沙漠研究 19-1, 261- 264 (2009 )Journal of Arid Land Studies“Desert Technology IX ”
Refereed Paper

Environmental Risk Assessment of Water Resources in Arid and Semi-arid Lands
Using Bioassays Systems
Fahmi BEN FREDJ1, Mitsuteru IRIE1, Junkyu HAN1, Parida YAMADA1,Atef LIMAM2, Ahmed GHRABI2, Hiroko ISODA1*
Abstract: Control of effluents has traditionally been regulated using methods with unspecified
responses and measuring global parameters, such as the chemical oxygen demand (COD) or Biological
Oxygen Demand (BOD). Moreover, information about biological effects is unknown. Thus, theintroduction of in vitro assays using mammalian cells to investigate the presence of environmental
contaminants is more than needed. At first, physicochemical parameters of municipal wastewater
samples from Tunisia were estimated. On a further step, in vitro bioassays systems were applied
intending to find out the biological effects of these samples on mammalian cells. Results using theE-screen assay reveal that the wastewater samples induced estrogenic activity at different concentrationsin 6 days incubation period. Besides, the same samples were able to generate stress response on
animal cells. These findings correlate with the physicochemical parameters data and thus evaluate theefficiency of treatment system to remove such toxic compounds.
Keywords: Estrogenic activity, Risk assessment, Stress response, Wastewater

1. Introduction
Water environments have to face solid or liquid discharges containing a complex mixture of variousorganic and inorganic substances. Wastewater reclamation and reuse projects have been developed in arid
and semiarid lands as an essential response to growing water needs. The treated water was used forrecharging the ground water 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 with the threat of residual pollutants in effluents. In arid and semiarid lands,
particularly where the water resources are scarce, the detection of contaminants and the measurement of theirharmful impact has become a major issue in environmental risk assessment. Therefore, in a primary step,
the quality of treated water, along the wastewater treatment process, was investigated by the mean ofconventional physicochemical parameters. Moreover, in vitro bioassays systems were used to evaluate thebiological effects of wastewater samples on mammalian cells. Heat Shock Protein 47 assay for stressresponse and E-screen assay for estrogenic activity were carried out to assess the risk of wastewater samples
from an activated sludge treatment system plant in semi-arid environment.
2. Materials and Methods
2.1. Sampling site
Water samples from the wastewater treatment system in Soliman, Tunisia, were obtained along thetreatment process from the influent input, the pretreatment pond, the oxidation channel based on activatedsludge treatment and the effluent pond (Fig. 1). Soliman STP system renewed in 2004, receives influents(12300 m3/day) from a population of 30 000 residents (www.onas.nat.tn, 2007). The primary treatment iscarried out to remove the coarse fraction inclusive of oil, fatty acids and suspended solids. The biologicalprocessing is used to remove microorganisms, nitrogen and phosphorus, and lower the organic load as
defined by the BOD. The biological treatment, based on the natural ability of wastewater containing
bacteria to degrade organic material, is roughly divided into aerobic and anaerobic treatments including
conventionally the oxidation pond and the activated sludge process.
2.2. Sampling procedures
Sterilized glass bottles served for the samples to be used for physicochemical parameters investigation,
1Alliance for Research on North Africa (ARENA), Graduate School of Life and Environmental Sciences, Universityof Tsukuba, Japan; *Corresponding Author: Fax. +81298535775, E-mail: isoda@sakura.cc.tsukuba.ac.jp
2Water Treatment and Recycling Laboratory, CERTE. B.P. 95-2050. Hammam-Lif, Tunisia;

262

sterilized plastic bottles were used for thesamples intended for the bioassays. For thelatter samples, a primary filtration using a 0.45
μm filter was carried out on the sampling day.
Samples were stored at - 20 ˚C until transport toJapan inside an icebox to the laboratory wherethe same conditions were maintained. A
secondary filtration using a 0.22 μm filter isrequired prior to use in the bioassays following
pH adjustment between 3 and 6.
Fig. 1. Sampling points from Soliman activated sludge
treatment system plant in Tunisia.
2.3. Water quality parameters
The following in situ parameters were immediately determined prior to filtration as follows: Temperatureand pH using a pH meter 330i, dissolved oxygen using an oxymeter 340i, and electric conductivity using a
conductimeter 330i (all fromWissenschaftlich-Technische-Werkstätten, Weilheim, Germany).
Other
physicochemical parameters, such as Chemical Oxygen Demand (COD), 5d-Biological Oxygen Demand(BOD5), suspended sludge (SS) were analyzed in the laboratory according to standard methods (Rodier, 1984).

2.4. E-screen assay
The estrogenic activity of the samples was investigated using E-screen assay. Human breast cancer MCF-7
cells were initially cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS). They werethen plated onto 96-well plates at 1×103 cells/well and allowed to attach for 24 h. The medium was removed
and the wastewater samples at 5, 10 and 20% (V/V) final concentrations, in addition to 17-β-estradiol (E2) (29nM final concentration) as positive control, were diluted in phenol-free RPMI medium supplemented with 10%
charcoal-treated FBS and then added to the cells. A separate experiment was carried out to adjust the effects ofvarious concentrations (Phosphate Buffer Solution PBS (-) at 5%, 10% and 20%) on the survival andproliferation of MCF-7 cells. The cells were incubated for 6 days and the medium changed, after which 10 μLof 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H -tetrazolium bromide (MTT) (Dojindo) was added toeach well followed by incubation for 24 h. Sodium dodecyl sulfate (SDS, 10%) was then added at 100 μL per
well, followed by incubation for another 24 h. The absorbance was then determined at 570 nm using a
multidetection microplate reader (Powerscan® HT, Biotek Instruments).
2.5. HSP 47 assay
The HSP 47 assay based on the enzymatic activity of β-galactosidase (Isoda et al., 2003), driven by thehsp47 gene promoter, aims to determine the stress response of HSP 47-transformed cells due to samples
addition. HSP 47-transformed cells were trypsinized and plated onto 96-well plates at initialconcentrations of 1×104 cells per well in 100 µL of F12 Medium (Gibco®, Invitrogen) supplemented with
10% FBS, 200 µg/mL of G418 (Gibco BRL) and 0.1 µg/mL kanamycin solution (Sigma). The cells wereallowed 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 wastewater samples were added at 5concentrations 0.01, 0.1, 1, 5, 10 and 20% (V/V). For the highest concentrations, additional controls (5,10 and 20% PBS(-), data not shown) were considered as references in order to consider the sample effectexclusively. The medium was then carefully removed and the cells washed twice with PBS(-). Fiftymicroliters of lysis buffer (Promega) was then added and the plates incubated for 30 min at room
temperature (RT). Twenty microliters of cell 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-galactose (MUG), pH 7.0) was added in order to trigger the conversion ofMUG into β-galactose and methylumbelliferyl. After allowing the reaction to occur in the dark for 30
min at RT, 60 µl of reaction stop buffer (1 M glycine-NaOH, pH 10.3) was added and the fluorescence at
365 nm excitation/ 450 nm emission was then determined using a multidetection microplate reader.

263

2.6 Statistical analyses
Statistical analyses such as Student’s t-test were carried out. Differences in means were consideredsignificant at p<0.05. All experiments were conducted at least three times.
3. Results and Discussion
3.1 Physicochemical parameters
Table 1 clearly shows that the wastewater samples had a higher electric conductivity (EC) ranging from
5.26 to 8.72 ms/cm as a sign of presence of salts and impurities. The sample obtained from the oxidation
pond exhibited the highest concentration of organic matter, as represented by the chemical oxygen demand(COD), reaching 2 to 3 fold the levels of influent and effluent samples, respectively. On the other hand, theratio of BOD5 to COD for both the influent and the effluent attained 80%. This is explicit a relatively highbiodegradability for the treatment system. Nevertheless, the removal rate of organic content did not exceed
35%. Thus, the residual levels of COD and BOD were broadly above the Tunisian guidelines (Table 1).
Overall, the suspended sludge (SS) for all the samples was lower than the guidelines.
3.2 E-screen assay
The E-screen assay is widely used to determine the estrogenicity of environmental compounds through theability of a substance to stimulate the growth of an estrogen-dependent cell line, commonly MCF-7. This humanbreast cancer cell line possesses estrogen receptors (ERs) and responds in culture to the presence of estrogens or
xenoestrogens with a proliferative response (Jones et al., 1998). Indeed, Estrogens play important roles ingrowth, development, reproduction and maintenance of a diverse range of mammalian tissues. The
physiological effects of estrogens are mediated by the intracellular ERs, which regulate transcription of target
genes through binding to specific DNA target sequences. As illustrated in Figure 2, the coregulators PELP1(Proline Glutamic Acid-Rich Nuclear Protein) and Steroid Receptor Coactivator (SRC) act as coactivators of ER(Mishra et al., 2003) to promote cell proliferation. This leads to the subsequent activation of Extracellular Signal
Regulated Kinases (ERK)1/2.
The endpoint of the E-screen assay used in this study is the number of cells as determined by the absorbanceof the purple formazan product, which is produced by the metabolic conversion of MTT (Mosmann, 1983). The
proliferation levels of the human breast cancer MCF-7 cells treated with Soliman wastewater samples weresignificant in comparison with the nontreated control (T-test, p<0.05). Overall, the wastewater samples containestrogenic or xenoestrogenic compounds, hormones or chemicals known to act like estrogen. In fact, Steroidestrogens have been detected in influents and effluents of STPs in different countries such as Italy, Brazil,Netherlands and Japan (Cui et al., 2006; references cited in there). The removal rate of these estrogenic
compounds in such municipal STPs were approximately above 80%. Drewes et al. (2005) reported asignificant correlation between the BOD5 loading and the total estrogenic activity exhibited in the E-screen inseven full-scale water reclamation facilities. Moreover, it is difficult for these chemicals to be degraded bymicroorganisms because of complexity and anti-biodegradation of estrogen containing wastewater (Cui et al.,2006). Indeed, in a recent investigation, Narita et al. (2007) found out that the by-product of activated sludge
process induced higher activity than the influent due to the production of some metabolites during biologicalprocess. On the other hand, the estrogenic activity shown by the samples correlate with the BOD5 loading andwith previous investigations by Limam et al. (2007) and consequently, might put under question theefficiency of the treatment system.
EstorgenTable 1. Main physicochemical parameters of wastewater.
SampleInfluent

pH
7.96

ECTDO

COD

BOD5SS(ms/cm)(°C)(mg/L)(mg/L)(mg/L)(µg/L)

8.72

18.7

1.31

900

720

224

Pretreatment

8.09

5.26

18.6

0.93

994

795

38

Oxidation pond

7.75

6.20

17.2

1.02

1996

1596

86

Effluent

7.58

8.32

16.7

0.76

602

482

60

Guidelines (NT 106.002, 1989)

7-8---

< 90

< 30

<3000

Estorgen
receptor

Extracellular

PELP1
ERK1

Nucleus
ERK2

SRC

Cell proliferation
Cytoplasm

Fig. 2. Estrogen Pathway Overview.

264

Stressor
3.3 HSP 47 assay
Heat shock proteins (HSPs) are a distinctive classof molecules that protects cells against a wide range ofinjuries (Georgopoulos and Welch, 1993). They assist
hsp 47 promoter
β-galactosidase proteinin the recovery of stress either by repairing damaged
coding region
proteins or by removing them to restore protein
Expression ofhomeostasis (Hartl, 1996). Heat shock protein 47
β-galactosidase protein(HSP47) is a collagen-specific molecular chaperone CHO cells carrying hsp47 :Detection of expression bythat is required for molecular maturation of various β-galactosidase gene
β-galactosidase activity genetypes of collagens. HSP47 activity can be determined
Fig. 3. HSP 47 assay overview.
using HSP47-transformed Chinese Hamster Ovary(CHO) cells by incubating the latter with the test
compound and measuring the enzymatic activity of β-galactosidase upon stress induction. In the presence of
ß-galactosidase, methylumbelliferyl-β-galactose (MUG) is converted to β- galactose and methylumbelliferyl.
Since it is difficult to measure HS protein activity, the gene protein ß-galactosidase activity is thus measured
spectrophotometrically to detect chemicals or toxins which may disrupt or disturb homeostasis in organisms(Isoda et al., 2003).
It has already been revealed that the production of stress proteins is induced as a result of the reaction of cellswith a stressor such as heat, a chemical substance or a heavy metal (Fig. 3). Isoda et al. (2003) developed thishighly sensitive system, HSP 47 assay, for detecting trace amounts of environmental pollutants and natural toxins.
Following 3 h treatment time, the combination of the stress response and the cytotoxicity effect of Soliman STP
samples on HSP cells showed that they induced a significant increase in ß-galactosidase activity in comparison
with the control. This result can be correlated with the estrogenic activity and the physicochemical parameters of
the same samples as shown by the E-screen assay.

4. Conclusion
These specific bioassays systems allowed the detection of wastewater samples components responsible forstress response or estrogenic activity in order to evaluate the efficiency of the treatment system. These findings
will help establish, in a future purpose, some specific environmental biomarkers for wastewater pollutants
Aknowledgement
This study was supported in part by funds from the Grant-in-Aid for Scientific Research (Grant No. 17255011).
References
Cui C.W., JI S.L., Ren H.Y. (2006): Determination of steroid estrogens in wastewater treatment plant of a controceptives producing
factory. Environ. Monitoring and Assessment, 121: 409-419.
Drewes J.E., Hemming J., Ladenburger S.J., Schauer J., Sonzogni W. (2005): An assessment of endocrine disrupting activity changes
during wastewater treatment through the use of bioassays and chemical measurements. Water Environ. Res., 77 (1): 12–23.
Frecheney R.I. (2000): Culture of animal cells-A manual of basic techniques- Fourth Edition, Wiley-Liss: 336.
Georgopoulos C., Welch W.J. (1993): Role of the major heat shock proteins as molecular chaperones. Cell Biol., 9: 601–634.
Hartl F.U. (1996): Molecular chaperones in cellular protein folding. Nature, 381: 571–579.
Isoda H., Koyama T., Tasaki M., Oka S., Sugiura N., Maekawa T., Inamori Y., Yokota S., Kiktahara M., Nagata K. (2003): High-sensitve detection of environmental pollutants. US Patent and Trademark office, 6740521.
Jones P.A., Baker V.A., Irwin A.J.E., Earl L.K.(1998): Interpretation of the in vitro proliferation response of MCF-7 cells to potential
estrogens and non-estrogenic substances. Toxicol. In Vitro, 12: 373-382.
Limam A., Talorete T.P.N., Ben Sik Ali M., Kawano M., Jenhani A., Abe Y., Ghrabi A., Isoda H. (2007): Assessment of Estrogenic
activity in Tunisian water and wastewater by E-screen assay. Environmental Sciences, 14: 043-052.
Mishra S.K., Mazumdar A., Vadlamudi R.K., Li F., Wang R.A., Yu W., Jordan V.C., Santen R.J., Kumar R. (2003): MICoA, a novel
metastasis-associated protein 1 (MTA1) interacting protein coactivator, regulates estrogen receptor-alpha transactivation functions.
J Biol Chem., 278(21): 19209-19219.
Mosmann T. (1983): Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J.
Immunol. Methods, 65: 55-63.
Narita H., Talorete T.P.N., Han J., Funamizu N., Isoda H. (2007): Human intestinal cells incubated with activated sludge andlipoposaccharide express Hsp90b. Environmental Sciences, 14: 035-039.
N. T. (Tunisian Standards) 106.002 (1989): Environmental protection against effluent disposal in the water environment. INNORPI.
Rodier J. (1984): L’analyse de l’eau: Eaux naturelles, Eaux résiduaires, Eau de Mer, 7th ed. Dunod, Paris, France.