrupting chemicals in the

Portuguese marine environment utilizing large volume injection-

capillary gas chromatography-mass spectrometry combined with

retention time locking libraries (LVI-GC-MS-RTL). Anal Bio-

anal Chem 387:2569–2583

Azevedo DA, Lacorte S, Viana P, Barcelo D (2001) Analysis of

priority pesticides and phenols in Portuguese river water by

liquid chromatography-mass spectrometry. Chromatographia

53:113–118

Ballesteros O, Zafra A, Navalon A, Vilchez JL (2006) Sensitive gas

chromatographic-mass spectrometric method for the determina-

tion of phthalate esters, alkylphenols, bisphenol A and their

chlorinated derivatives in wastewater samples. J. Chromatogr A

1121:154–162

Belfroid AC, Van der Horst A, Vethaak AD et al (1999) Analysis and

occurrence of estrogenic hormones and their glucuronides in

surface water and waste water in The Netherlands. Sci Total

Environ 225:101–108

Benassayag C, Perrot-Applanat M, Ferre F (2002) Phytoestrogens as

modulators of steroid action in target cells. J Chromatogr B

777:233–248

Booth NL, Overk CR, Yao P et al (2006) Seasonal variation of red

clover (Trifolium pratense L., Fabaceae) isoflavones and estro-

genic activity. J Agric Food Chem 54:1277–1282

Bordalo AA, Teixeira R, Wiebe WJ (2006) A water quality index

applied to an international shared river basin: the case of the

Douro river. Environ Manage 38:910–920

Bordalo AA, Vieira MEC (2005) Spatial variability of phytoplankton,

bacteria and viruses in the mesotidal salt wedge Douro Estuary

(Portugal). Est Coast Shelf Sci 63:143–154

Cararmelo L, Orgaz MDM (2007) A study of precipitation variability

in the Duero Basin (Iberian Peninsula). Int J Climatol 27:327–339

Cespedes R, Lacorte S, Raldua D, Ginebreda A, Barcelo D, Pina B

(2005) Distribution of endocrine disruptors in the Llobregat

River basin (Catalonia, NE Spain). Chemosphere 61:1710–1719

Ellis J, Kanamori S, Laird PG (1977) Water pollution studies on Lake

Illawarra. I. Salinity variations and estimation of residence time.

Marine Freshwater Res 28:467–477

Ferreira M, Antunes P, Gil O, Vale C, Silva L, Reis-Henriques MA

(2002) Testis-ova in mullet (Mugil cephalus) exposed to organic

contaminants in the Douro Estuary, Portugal. In: Proceedings of

the 21th conference of European comparative endocrinologists,

Germany

Ferreira M, Moradas-Ferreira P, Reis-Henriques MA (2006) The

effect of long-term depuration on phase I and phase II

biotransformation in mullets (Mugil cephalus) chronically

exposed to pollutants in River Douro estuary, Portugal. Marine

Environ Res 61:326–338

Gutendorf B, Westendorf J (2001) Comparison of an array of in vitroassays for the assessment of the estrogenic potential of natural

and synthetic estrogens, phytoestrogens and xenoestrogens.

Toxicology 166:79–89

International Conference on Harmonisation (ICH) (1996) Validation of

analytical procedures: methodology, Q2B (CPMP/ICH/281/96)

Johnson AC, Belfroid A, Di Corcia A (2000) Estimating steroid

oestrogen inputs into activated sludge treatment works and

observations on their removal from the effluent. Sci Total

Environ 256:163–173

Kiparissis Y, Balch GC, Metcalfe TL , Metcalfe CD (2003) Effects of

the isoflavones genistein and equol on the gonadal development

of Japanese medaka (Oryzias latipes). Environ Health Perspect

111:1158–1163


123

Komori K, Tanaka H, Okayasu Y, Yasojima M, Sato C (2004)

Analysis and occurrence of estrogen in wastewater in Japan.

Water Sci Technol 50:93–100

Lagana A, Bacaloni A, De Leva I, Faberi A, Fago G, Marino A (2004)

Analytical methodologies for determining the occurence of

endocrine disrupting chemicals in sewage treatment plants and

natural waters. Anal Chim Acta 501:79–88

Lee SH, Jung BH, Kim SY, Chung BC (2004) Determination of

phytoestrogens in traditional medicinal herbs using gas chroma-

tography-mass spectrometry. J Nutr Biochem 15:452–460

Liggins J, Bluck LJC, Runswick S, Atkinson C, Coward WA,

Bingham SA (2000a) Daidzein and genistein content of fruits

and nuts. J Nutr Biochem 11:326–331

Liggins J, Bluck LJC, Runswick S, Atkinson C, Coward WA,

Bingham SA (2000b) Daidzein and genistein contents of

vegetables. Br J Nutr 84:717–725

Liggins J, Mulligan A, Runswick S, Bingham SA (2002) Daidzein

and genistein content of cereals. Eur J Clin Nutr 56:961–966

Lintelmann J, Katayama A, Kurihara N, Shore L, Wenzel A (2003)

Endocrine disruptors in the environment (IUPAC Technical

Report). Pure Appl Chem 75:631–681

Metcalfe CD, Metcalfe TL, Kiparissis Y et al (2001) Estrogenic

potency of chemicals detected in sewage treatment plant

effluents as determined by in vivo assays with Japanese medaka

(Oryzias latipes). Environ Toxicol Chem 20:297–308

Mills LJ, Chichester C (2005) Review of evidence: are endocrine-

disrupting chemicals in the aquatic environment impacting fish

populations? Sci Total Environ 343:1–34

Mucha AP, Bordalo AA, Vasconcelos M (2004) Sediment quality in

the Douro river estuary based on trace metal contents, macro-

benthic community and elutriate sediment toxicity test (ESTT). J

Environ Monit 6:585–592

Noppe H, Verslycke T, De Wulf E et al (2007) Occurrence of

estrogens in the Scheldt estuary: a 2-year survey. Ecotoxicol

Environ Safety 66:1–8

Quiros L, Cespedes R, Lacorte S et al (2005) Detection and

evaluation of endocrine-disruption activity in water samples

from Portuguese rivers. Environ Toxicol Chem 24:389–395

Ramalhosa E, Pereira E, Vale C, Valega M, Monterroso P, Duarte AC

(2005) Mercury distribution in Douro estuary (Portugal). Marine

Pollut Bull 50:1218–1222

Ribeiro C, Tiritan ME, Rocha E, Rocha MJ (2007) Development and

validation of a HPLC-DAD method for determination of several

endocrine disrupting compounds in estuarine water. J Liq

Chromatogr Relat Technol 30:2729–2746

Segner H, Navas JM, Schafers C, Wenzel A (2003) Potencies of

estrogenic compounds in in vitro screening assays and in life

cycle tests with zebrafish in vivo. Ecotoxicol Environ Safety

54:315–322

Shareef A, Angove MJ, Wells JD (2006) Optimization of silylation

using N-methyl-N-(trimethylsilyl)-trifluoroacetamide, N,O-bis-

(trimethyl.silyl)-trifluoroacetamide and N-(tert-butyldimethylsi-

lyl)-N-methyltrifluoroacetamide for the determination of the

estrogens estrone and 17 alpha-ethinylestradiol by gas chroma-

tography-mass spectrometry. J Chromatogr A 1108:121–128

Silveira M (2007) Identification of endocrine disruptors in superficial

waters of Douro River estuary, University of Porto, Portugal

Staples CA, Woodburn K, Caspers N, Hall AT, Klecka GM (2002) A

weight of evidence approach to the aquatic hazard assessment of

bisphenol A. Hum Ecol Risk Assess 8:1083–1105

Vasconcelos RP, Reis-Santos P, Fonseca V et al (2007) Assessing

anthropogenic pressures on estuarine fish nurseries along the

Portuguese coast: a multi-metric index and conceptual approach.

Sci Total Environ 374:199–215

Vethaak AD, Lahr J, Schrap SM et al (2005) An integrated

assessment of estrogenic contamination and biological effects

in the aquatic environment of The Netherlands. Chemosphere

59:511–524

Vieira MEC, Bordalo AA (2000) The Douro estuary (Portugal): a

mesotidal salt wedge. Oceanol Acta 23:585–594

Waring RH, Harris RM (2005) Endocrine disrupters: a human risk?

Mol Cell Endocrinol 244:2–9


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