Biological Analysis of Endocrine-Disrupting Compounds in Tunisian Sewage Treatment Plants

Biological Analysis of Endocrine-Disrupting Compoundsin Tunisian Sewage Treatment Plants

Wissem Mnif • Sonia Dagnino • Aurelie Escande • Arnaud Pillon •

Helene Fenet • Elena Gomez • Claude Casellas • Marie-Josephe Duchesne •

Guillermina Hernandez-Raquet • Vincent Cavailles • Patrick Balaguer •

Aghleb Bartegi

Received: 15 July 2009 / Accepted: 29 November 2009 / Published online: 22 December 2009

� Springer Science+Business Media, LLC 2009

Abstract Endocrin-disrupting compounds (EDCs) are

frequently found in wastewater treatment plants (WWTPs).

So far, research has been mainly focused on the detection

of estrogenic compounds and very little work has been

carried out on other receptors activators. In this study, we

used reporter cell lines, which allow detecting the activity

of estrogen (ERa), androgen (AR), pregnane X (PXR),

glucocorticoid (GR), progesterone (PR), mineralocorticoid

(MR), and aryl hydrocarbon (AhR) receptors, to charac-

terise the endocrine-disrupting profile of the aqueous,

suspended particulate matter, and sludge fractions from

three Tunisian WWTPs. The aqueous fraction exhibited

estrogenic and androgenic activities. Suspended particulate

matter and sludge extracts showed estrogenic, aryl hydro-

carbon and pregnane X receptor activities. No GR, MR, or

PR (ant) agonistic activity was detected in the samples,

suggesting that environmental compounds present in

sewage might have a limited spectrum of activity. By

performing competition experiments with recombinant

ERa, we demonstrated that the estrogenic activity detected

in the aqueous fraction was due to EDCs with a strong

affinity for ERa. Conversely, in the sludge fraction, it was

linked to the presence of EDCs with weak affinity. More-

over, by using different incubation times, we determined

that the EDCs present in suspended particulate matter and

sludge, which can activate AhR, are metabolically labile

compounds. Finally, we showed in this study that envi-

ronmental compounds are mainly ER, AR, PXR, and AhR

activators. Concerning AR and PXR ligands, we do not to

know the nature of the molecules. Concerning ER and AhR

compounds, competition experiments with recombinant

receptor and analysis at different times of exposure of the

AhR activation gave some indications of the compound’s

nature that need to be confirmed by chemical analysis.

P. Balaguer and A. Bartegi should be considered as last co-authors.

W. Mnif � A. Bartegi

Laboratoire de Biochimie, Unite de Recherche 02/UR/09-01,

Institut Superieur de Biotechnologie de Monastir, 5000,

Monastir, Tunisie

W. Mnif � S. Dagnino � A. Escande � A. Pillon �M.-J. Duchesne � V. Cavailles � P. Balaguer (&)

IRCM, Institut de Recherche en Cancerologie de Montpellier,

34298 Montpellier, France

e-mail: [email protected]

W. Mnif � S. Dagnino � A. Escande � A. Pillon �M.-J. Duchesne � V. Cavailles � P. Balaguer

INSERM, U896, 34298 Montpellier, France


Universite Montpellier 1, 34298 Montpellier, France


CRLC Val d’Aurelle Paul Lamarque, 34298 Montpellier, France

S. Dagnino � A. Escande � H. Fenet � E. Gomez � C. Casellas

UMR 5569 Hydrosciences Montpellier, Universite Montpellier

1, 15 Avenue Charles Flahault, B.P. 14491-34093 Montpellier,

France

G. Hernandez-Raquet

INRA UR050, Laboratoire de Biotechnologie de

l’Environnement, 11100 Narbonne, France

123

Arch Environ Contam Toxicol (2010) 59:1–12

DOI 10.1007/s00244-009-9438-0

In the last two decades, the development of human,

industrial and agricultural activities have generated an

increasing amount of organic pollutants such as natural and

synthetic hormones, alkyl phenols, polycyclic aromatic

hydrocarbons (PAHs), polychlorinated biphenyls (PCBs),

brominated flame retardants (BFRs), organochlorine pes-

ticides, and many more. Most of these chemicals have

toxic, carcinogenic, and/or mutagenic properties that can

disrupt the normal functioning of the endocrine system

(Kavlock et al. 1996). Hence, they are recognised as

endocrine-disrupting compounds (EDCs) and they have

been associated with degenerative effects such as femini-

zation, reduction of fertility, changes in sex ratio, devel-

opmental alteration in fish populations (Jobling et al. 2002;

Purdom et al. 1994; Routledge et al. 1998) as well as breast

cancer cell proliferation in humans (Soto et al. 1995). Most

of these chemicals might therefore have a deleterious

impact on the aquatic fauna and more generally on wildlife

and human health.

The main sources of EDCs are domestic, industrial, and

agricultural wastes that end up in wastewater treatment

plants (WWTPs), where they should be eliminated. How-

ever, the efficacy of sewage treatment in WWTPs is

measured using parameters, such as BOD (biological

oxygen demand) and COD (chemical oxygen demand),

which do not take into account EDC removal. In fact,

during sewage treatment, EDCs are only partially elimi-

nated and estrogenic compounds are frequently detected in

WWTP effluents (Gomez et al. 2007; Johnson et al. 2005;

Svenson et al. 2003; Tan et al. 2007; Ternes et al. 1999a).

As WWTP effluents are discharged in the environment, the

presence of endocrine disruptors has been reported in the

receiving waters, rivers, and sediments (Fenet et al. 2003)

and in the marine environment (Noppe et al. 2007).

The study of the presence of EDCs in environmental

samples classically involves chemical analyses that detect

all kinds of pollutants. However, in the case of complex

mixtures, such as sewage and WWTP effluents, this

approach is limited, as only known substances can be

analyzed and its costs strongly limit the number of sub-

stances that can be assessed. Furthermore, the biological

effects of chemical mixtures, such as additivity, synergy, or

antagonism, cannot be predicted from individual analytical

chemical results.

In vitro bioassays appear to be more suitable for the

evaluation of chemical mixtures extracted from environ-

mental samples. In vitro bioassays, based on EDC mech-

anisms of action, might be very useful for rapidly and

inexpensively determining the total biological potency of

distinct substances or chemical mixtures. These tests are

based on the capacity of genetically modified cells to

express the luciferase enzyme following the activation of

nuclear receptors by natural or xenobiotic ligands. Such

bioluminescent cell lines have been widely used to evaluate

dioxin or estrogenic activities in food or complex envi-

ronmental mixtures (Gomez et al. 2007; Pillon et al. 2005;

Riu et al. 2008). Cell bioassays can assess the overall

endocrine-disrupting potential of a sample and gives an

information on the nature of the ligands (agonistic or

antagonistic) for the complex mixtures of pollutants found

in wastewater. EDCs found in WWTPs have many ways of

action; one of them is binding to nuclear receptors, such as

ERs (estrogenic receptors), GR (glucocorticoid receptor),

MR (mineralocorticoid receptor), PR (progesterone recep-

tor), AR (androgen receptor), and PXR (pregnane X

receptor) or to receptors present in the cytoplasm, like the

AhR (aryl hydrocarbon–dioxin receptor).

We developed luciferase reporter cell lines that can be

used to perform large screens to detect the activity of

modulators of nuclear and dioxin receptors in WWTP

samples (Balaguer et al. 1999, 2001; Terouanne et al.

2000). The occurrence of these EDCs in sewage treatment

plants has been well documented by several studies around

the world (Bila et al. 2007; Dagnino et al. 2009; Desbrow

et al. 1998; Kim et al. 2007; Stasinakis et al. 2008; Svenson

et al. 2003; Ternes et al. 1999b). However, there are only

few figures for the Mediterranean area (Gomez et al. 2007;

Stasinakis et al. 2008) and no data at all concerning

Tunisian WWTPs. Therefore, in this study we decided to

characterize the endocrine-disrupting potential of Tunisian

WWTP samples using the luciferase reporter cell lines we

developed. Moreover, despite the existence of several

studies investigating the presence of EDCs in wastewater,

most of them were focused exclusively on WWTP aqueous

fractions. Suspended particulate matter is often not inclu-

ded in wastewater analysis, and concentrations of EDCs in

sludge are rarely determined. On the other hand, due to the

relatively high octanol–water partition coefficients of these

compounds, it is reasonable to expect that a significant part

of EDCs is adsorbed to the suspended particulate matter or

accumulated in biosolids. Therefore, we collected aqueous,

suspended particulate matter, and sludge samples from

three Tunisian WWTPs to characterize the endocrine-dis-

rupting potential of these three fractions.

Materials and Methods

Materials

Cell culture products were obtained from Life Technolo-

gies (Cergy-Pontoise, France). Luciferin was purchased

from Promega (Saint-Quentin-Fallavier, France). Aldoste-

rone, benzo(a)pyrene (BaP), dexamethasone, and E2 and

3-methylcholanthrene (3MC) were purchased from

Sigma Chemical Co (St. Louis, MO, USA). R5020

2 Arch Environ Contam Toxicol (2010) 59:1–12

123

(promegestone) was a gift from Sanofi-Aventis (Romain-

ville, France). R1881 (methyltrienolone) and SR12813

were purchased from NEN Life Science Products (Paris,

France) and Tebu-bio (LePerray en Yvelines, France),

respectively. Dioxin was obtained from Promochem

(Molsheim, France). For bioassays experiments, stock

solutions were prepared in dimethyl sulfoxide (DMSO) at

10 mM and stored at -20�C.

Generation of Stably Transfected Reporter Cell Lines

The stably transfected luciferase reporter cell lines were

obtained as already described (Balaguer et al. 2001).

Briefly, the MELN cell line, to measure ER transcriptional

activation, was obtained by transfecting MCF-7 cells (ERa-

positive breast cancer cells) with the ERE-bGlob-Luc-

SVNeo construct in which an estrogen responsive element

(ERE) was cloned upstream of the luciferase reporter gene

(Balaguer et al. 1999).

PALM cells, to characterize AR transcriptional activa-

tion, are human prostate adenocarcinoma PC-3 cells stably

transfected with human AR and an AR-responsive element

cloned upstream of a luciferase reporter gene construct

(Terouanne et al. 2000).

HAhLP cells, used to detect dioxin receptor-mediated

activity, were obtained by transfecting HeLa cells with the

CYP1A1-Luc and pSG5-puro plasmids (Pillon et al. 2005).

HG5LNGal4-PR, HG5LNGal4-GR, HG5LNGal4-MR,

and HG5LNGal4-PXR cells, used to detect progesterone-,

glucocorticoid-, mineralocorticoid-, and PXR-like activi-

ties, respectively, were derived from HeLa cells in two

steps. HG5LN cells were obtained by stably transfecting a

Gal4-responsive reporter gene into HeLa cells. The

resulting cells were then stably transfected with the Gal4

DNA binding domain (DBD)–hPR ligand binding domain

(LBD), DBD-hGR (LBD), DBD-hMR (LBD), and DBD-

hPXR (LBD) expressing plasmids, respectively (Lemaire

et al. 2006; Molina-Molina et al. 2006).

Cell Culture Conditions

HG5LN and MELN cell lines were grown in Dulbecco’s

modified Eagle’s medium (DMEM) with phenol red, 1 g/L

glucose, 5% of fetal calf serum (FCS) (culture medium)

and 1 mg/mL geneticin in a 5% CO2 humidified atmo-

sphere at 37�C. HG5LN Gal-GR, -MR, -PR, and -PXR cells

were cultured in culture medium supplemented with 1 mg/

mL geneticin and 0.5 lg/mL puromycin. The HahLP cell

line was cultured in culture medium supplemented with

0.5 lg/mL puromycin. Because of phenol red and FCS

steroid activity, in vitro experiments were carried out in

DMEM without phenol red and supplemented with 5%

dextran-coated, charcoal-treated FCS (DCC) (test culture

medium). PALM cells were maintained in Ham’s F-12

medium supplemented with 10% FCS, 1 mg/mL geneticin,

and 1 lg/mL puromycin in a 5% CO2 humidified atmo-

sphere at 37�C. The test culture medium was Ham’s F-12

medium supplemented with 3% DCC.

Sample Collection

Wastewater, suspended particulate matter, and sludge were

sampled in three WWTPs with similar treatment capacities

(Sites A, B, and C) located along the Tunisian coastal area

near the towns of Sousse and Sfax (Fig. 1). The main

characteristics of each WWTP are described in Table 1.

Site A influents were from domestic and industrial origins

(i.e., plastic, detergent, paint, and other chemical waste).

Site B treated only domestic sewage waste from an area

with intense tourist activity. Finally, Site C received both

domestic and industrial (mainly textile industry) influents.

The three WWTPs performed a primary (sedimentation),

secondary (activated sludge), and tertiary (chlorination)

treatment before discharging the effluents into the Medi-

terranean Sea. Site B used anaerobic sludge, whereas Sites

A and C used aerobic conditions. Water grab samples were

collected in glass bottles at influent and effluent within the

Fig. 1 Location of the three WWTPs (Sites A, B, and C) along the

Tunisian coastal area

Arch Environ Contam Toxicol (2010) 59:1–12 3

123

WWTPs and stored at 4�C until filtration within 24 h.

Dried sludge grab samples were collected in aluminum

containers and frozen at –20�C until extraction.

Sample Preparation

Samples from Sites A, B, and C were collected between

November 2003 and March 2004. In a first group of

experiments, a few milliliters of influent and effluent

wastewaters from Sites A and B were collected. They were

filtered through a solvent-rinsed Whatman GF/C filter

(Whatman) to eliminate the suspended solids and were then

stored at –20�C. When needed, they were thawed and fil-

tered through a 0.22-lm nitrocellulose filter (Corning).

Different amounts (up to 50 lL) of filtered samples were

directly added to the culture medium.

In a second group of experiments, wastewater samples

(1.5 L) were collected from Sites A, B, and C influents.

Raw water samples were centrifuged (2000 g, 15 min) to

eliminate the suspended solids. Supernatants were then

extracted by solid-phase extraction as described in Pillon

et al. (2005). Briefly, aqueous samples were concentrated

on reverse- phase C18 (5 g, 20 mL) cartridges (Sigma

Aldrich, France) preconditioned with methanol. Elutions

from columns were performed with methanol followed by

hexane. Eluates were dried at 37�C in a rotary evaporator

and residues were taken up in 2 mL methanol (concentra-

tion factor: 750).

From the same sites, larger influent wastewater samples

(5 L from Site A, 9 L from Site B, and 6 L from Site C) and

sludge were also collected. Suspended particulate matter

was recovered by centrifugation of these samples. Sus-

pended particulate matter and sludge were homogenized

before lyophilization. Lyophilized samples (2.7 g, 5.7 g,

and 0.84 g of suspended particulate matter from Sites A, B,

and C, respectively, and 20 g of sludge for each site) were

then extracted twice with dicholoromethane/methanol (2:1

v/v) (for 20 and 30 min). Extracts were dried by passing

through anhydrous sodium sulfate on glass microfibers, and

after evaporation in a rotary evaporator, they were taken up

with 2 mL methanol. Concentrations factors for suspended

matters were 2500, 4500, and 3000 for Sites A, B, and C,

respectively. Methanol extracts were stored at –20�C.

Filtered aqueous samples and concentrated samples from

the aqueous, suspended particulate matter, and sludge

fractions were then added to stably transfected reporter cell

lines, and receptor activation was measured by luciferase

assay.

A water blank extraction was performed with 1.5 L of

Milli-Q water. A suspended solids blank extraction was

done with clean filters. No activity was detected in blank

extraction samples.

Luciferase Assay

For luciferase assay, 5 9 104 cells per well were seeded in

96-well white opaque tissue culture plates (Greiner,

France) with 150 lL test culture medium/well. Eight hours

after plating, 50 lL of filtered aqueous samples or con-

centrated extracts diluted in test culture medium were

added to each well for 16 h except in HAhLP cells, in

which WWTP samples were left for 8 h or 24 h. Experi-

ments were performed in quadruplicate. At the end of the

incubation time, the test culture medium was removed and

replaced by fresh test culture medium containing 0.3 mM

luciferin. Intact living cell luminescence was measured

with a Microbeta Wallac luminometer (E & G) for 2 s and

expressed as relative luminescence units (RLUs). Sample

activities were expressed as the percentage of the maximal

activity obtained with the reference ligand at saturating

concentration: 10 nM for E2 and R1881; 100 nM for

dexamethasone, aldosterone, and R5020; 1 lM for

SR12813, and 10 nM for dioxin, in MELN, PALM,

HG5LN Gal-GR, -MR, -PR, -PXR, and HahLP cells,

respectively.

In parallel, tests were performed to ascertain that the

agonistic activities were specific. For MELN, PALM, and

HAhLP cells, the activation of luciferase expression was

measured in the presence of the saturating concentration of

the specific agonist and increasing concentrations of sam-

ple extracts. If no superactivation was observed with the

sample, the sample activity was considered specific. For

HG5LN derivatives cells, the HG5LN parent cell line was

used because it contains the same reporter gene as the

derivative cells. In addition, the reporter gene is integrated

at the same site in the genome and its expression is

Table 1 Characteristics of three WWTPs

Population

equivalents

Flow (m3/day) BOD5 influent

(mg/L)

Suspended solids

influent (mg/L)

HRT (h)

Site A 300,000 24,000 500 400 24

Site B 132,000 17,430 400 380 12

Site C 323,000 18,700 420 450 8

BOD biological oxygen demand, HRT hydraulic retention time


123

constitutive. HG5LN cells show a basal activity in the

absence of any tested compound. Any modification of this

baseline activity by a sample indicates that this activity is

nonspecific. On the other hand, the activity of a sample,

which induces luciferase expression in the derivative cells

but not in the parental HG5LN cells, is considered specific.

Tests were also performed to detect antagonistic activities

of samples. To this aim, cells were activated with the ref-

erence agonist at a concentration yielding 80% of the

maximal activity in the presence of increasing concentra-

tions of sample extracts.

Inhibition Test of MELN Activation

To assess whether high-affinity estrogens were present,

even at low concentration, we used a method described

earlier and called the ‘‘inhibition test of MELN cell acti-

vation’’ (Pillon et al. 2005). In this test, transactivation of

cellular ERa by high-affinity estrogenic compounds is

competitively inhibited by limited amounts of recombinant

ERa. MELN cells were seeded in 96-well white opaque

tissue culture plates, as described earlier. In separate tubes,

WWTP-filtered aqueous samples and methanol extracts

were diluted in test culture medium at the concentration

needed to obtain 80% of MELN cell maximal activity and

preincubated without or with concentrated purified

recombinant ERa (1, 10, and 100 nM final concentration)

in test culture medium at 4�C for 16 h. Then culture

medium of MELN cells was removed and cells were

incubated with the different preincubated mixtures for 6 h

at 37�C before luciferase assay.

Statistics and EC50 Values

Each agonist concentration response curve was fitted using

the sigmoidal dose–response function of a graphic and

Graph-Pad Prism, version 4.0, 2003 (Graphpad Incorpo-

rated, San Diego. CA). EC50 (the concentration yielding

half of the maximum activity) values were calculated from

the same dose–response curves. For compounds that pro-

vided incomplete dose–responses curves, EC25 (the con-

centration yielding 25% of the maximum activity) values

were calculated from the same dose–response curves.

Expression of WWTP Samples’ Activities

The WWTP samples’ activities were determined by

dividing the EC50 of the reference chemicals (expressed in

nanograms per liter for E2 and micrograms per garm for

dioxin and SR12813) by that of the sample (expressed as

equivalent per liter of water or per gram of dry suspended

particulate matter or sludge). The calculation takes into

account the fold concentration of the methanol extracts

over the crude samples. Three reference EC50 values were

taken from published results: 0.018 nM (4.9 ng/L) for E2 in

MELN cells (Pillon et al. 2005), 140 nM (70.63 lg/L) for

SR12813 in HG5LN Gal4-PXR cells (Lemaire et al. 2004),

and 0.1 nM (28.4 ng/L) for R1881 in PALM cells (Molina-

Molina et al. 2006). The reference EC50 value for dioxin in

HAhLP cells (0.2 nM or 64.4 ng/L) was derived from the

present experiments in which cells were incubated for 8 h

(see the Results section).

Results

Estrogenic Activity of WWTP-Filtered Aqueous

Samples

In the first phase of this study, estrogenic activity was

assessed in filtered influent and effluent aqueous samples

from Sites A and B by measuring luciferase activation in

MELN cells. Filtered influent samples from both sites

showed more than or around 70% of the maximum estro-

genic activity when present at 10% concentration in test

culture medium (Fig. 2). The effluent sample from Site A

was nearly as active as the corresponding influent, whereas

the effluent from Site B exhibited very little activity.

10-1 10 0 101

0

25

50

75

100

% of water

Tra

nsac

tivat

ion

(%)

Fig. 2 Relative percentage of estrogenic activity of filtered influent

and effluent aqueous samples from Sites A and B measured using

MELN cells. Nonconcentrated influent (j) and effluent (d) samples

from Site A and influent (h) and effluent (s) samples from Site B.

The percentage of luciferase activity measured per well (mean ± SD

of quadruplicates) is represented as a function of the percentage of

aqueous sample in the test culture medium. . The value obtained in

the presence of 10 nM E2 was taken as 100% transactivation


123

Taking into account the EC50 of the reference ligand E2

(4.79 ng/L), we evaluated the concentration of estrogenic

compounds in the water samples. Concentration ranged

between 79 and 119 ng/L E2 eq for the influents and cor-

responded to 58.5 ng/L E2 eq for the effluent from Site A,

whereas it was below the quantification limit for Site B

(Table 2). For Site B, the limit of quantification (around

47 ng/L E2 eq for this experiment) was used to calculate

the removal percentage. Therefore WWTP removal effi-

ciency of estrogenic compounds was only 26% for Site A

and above 88% for Site B.

Estrogenic Activity of WWTP-Concentrated Aqueous,

Suspended Particulate Matter, and Sludge Methanol

Extracts

In the second phase of the study, we analyzed the influent

aqueous, suspended particulate matter, and sludge metha-

nol extracts from the three WWTPs. Taking into account

the concentration factor, the estrogenic activity of influent

samples ranged between 54.5 and 81.7 ng/L E2 eq for the

aqueous, 8 and 24.5 ng/g E2 for the suspended particulate

matter, and between 5.4 and 19 ng/g E2 eq for the sludge

extracts (Fig. 3a). When taking into account the weight of

particulate matter per liter (0.54, 0.63, and 0.14 g) for Sites

A, B, and C, respectively, the activity was estimated to be

8.4, 5.7, and 3.5 ng/L E2 eq for Sites A, B, and C,

respectively, which represents less than 15% of the corre-

sponding value for aqueous samples.

The specificity of the measurements was confirmed by

performing experiments in which cells were incubated with

10 nM of saturating E2 and increasing concentration of

WWTP extracts. There was no or negligible superactiva-

tion with 0.03% and 0.01% extracts (100–120%, not

shown) except for the sludge extract from Site C (145%,

not shown). In conclusion, the estrogenic activity of

WWTP influents from Sites A, B, and C was mainly

present in aqueous samples rather than in suspended par-

ticulate matter or sludge.

High-Affinity Estrogens Are Present in Concentrated

Aqueous Samples

To determine whether the estrogenic activity detected in

WWTP samples was due to low- or high-affinity compounds,

Table 2 Estrogenic activity of influents and effluents of Sites A and

B

Concentration (pmol E2 eq/l)

Site A Site B

Influents 291 ± 18.5 400 ± 56.3

Effluents 215 ± 18.8 nd

Note: Results were determined from the dose–response curves shown

in Fig. 2 and expressed as a ratio of E2 (reference agonist) equivalent

per liter of water

nd not determined values because the activity of Site B effluents was

to low to allow EC50 calculation Site A Site B Site C0

50

100

150Water

Particulate matter

Sludge

ng

of

E2

Eq

. /L

or

/g

Site A Site B Site C0

1

2

3

4

5

µg

of

Dio

xin

Eq

. / L

or

/g

Site A Site B Site C0.0

0.1

0.2

0.3

0.4

0.5

mg

of

SR

1281

3 E

q. /

L o

r /g

Fig. 3 Endocrine-disrupting activities in influent aqueous, influent

suspended particulate matter, and sludge extracts from Site A, Site B,

and Site C. The reference ligands were E2 in MELN cells, dioxin in

HAhLP cells, and SR12813 in HGPXR cells. The activities are

expressed as reference ligand equivalent per liter in aqueous extracts

(light gray bars) and as ligand equivalent per gram in suspended

particulate matter (medium gray bars) and sludge (dark gray bars)

extracts. They represent the ratio between the EC50 values given by

the dose–response curves of WWTP samples and the EC50 values of

the reference compounds ± SD and they take into account the fold

concentration of the methanol solution over the WWTP crude sample


123

we performed the ‘‘inhibition test of MELN cell activation,’’

in which ERa transactivation by high-affinity estrogenic

compounds is competitively inhibited by limited amounts of

recombinant ERa (0–100 nM) (Pillon et al. 2005). We

observed that in the presence of 100 nM recombinant ERa,

the luciferase activity induced by the aqueous samples from

the three sites was close to background level (20%, as shown

in Fig. 2), indicating that it was strongly inhibited by

recombinant ERa (Fig. 4 for Site A and data not shown).

This result shows that the aqueous samples contained high-

affinity compounds, such as natural and synthetic hormones;

by contrast, suspended particulate matter and sludge extracts

contained lower-affinity estrogens like alkylphenols.

PR, GR, and MR Activities Were Not Detected in

WWTP Samples

HG5LN-Gal4 PR, -Gal4 GR, and -Gal4 MR cell lines were

used to measure the respective (ant)agonistic activities in

Site A, B, and C aqueous, suspended particulate matter,

and sludge extracts. No agonistic activity was detected (not

shown). As a number of endocrine disruptors have PR, GR,

and MR antagonistic activities (Molina-Molina et al. 2006;

Willemsen et al. 2004), the same cell lines were used to

assess the antagonistic activities in the same extracts.

Again, no activity was detected (data not shown).

Weak AR Agonistic Activity Is Detected in WWTP

Samples

PALM cells were used to measure androgenic agonistic

and antagonistic activities in aqueous, suspended

particulate matter, and sludge extracts from Sites A, B,

and C. Cells were either incubated with the different

extracts in the absence of R1881 to assess agonistic

activities or in presence of R1881 to assess antagonistic

activity. Weak agonistic activity was only observed in

aqueous extracts (Fig. 5). Taking into account the con-

centration factor, the activity ranged between 37.9 and

75.7 ng/L. This androgenic activity was specific because

preincubation with 100 nM R1881 and 0.03% and 0.1%

aqueous extracts did not increase luciferase activation

(data not shown). No antagonistic activity was detected

(not shown).

Evaluation of AhR Activity Induced by 3MC, BaP,

Dioxin, and WWTP Samples in HAhLP Cells After

Two Different Incubation Times

In order to better characterize AhR activity, HAhLP cells

were exposed to the different samples for 8 h and 24 h.

Previous works (Jones et al. 2000; Machala et al. 2001), in

which the CYP1A1 gene reporter cell line was used to study

the mechanisms of AhR-mediated induction, reported that,

due to degradation, luciferase activation by PAHs

decreased after 6 h of incubation, whereas it did not change

when induced by dioxin-like compounds.

0 1 10 1000

25

50

75

100

Recombinant ER concentration (nM)

Tra

nsac

tivat

ion

(%)

Fig. 4 Inhibition test of MELN cell activation. Luciferase activity

induced by influent aqueous (j), influent suspended particulate

matter (�), and sludge extracts (D) from Site A was measured in the

presence of 0–100 nM recombinant ERa. The percentage of luciferase

activity measured per well (mean ± SD of quadruplicates) is

represented as a function of the percentage of WWTP extract in the

test culture medium. The value obtained in the presence of 10 nM E2

was taken as 100% activity10-3 10-2 10-1

0

25

50

75

100

% of ExtractT

rans

activ

atio

n (%

)Fig. 5 Relative percentage of androgenic activity in influent aqueous

extracts from the three Tunisian WWTPs measured with PALM cells.

The percentage of luciferase activity of influent aqueous extracts from

Site A (j), Site B (�), and Site C (D) per well (mean ± SD of

quadruplicates) is represented as a function of the percentage of

WWTP extract in the test culture medium. The value obtained in the

presence of 100 nM R1881 was taken as 100% transactivation


123

HAhLP cells were incubated with 3MC, BaP, dioxin,

aqueous, suspended particulate matter, or sludge extracts

for 8 h and 24 h. 3MC and BaP induction of luciferase was

stronger after 8 h of incubation than after 24 h, and their

EC50 values were higher after 24 h than after 8 h (3MC: 37

nM at 24 h vs. 13 nM at 8 h; BaP: 130 nM vs. 35 nM)

(Fig. 6b, c). In the case of dioxin, activation was lower after

24 h, but the EC50 did not change much (0.56 nM at 8 h vs.

0.8 nM at 24 h) (Fig. 6a). Finally, luciferase induction by

WWTP extracts from Site A was stronger after 8 h than

after 24 h of incubation and EC50 values were much higher

after 24 h than after 8 h like for 3MC and BaP (Fig. 7).

These results indicate that Site A contained mainly meta-

bolically labile agonistic compounds. The same findings

were obtained for Site B and C (data not shown).

AhR Activity in WWTP Samples

HAhLP cells were used to detect AhR agonistic activity in

WWTP samples. No AhR agonistic activity was detected in

filtered influent samples (not shown). At all sites, AhR

activity was stronger in suspended particulate matter and

sludge extracts than in concentrated aqueous samples

(Fig. 8), in which it was too low to allow quantification

(Fig. 8a). AhR activity in suspended particulate matter

extracts, as measured after 8 h of incubation (Fig. 8b), was

41.29 lg/g dioxin eq at Site A, 0.64 lg/g dioxin eq at Site

B, and 3.22 lg/g at Site C (Fig. 3b). In sludge extracts,

AhR activity corresponded to 0.64 lg/g dioxin eq at Site B,

1.28/g at Site B, and 32.2 ng/g at Site C. When specificity

was assessed, we observed that all responses were specific

except those due to Site C sludge extracts at concentrations

higher than 0.03% (not shown). In conclusion, AhR

activity was mainly observed at Site C (domestic origin and

textile industry).

10-9 10-8 10-7 10-60

25

50

75

100

125

Bap 24 hr

Bap 8 hrc

Concentration (M)

RL

U

10-10 10-9 10-8 10-70

25

50

75

100

125

150

3MC 24 hr

3MC 8 hrb

Concentration (M)

RL

U

10-11 10-10 10-9 10-8 10-70

25

50

75

100

125

150

175

200

Dioxin 24 hr

Dioxin 8 hra

Concentration (M)

RL

U

Fig. 6 Induction of luciferase activity by dioxin, 3MC, and BaP in

HAhLP cells after two different incubation times. HahLP cells were

incubated with dioxin (j, h), 3MC (m, 4), or BaP (d, �) for 8 h

(solid symbols) and 24 h (open symbols). The luciferase activity,

expressed as RLUs per well (mean ± SD of quadruplicates), is

represented as a function of the ligand concentration

10-5 10-4 10-3 10-2 10-1

0

25

50

75

100

125

150

175

Concentration(% of methanol solution)

RLU

Fig. 7 Induction of luciferase activity by WWTP extracts from Site

A in HAhLP cells after two different incubation times. HAhLP cells

were incubated with influent aqueous (d, �), influent suspended

particulate matter (m, 4), or sludge (j, h) extracts for 8 h (solidsymbols) and 24 h (open symbols). The luciferase activity, expressed

as RLUs per well (mean ± SD of quadruplicates), is represented as a

function of the percentage of WWTP extract in the test culture

medium


123

PXR Activity in WWTP Samples

HG5LN-Gal4 PXR cells were used to detect PXR activity

in the different samples. As for AhR activity, we did not

observe any PXR agonistic activity in influent filtered

samples (not shown) and aqueous extracts (Fig. 9a). Con-

versely, we detected a strong PXR activity in suspended

particulate matter and sludge extracts (Fig. 9b,c). PXR

activity reached 0.2–0.3 mg/g SR12813 eq in suspended

particulate matter extracts from all sites, 0.22 lg/g

SR12813 eq in Site A sludge extracts, and only around

0.05 mg/g SR12813 eq in Sites B and C sludge extracts

(Fig. 3c). Moreover, we observed that all responses were

specific except those induced by incubation with sludge

extracts from Site C (not shown) at concentrations higher

than 0.03%.

Discussion

In this work, we characterised the EDCs present in three

Tunisian WWTPs (Site A, B and C) by using several

0

25

50

75

100b

Tra

nsac

tivat

ion

(%)

0

25

50

75

100a

Tra

nsac

tivat

ion

(%)

10-4 10-3 10-2 10-10

25

50

75

100

125c

% of Extract

Tra

nsac

tivat

ion

(%)

Fig. 8 Relative percentage of xenobiotic activity of influent aqueous,

influent suspended particulate matter, and sludge extracts from the

three Tunisian WWTPs measured with HahLP cells. Influent aqueous

(a), influent suspended particulate matter (b), and sludge extracts (c)

from Site A (j), Site B (�), and Site C (D). The percentage of

luciferase activity per well (mean ± SD of quadruplicates) is


test culture medium. The value obtained in the presence of 100 nM

dioxin was taken as 100% transactivation

0

25

50

75

100a

Tra

nsac

tivat

ion

(%)

0

25

50

75

100b

Tra

nsac

tivat

ion

(%)

10-4 10-3 10-2 10-10

25

50

75

100

125

150c

% of Extract

Tra

nsac

tivat

ion

(%)

Fig. 9 Relative percentage of xenobiotic activity of influent aqueous,

influent suspended particulate matter, and sludge extracts from the

three Tunisian WWTPs measured with HGPXR cells. Influent

aqueous , influent suspended particulate matter , and sludge extracts

from Site A (j), Site B (�), and Site C (D). The percentage of

luciferase activity per well (mean ± SD of quadruplicates) is


test culture medium. The value obtained in the presence of 1 lM

SR12813 was taken as 100% transactivation


123

luciferase reporter cell lines we developed to detect the

activity of modulators of nuclear and dioxin receptors in

WWTP samples (Balaguer et al. 1999, 2001; Terouanne

et al. 2000).

We first assessed the estrogenic-disrupting potential of

filtered influent and effluent wastewater samples from Sites

A and B using MELN cells. The results show that influents

from both sites had a significant estrogenic activity and

that, at Site A, these compounds were not totally eliminated

upon wastewater treatment. On the other hand, the estro-

genic activity was found to be insignificant in Site B

effluent sample, indicating a very efficient biodegradation

upon treatment. Our results are in agreement with pub-

lished studies showing that estrogenic activity is present in

WWTP waters (Aerni et al. 2004; Desbrow et al. 1998;

Fernandez et al. 2007, 2008; Gomez et al. 2007; Harries

et al. 1999; Korner et al. 1999; Svenson et al. 2003; Tan

et al. 2007).

Aqueous, suspended particulate matter, and sludge

extracts also presented estrogenic activity. Using compe-

tition experiments (Pillon et al. 2005), strong-affinity

compounds were shown to be present in aqueous extracts,

whereas lower-affinity compounds were mainly in sus-

pended particulate matter and sludge extracts. These dif-

ferences could be due to the fact that high-affinity

compounds (natural estrogens such as E2, E1, and E3, or

synthetic estrogens like EE2) coming from human rejec-

tions (Baronti et al. 2000; D’Ascenzo et al. 2003) are

present mainly in aqueous extracts, whereas weak-affinity

chemicals like nonylphenols, which show higher adsorp-

tion properties (Fenet et al. 2003; Hernandez-Raquet et al.

2007), could be adsorbed to sludge.

We then tested whether other EDCs were present in the

different extracts and detected a weak androgenic agonist

activity. This activity, however, was only found in aqueous

extracts. Other studies also found weak androgenic activity

in wastewater (Kirk et al. 2002; Leusch et al. 2006; Liu

et al. 2009; van der Linden et al. 2008). An earlier Cana-

dian study characterized AR activity as being mediated by

testosterone compounds from animal feedings and human

feces (Lee et al. 2004). No progesterone, glucocorticoid,

and mineralocorticoid (ant)agonistic activities were

observed. Similar findings about progesterone activity were

reported by van der Linden et al. (2008), who, however,

found 11–38 ng/L dexamethasone eq in wastewater. Our

results also disagree with those by Chang and Shao (2007),

who detected six glucocorticoids (prednisone, predniso-

lone, cortisone, cortisol, dexamethasone, and 6 alpha-

methylprednisolone) in sewage treatment plants. These

discrepancies could be due to the extraction procedures we

used, which might not be efficient enough for this type of

compounds.

Concerning AhR activity, we observed dioxin-like

activity mainly in suspended particulate matter extracts and

determined that the dioxin-like activity was mostly due to

metabolically labile compounds such as PAHs. Theses

results are in agreement with another study concerning the

Tunisian region that showed that AhR activity is mediated

by the same kind of compounds (Louiz et al. 2008).

Because PAHs have high log Kow coefficients, they tend to

accumulate in sludge and suspended solids, as observed

also in this work. Finally, PXR activity could be measured

only in suspended particulate matter and sludge extracts

and the values were similar to those obtained in sludge

extracts in France (Patureau et al. 2008). PXR activity

could be due to some environmental compounds like

estrogens, nonylphenols, or pesticides (Creusot et al. 2010;

Kinani et al. 2010; Lemaire et al. 2006; Mnif et al. 2007).

Finally, we showed in this study that environmental

compounds are mainly ER, AR, PXR, and AhR activators.

Concerning ER and AhR compounds, competition experi-

ments with recombinant receptor and analysis at different

times of exposure of the AhR activation gave some indi-

cations of the compound’s nature that need to be confirmed

by chemical analysis. On other environmental samples, we

used a combined approach involving targeted chemical

analyses of more than 50 chemicals selected on the basis of

both their environmental occurrence and their known EDC

potency, and a similar panel of in vitro bioassays that

allowed the detection of ERa, AR-, AhR-, and PXR-med-

iated activities (Creusot et al. 2010; Kinani et al. 2010).

The contribution of analyzed EDCs in the biological

activities detected by the bioassays was estimated by

comparing toxic-equivalent quantities from both approa-

ches. The natural estrogens bE2 and E1 and of PAH-like

compounds were identified as main contributors to estro-

genic and dioxin-like activities, respectively, as determined

by the bioassays. Conversely, (anti)androgenic and PXR-

mediated activities were detected, but the responsible

compounds could not be identified using targeted chemical

analyses.

To precisely identify the compounds responsible for the

PXR activity in our samples, we are currently testing a

recombinant PXR-based purification procedure (Dagnino

et al. unpublished data) similar to the ERa-based affinity

columns we previously used for the isolation of estrogen

compounds (Pillon et al. 2005; Riu et al. 2008). Further-

more, this technique could be also applied to emerging

environmental nuclear receptors targets such as androgen

receptor (Creusot et al. 2010; Kinani et al. 2010; Liu et al.

2009), peroxysome proliferator activated receptor c (Grun

and Blumberg 2006), estrogen related receptor c (Li et al.

2010; Okada et al. 2008), or retinoid X receptors (le Maire

et al. 2009).


123

Conclusion

We studied the EDC activity of three Tunisian sewage

treatment plants that receive wastewater from different

sources (domestic and industrial). Our results show a dif-

ference in the efficiency of wastewater treatment between

Site A (26%) and Site B (stronger percentage) possibly due

to the nature (aerobic in Site A; anaerobic in Site B) and

duration of treatment at each site (shorter at Site A than at

Site B). In aqueous extracts, activities were roughly com-

parable. In suspended particulate matter extracts, the

strongest estrogen and AhR activities were detected at Site

C, and PXR activity was more important at the two

industrial sites (Sites A and C). Sludge extracts from Sites

A and C presented more toxicity at 0.1% methanol con-

centration. Finally, PR, GR, and MR agonist and/or

antagonist activities were not detected in the samples from

the three sewage treatment plants. To precisely identify the

compounds responsible for the ER, AR, AhR, and PXR

activities in these samples, more investigation using

nuclear receptor affinity columns and chemical analyses by

mass spectrometry techniques will be needed.

Acknowledgments This study was co-funded by the Tunisian

Ministry for the Scientific Research, the Technology and the Devel-

opment of Competences, to the Research Unit 02/UR/09-01 of the

Higher Institute of Biotechnology of Monastir, and by the Embassy of

France in Tunisia/EGIDE Montpellier, France (SSHN, 2008). WM is

currently at the Department of Biology of the Higher Institute of

Biotechnology of Sidi Thabet, University of Manouba, Tunisia.

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Biological Analysis of Endocrine-Disrupting Compounds in Tunisian Sewage Treatment Plants

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