沙漠研究 22-1, 319 -322 (2012 )"ICAL 1 / DT X"

Journal of Arid Land Studies－Refereed Paper－Sensitivity of in Vitro Bioassays towards Several Water Origins
in Tunisian Arid and Semi-arid Area
Fahmi BEN FREDJ1), Mitsuteru IRIE1), Junkyu HAN1), Atef LIMAM2),
Ahmed GHRABI2) and Hiroko ISODA*1)

Abstract: Arid and semi-arid lands are confronted to water resources scarcity. Thus, it is essential to prevent their contamination.
Hazardous compounds present in the effluents or solid waste as single compounds or mixture may settle in receptor sites anddrastically affect the human health through drinking from aquifers or the food chain. Control of effluents has traditionally beenregulated using physicochemical parameters. Moreover, information about biological effects is unknown. Thus, the introduction ofin vitro assays using mammalian cells to investigate the presence of environmental contaminants is necessary. The aim of this study
is to investigate the biological effects on mammalian cells of potential environmental contaminants present in river, wastewater,leachate, channel, and dam samples obtained from semi-arid area in Tunisia. This new approach for risk assessment in water
environments used a combination of physicochemical parameters with in vitro bioassays relative to stress response and endocrine
disrupters. Among all, the leachate sample (2520 mg/L), the river sample R1 (1792 mg/L) on the far downstream of the watershednetwork and the wastewater sample W5 (1056 mg/L) from Grombalia STP showed the highest levels of organic content. Tarhouna
and El Bey rivers have very high estrogenic activity because they receive wastewater effluents from Beni Khalled and Grombalia
STPs. As expected the highest stress response levels were observed for the industrial wastewater W3 and the leachate L samples.
These specific bioassays systems, sensitive to wastewater and water components responsible for stress response or estrogenic activity
are very useful in evaluating the associated hazardous risk.
Key Words: Endocrine disruptors, Leachate, Risk assessment, Stress response, Wastewater

1. Introduction
Water environments have to face solid or liquid discharges
containing a complex mixture of various organic and inorganicsubstances. Wastewater reclamation and reuse projects havebeen developed in semi-arid lands as an essential response to
growing water needs. The treated water was mainly
discharged into natural bodies and eventually reused forrecharging the groundwater or irrigating some specified
cultures, public green areas and sports facilities such as golf
courts or football fields. Through pathways such as drinking
from aquifers or the food chain, humans are confronted withthe threat of residual pollutants in effluents. In semi-arid
lands, particularly where the water resources are scarce, thedetection of contaminants and the measurement of theirharmful impact has become a major issue in environmental
risk assessment. Therefore, the aim of this study is toinvestigate the biological effects on mammalian cells ofpotential environmental contaminants including endocrine
disrupters and heavy metals present in river, wastewater,leachate, channel, and dam samples obtained from arid andsemi-arid area in Tunisia. Hence, in vitro bioassays systems
to evaluate the biological effects on mammalian cells of water
samples obtained from rivers, wastewater effluents, leachate,channels, and a dam. Heat Shock Protein 47 assay for stressresponse and E-screen assay for estrogenic activity werecarried out to investigate the sensitivity of these in vitro
bioassays toward several water origins.

2. Materials and Methods
2.1. Sampling sites
Water samples from several water origins in Soliman andGrombalia (lat. between N36:31:55.38 and N36:43:21.96, long.
between E10:28:21.54 and E10:32:06.06) semi-arid area inTunisia were obtained on March 24th and 25th, 2008 (Fig. 1).
The samples includes mainly six river samples (R1 to R6)from El Bey river watershed and six wastewater effluents (W1to W6) from Grombalia and Beni Khaled sewage treatment
plants (STPs) as well as from industrial installations.
Besides, two samples from a drainage channel (D1 and* Corresponding Author: isoda.hiroko.ga@u.tsukuba.ac.jp305-8572 Tennodai 1-1-1 Tsukuba Ibaraki, University of Tsukuba, Cooperative Research Bulding A411
1) Alliance for Research on North Africa, University of Tsukuba, Japan2) Water Treatment and Recycling Laboratory, CERTE, Borj Cedria Technopark, University of Tunis El Manar, Tunisia

STtB. KhalledDrainage
channelIndustry
W3 W4 W

5 W6

R3W2
W1R5Mediterranean
sea
R1R2
D1
D2landfillL

R44R
Channel DRaedjerda
Cap bonDinking water
Urban Wastewater
Drainage
Leachate
Industrial wastewater
Irrigation

R6IR
Masri
Dam

STt
Drombalia

Fig. 1. Sampling sites in Soliman and Grombalia areaD2), a leachate sample from Soliman urban landfill (L) inaddition to a sample (IR) from El Masri dam for irrigation
purpose were collected. Moreover, a sample (DR) takenfrom Medjerda-Cap bon channel situated on the upstream of adrinking water treatment plant was considered. A primary
filtration using a 0.45 μm filter was carried out on the samplingday. Samples were stored at -20°C until transport inside anicebox to the laboratory where the same conditions weremaintained. A secondary filtration using a 0.22 μm filter wasperformed prior to use in the bioassays following pH
adjustment between 5 and 6 in order to maintain optimum
conditions for cells culture.
2.2. Water quality parameters
The following in situ parameters were immediately
determined as follows: Temperature and pH using a pH meter
330i, dissolved oxygen using an oxymeter 340i, and electric
conductivity using a conductimeter 330i (all fromWissenschaftlich-Technische-Werkstӓtten,Weilheim,
Germany). Other physicochemical parameters, such as
Chemical Oxygen Demand (COD), 5d-Biological Oxygen
Demand (BOD5), and suspended sludge (SS) were analyzed in
the laboratory according to standard methods (APHA, 1989).
2.3. Modified E-screen assay
The estrogenic activity of the samples was investigatedusing E-screen assay. The endpoint of the E-screen assay
used in this study is the number of cells as determined by theabsorbance of the purple formazan product, which is producedby the metabolic conversion of MTT (Mosmann, 1983).
Estrogen receptor-positive human breast cancer MCF-7 cells
were initially cultured in RPMI medium supplemented with
10% fetal bovine serum (FBS). They were then plated onto
96-well plates at 1×103 cells/well and allowed to attach for 24
h. The medium was removed and the water samples at 0.01,
0.1, 1, 5, 10 and 20% (V/V) final concentrations in addition to17-β-estradiol (E2) (29 nM final concentration) as positive

control were diluted in phenol-free RPMI medium
supplemented with 10% charcoal-treated FBS and then added
to the cells. For the highest concentrations, additional
controls (5, 10 and 20% PBS(-), data not shown) wereconsidered as references in order to consider the sample effectexclusively on the survival and proliferation of MCF-7 cells.
The cells were incubated for 6 days and the medium changed,
after which 10 μL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Dojindo,Japan) was added to each well followed by incubation for 6 h.
Sodium dodecyl sulfate (SDS, 10%) was then added at 100 μL
per well, followed by incubation for another 18 h. The
absorbance was then determined at 570 nm using a
multidetection microplate reader (Powerscan® HT, Biotek
Instruments, USA).
2.4. HSP 47 assay
The HSP 47 assay based on the enzymatic activity of
β-galactosidase (Isoda et al., 2003) aims to determine the stressresponse of HSP 47-transformed cells due to samples addition.
Chinese hamster ovary (CHO) cells stably transfected with (+)
or without (-) a HSP 47 promoter were used for thisexperiment. HSP 47-transformed cells were plated onto
96-well plates at initial concentrations of 1×104 cells per wellin 100 µL of F12 Medium (Gibco®, Invitrogen, Tokyo, Japan)
supplemented with 10% FBS, 200 µg/mL of G418 (GibcoBRL) and 0.1 µg/mL kanamycin solution (Sigma, USA).
The cells were allowed to attach for 48 h before removing
medium and adding 100 µL of samples diluted with medium
followed by incubation for 3 h in a 5% CO2 incubator at 37°C.
The water samples were added at the same concentrations asthe E-screen assay. The medium was then carefully removed
and the cells washed twice with PBS(-). Fifty microliters oflysis buffer (Promega) was then added and the plates incubatedfor 30 min at room temperature (RT). Twenty microliters ofcell lysate was transferred to a new plate, to which 100 µL ofsubstrate solution (10 mM NaH2PO4•2H2O, 100 mM NaCl,1% BSA, 0.005% NaN3, 1 mM MgCl2•6H2O, 1%
4-methylumbelliferyl-β galacticose (MUG), pH 7.0) wasadded in order to trigger the conversion of MUG into galactose
and methylumbelliferyl. After allowing the reaction to occur
in the dark for 30 min at RT, 60 µl of reaction stop buffer (1 Mglycine-NaOH, pH 10.3) was added and the fluorescence at
365 nm excitation/ 450 nm emission was then determinedusing a multidetection microplate reader.
2.5. Statistical analyses
Statistical analyses such as Student’s t-test were carried out.
Differences in means were considered significant at p<0.05 (*or X) or highly significant at p<0.01 (** or XX). All

Table 1. Main physicochemical parameters of water samples.
OriginSampleR1R2R3River
R4R5R6W1W2W3Wastewater
W4W5W6D1Drainage
D2Irrigation IR
Upstream
Drinikingwater plant DR
Leachate L
Guidelines (NT106.002, 1989)ECT
D O COD BOD(mg/L)(mS/cm) (°C)
7.44
3.10 16.9 0.45 1792 1433
7.89
3.26 15.9 7.60
278
60
7.43
2.20 18.4 0.97
580
220
7.62
2.72 16.2 7.53
232
40
7.64
3.12 16.3 7.43492
120
7.41
1.71 14.5 7.83
120
168.104.17 17.3 2.12354
70
7.60
2.05 17.5 7.47
492
160
7.89
14.70 21.7 0.82
926
310
7.29
2.62 17.7 0.36
646
320
7.46
2.18 17.5 2.54 1056300
7.20
3.83 22.3 0.53
808
280
7.99
6.64 13.0 8.08
142
80
8.22
7.17 16.1 9.88
29
16
8.13
1.56 14.4 8.75
29
16

0.050
0.066
0.008
0.072
0.048
0.060
0.020
0.094
0.145
0.132
0.120
0.100
0.118
0.040
0.050

7.94
7.62

1.78
11.32

15
17.7

7.46
0.05

120
2520

0.070
0.010

6.5-8.5

7.00

--90

pH18350SS30 30.000

experiments were conducted at least three times.

Table 2. Estrogenic activity of water samples.
OriginConcentration

Sample0.01% 0.1% 1% 5% 10% 20%
R1
R2R3RiverR4R5R6W1
W2
W3
Wastewater
W4
W5W6
D1
Drainage
D2
Irrigation IR
Upstream
Driniking DRwater plant
Leachate L

-**-

ND

ND

** **- **
ND **
** **
ND **- X
** *
ND **

**
**
**
**
**

**
****
*
**X

***X

***X

X

XXXX-

**ND

ND--ND
X

NDX

ND

ND*-

** **
ND * ***---*-XX-**

**

**

***--

**

**
*-

(*) Significant estrogenicity(-) Non significant effect(X) cytotoxic effect(ND) Non determined

****X
-

3. Results and Discussion
3.1. Physicochemical parameters of the water samples
The physicochemical parameters of the water samples arepresented in Table 1. For most of the samples, the EC wasbelow the Tunisian standards (i.e., 7 mS/cm) indicating thepresence of low amounts of salts and impurities. However,drainage D2, industrial wastewater W3 and leachate samples
showed EC levels exceeding the guidelines by 2-fold. Eventhough, expected for the drainage sample, this is might be
explained by the textile wastes for the industrial sample and themixture of discharge for the leachate sample.
Apart from the drainage sample D2 and the sample IR
taken from Masri dam, all the samples exhibited highconcentrations of organic matter, as represented by the COD
content. Among all, the leachate sample (2520 mg/L), theriver sample R1 (1792 mg/L) on the far downstream of thewatershed network and the wastewater samples W5 (1056mg/L) from Grombalia STP showed the highest levels widely
exceeding the Tunisian guidelines. Interestingly, the COD
concentration of R1 sample reached 60-fold of IR sample
located on the far upstream. On the other hand, the same
trend was observed for the BOD concentrations that did notfeet the guidelines for most of the samples. Moreover, theratio of BOD to COD for R1 sample was equal to 0.8, whileother samples showed a relatively low biodegradability below
0.6. The average SS content of the wastewater samples was
twice the average of river sample level and 10-fold of theleachate sample level. However, none of them exceeded theguidelines.

3.2. Estrogenic activity of the water samples
The modified E-screen results clearly show that the river,wastewater and leachate samples had a high estrogenic activity(Table 2) while samples R6 and W3 induced a cytotoxic effect.
Among the river samples, R3 sample was able to generate avery high estrogenicity (more than 180% of the control cells at
10% sample concentration) whereas other river samples
reached a maximum estrogenic activity of 140 or 150%. In
the case of wastewater samples, the highest estrogenicity wasshown by W4 sample from Gromabalia STP (170% of thenegative control). On the other hand, other wastewater
effluents induced estrogenic activities between 120 and 137%.
Besides, the leachate sample taken from Soliman landfill
showed an estrogenicity of about 150% at 20% sample
concentration.
The presence of numerous endocrine-disruptor compounds(EDCs) such as estrogens in natural waters and sediments has
been attributed to the incomplete removal of these substances
during wastewater treatment (Farré et al., 2005). This was
further confirmed by the results of this study, which showedthat the activated sludge treatment system, implemented inGrombalia and Beni Khalled STPs, was not capable ofcompletely removing the estrogenic compounds from theeffluent. Natural and synthetic estrogens that are not removed
by wastewater treatment eventually end up in the aquaticenvironment. Tarhouna and El Bey rivers are typicalexamples of contaminated rivers. These rivers have very
high estrogenic activity because they receive wastewater
effluents from Beni Khalled and Grombalia STPs as well aTable 3. Stress response of water samples.
OriginConcentration

Sample0.01% 0.1% 1% 5% 10% 20%
R1
R2
R3River
R4
R5R6W1
W2
W3
Wastewater
W4
W5
W6
D1
Drainage
D2
Irrigation IR
Upstream
Driniking DRwater plant
Leachate L

*
***
*
*
*
*

*
*
**
**
**
*
*
**
**

*
**
**
**
*
**
**
**
**
**
*
**
X
***

**

**

**

**
**
**
*
*
*
**

**
**
*
*
**
*
*

**
**
*
*

X

X

** ****

*

** **

****

X

******X-** ******X XX-(*) Significant stress response (-) Non significant effect(X) cytotoxic effect(ND) Non determined

myriad of industrial wastewater effluents.
The latterprocesses not only domestic wastewater from highly populatedareas but also wastewater from hospitals and various industries
from the industrial zone in Grombalia and Soliman.
According to Khanal et al. (2006), natural estrogen compounds
are mainly removed from the aqueous phase by adsorption
onto associated solid phases, such as sludge in wastewater
treatment. However, these findings showed that the levels ofestrogenic activity in the effluents form Beni Khalled andGrombalia are quite high.
3.3. Stress response effect of the water samples
The stress response of HSP(+) cells exposed to the watersamples was estimated using the HSP47 assay (Table 3).
Overall, the results showed that the water samples exhibited asignificant stress response with a peak registered at 1%
concentration or above. As expected the highest stressresponse levels were observed for the industrial wastewater
W3 and the leachate L samples (142% and 139% of the control
cells level). Interestingly, the river samples R1, R2, R3 andR6 induced a significant stress response of about 135%.
Surprisingly, the sample taken from Masri dam showed asimilar stress response (i.e. 134% of the control cells). Out of
17 water samples, 10 samples showed a correlation with themodified E-screen results. Indeed R1 and R2 samples wereable to induce a concentration dependent stress response whilea peak was observed for R4, R5, W1, W4, W6, D1, IR and DR
samples between 1 and 10% concentration depending on thesample. Moreover, the wastewater samples W2 and thedrainage sample D2 resulted cytotoxic for the HSP cells atConcentrations above 1%. The high stress response inducedby the river samples might be attributed to the discharge ofwastewater effluents from Beni Khalled and Grombalia STPs
in addition to the industrial wastewater effluents (Fig. 1).

4. Conclusion
The introduction of in vitro bioassays systems for water
environment risk assessment is relatively new in semi-arid landand particularly in Tunisia. The physicochemical parameters
results have shown high levels of BOD or COD in the leachateand wastewater samples. Moreover, an increase in theorganic contamination was observed from the upstream of El
Bey river watershed (IR, Masri dam) until the downstream
point (R1). The E-screen assay results elucidated thepresence of high amounts of estrogenic compounds in thewastewater samples and consequently in their receptor sites,
the rivers. Some alarming values of estrogenic activities insamples R3 or W4 could not be predicted by thephysicochemical parameters. The HSP 47 assay results
showed a correlation with the E-screen results. However, thisstress response system has shown higher sensitivity toward
trace compounds present in Masri dam (IR) or the channel
Medjerda-Cap bon (DR). It was thus concluded that thisstrategy developed is sufficiently sensitive to be applied alone
or coupled with physicochemical parameters in the water
preservation strategy.

Aknowledgement
This study was supported in part by funds from theSATREPS project of “Valorization of Bio-resources inSemi-Arid and Arid Land for Regional Development”.
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