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Toxicological & Environmental Chemistry, 2013
http://dx.doi.org/10.1080/02772248.2013.802793

Titanium dioxide mediated photo-degradation of methyl orange by

ultraviolet light
Hassen Trabelsia, Moncef Khadhraouia,b, Olfa Hentatia,b and Mohamed Ksibia,b

a
University of Sfax, Laboratoire Eau, Energie et Environnement ENIS, Sfax, Tunisia; bInstitut
Sup

erieur de Biotechnologie de Sfax, Tunisia

The photo-degradation of an aqueous solution containing the azo dye methyl orange
(MO) is reported. Disappearance of color and substrate together with decreases in
chemical oxygen demand (COD) and total organic carbon (TOC) content were
observed. The effects of some auxiliary dyeing additives were also evaluated. The
photo-decomposition followed pseudo-first-order kinetics. Some by-products and their
photo-degradation pathways were identified. In addition, the toxicity and phytotoxicity
of some by-products were tested using the earthworm (Eisenia andrei) avoidance test
and the Bromus ramosus seed germination test. High sensitivity of E. andrei towards
soil wetted with MO solution was observed, and germination of B. ramosus seeds
was inhibited by MO solution, while no effects were seen for the same solution after
photocatalytic oxidation.
Keywords: TiO2; methyl orange; photo-decomposition; intermediate products; toxicity

1. Introduction
Synthetic dyes are mainly used in textile and paper industries (Gupta et al. 1992; Wrobel,
Boguta, and Ion 2001), besides many other chemicals such as surfactants, chelating agents,
and pH regulators. The resulting, highly polluted wastewater is complicated to treat due to
the presence of non-biodegradable compounds. Various methods have been developed for
removal of synthetic dyes from such wastewaters with the aim of attenuating their impact on
the environment. The conventional water treatment methods such as sedimentation, filtra-
tion, chemical and membrane technologies have high operating costs and often lead to toxic
secondary pollutants that also have to be treated (Gaya and Abdullah 2008). Advanced oxi-
dation processes (AOPs) are promising for treatment of wastewaters containing recalcitrant
organic compounds. In this regard, processes utilizing hydrogen peroxide (Esplugas et al.
2002; Pera-Titus et al. 2004) or ozone as oxidants (Colindres,Yee-Madeira, and Reguera
2010; Shu and Chang 2005), semiconductors as photocatalysts (Bansal, Singh, and Sud
2010) and electrochemical oxidation (Mart
ınez-Huitle and Brillas 2009) have been thor-
oughly investigated. Among these, TiO2-mediated photo-oxidation is established for the
decolorization and the mineralization of dyes using either ultraviolet (UV) or solar light (Al-
Qaradawiand Salman 2002; Hachem et al. 2001; Lihong et al. 2012). However, more work
is required to identify by-products (Tanaka, Padermpole, and Hisanaga 2000), photo-
degradation pathways (Konstantinou and Albanis 2004) and toxicity evaluation of the
treated medium. Hence, in this work, a heterogeneous photocatalytic system has been used
to perform the oxidation of the water soluble azo dye methyl orange (MO) which

*Corresponding author. Email: Mohamed.ksibi@tunet.tn

Ó 2013 Taylor & Francis

2 H. Trabelsi et al.

corresponds to the 40 -sulfonic acid derivative (pKa ¼ 3.47) and the above-mentioned
requirements were addressed. This dye was selected as a model on the basis of its industrial
consumption, water solubility, and toxicity, while the tested concentrations were aimed to
simulate typical exhausted dye bath compositions following water rinse (Pisa Textile Dyeing
Co., Istanbul, Turkey, 2006, personal communication). The photocatalytic oxidation was
carried out in aqueous suspensions of TiO2 (Degussa P25) irradiated by UV light. The decol-
orization of MO aqueous solution, the evolution of total organic carbon (TOC) and chemical
oxygen demand (COD) were investigated for process efficiency evaluation. Determination
of the photocatalytic oxidation pathway and the identification of the main formed intermedi-
ates were performed. The effect of some auxiliary dyeing additives such as Cl, SO42 and
S2O82 ions on the dye photo-degradation rate was also investigated. Indeed, these ions are
usually present in real wastewater produced by textile industry (Bansal, Singh, and Sud
2010) as they are added to the liquor used to perform the operation of scouring, i.e. the treat-
ment that removes wax, dirt, grease and other natural impurities from the fibers before the
dyeing process. Finally, since new attractive options for a possible water reuse either for a
subsequent dyeing process after relatively short plants treatment for agricultural and/or other
industrial activities are of increasing concern (Bradley et al. 2002; Lapena, Cerezo, and
Garcıa-Augustin 1995), the toxicity and phytotoxicity of the raw and treated solutions were
also evaluated by means of earthworm avoidance tests and germination of B. ramosus seeds.

2. Experimental section
2.1 Photocatalytic reaction and dark adsorption
MO (purity >97%) was purchased from Merck (Darmstadt, Germany) as a commercially
available dye without any further purification. The dye’s chemical structure and its main
characteristics are shown in Table 1. Solutions were prepared using bidistilled water. The
TiO2 powder (P25) was obtained from Evonik Degussa (Parsippany, NJ, USA) and contains
anatase and rutile phases in a ratio of about 4:1. It has a Brunauer, Emmett and Teller (BET)
surface area of 50 m2 g1. An open air Pyrex immerged lamp reactor, equipped with a
plunging tube containing a 125 W UV lamp (HPK, Philips, Holland) was used emitting in
the range from 200 to 400 nm. A Corning 0.52 filter was used to cut off radiation below the
wavelength of 340 nm. The tube had a Pyrex cylindrical jacket with circulating water to
avoid overheating of the liquid to be treated. The intensity of the UV radiation reaching the
reactor (l > 290 nm) was measured by a radiometer (HD9021, Delta Ohm, City, Country)
and estimated to be 6.67  107 einstein s1 (mole of photons). An aqueous photocatalyst
suspension was prepared by adding 1000 mg TiO2 powder to 1000 mL MO solution. The
photo-degradation was carried out at 293 K and at neutral pH. Before any analysis, the
aqueous sample was filtered through a 0.45 mm membrane filter to remove TiO2

Table 1. The main properties of the methyl orange.

Molecule Formula Structure Molecular Molecular

weight size

Methyl C14H12N3O3NaS 327.34 1.19 nm

orange 0.67 nm
(MO) 0.38 nm
 Toxicological & Environmental Chemistry 3

agglomerates in the suspension. The effect of some auxiliary dyeing additives such as
Cl, SO42 and S2O82 on the photocatalytic decolorization was also evaluated. All experi-
ments were run in triplicate and the relative standard deviation (RSD) was used to express
the errors of analysis.

2.2 Monitoring of color, COD, and TOC

Photocatalytic reaction was followed by monitoring the fading of color, COD and TOC.
These parameters were analyzed according to Japanese International Standards (Japanese
Industrial Standards Committee, 1998). Absorbance was determined using a Ultraviolet–
visible (UV–vis) spectrophotometer (U-200 Hitachi, Ltd., Tokyo, Japan). TOC was
observed by a TOC analyzer (TOC-5000A, Shimadzu, City, Country) under the non-
purgeable organic carbon (NPOC) mode. Samples were acidified with HCl (p.a. quality) to
about pH ¼ 2 and purged with ultrapure synthetic air to remove inorganic carbon prior to
instrument analysis. Calibration samples were run before each experiment using potassium
phthalate as standard.

2.3 Gas chromatography–mass spectrometry

In order to identify photo-degradation products of MO, gas chromatographic–mass spectro-
metric (GC–MS) analyses were performed. After photocatalysis, 50 mL of the aqueous sol-
utions were filtered and extracted three times with 30 mL each of ethylacetate. The
combined extracts were concentrated by means of a solvent evaporation under reduced
pressure to a volume of about 2 mL. This solution was analyzed by means of a GC–MS
instrument (HP model 5975B inert MSD) equipped with a capillary coated with DB-5MS
(30 m length, 0.25 mm internal diameter, 0.25 mm film thickness, Agilent Technologies,
J & W Scientific Products, USA). Nitrogen was used as a carrier gas at a flow rate of 1 mL
min1. The oven temperature program was as follows: 1 min isothermal at 100 C, ramped
from 100 C to 260 C at 5 C min1, and 10 min hold at 260 C. The chromatograph was
equipped with a split/splitless injector used in the split mode at a split ratio of 100:1.

2.4 Avoidance tests with earthworms

Earthworm avoidance tests were carried out using adults of E. andrei 420–440 mg culti-
vated in our laboratory. The selected worms were first transferred to artificial soil 24 h
before the experiment for acclimatization. The used medium was an artificial soil (70%
fine sand, 20% kaolin, 10% sphagnum peat with a small amount of CaCO3 for pH adjust-
ment to neutrality) wetted with the same volume of the raw or the treated MO solutions
and giving a maximum water holding capacity (WHC max) of 30%–35%. The tests were
conducted in plastic trays (30 cm length  22 cm large  6 cm depth) divided into two
identical parts using a transversally inserted cards and each part was filled with the corre-
sponding soil, control or contaminated soil. Five replicates were conducted and each rep-
licate included a control side (wet soil with distilled water) and the other side with the
contaminated soil. The dividers were then removed and 10 earthworms were carefully
placed on the midline of each tray which was afterward covered by a transparent lid in
order to prevent evaporation. Tests were performed under a photoperiod of 16:8 h/light:
dark, at 20 C. Depending on the soil toxicity, earthworms will either avoid the contami-
nated soil or lose their life after certain time in case they choose to go further. After
48 hours, the number of worms existing in each part of the plastic tray was counted and
results were expressed in percentage of avoidance.
4 H. Trabelsi et al.

2.5 Germination tests

Phytotoxicity of MO was assessed before and after photocatalytic treatment using the ger-
mination of B. ramosus seeds (Tam and Tiquia 1994; Tiquia, Tam, and Hodgkiss 1996).
Twenty seeds were placed on each filter paper located in Petri dish and 6 mL of the
treated solution were then uniformly added to each dish. Dishes were then incubated in
the dark at 26 C for 5 days. Distilled water was used as control. All samples including
controls were triplicated. The germination index (GI) was calculated by counting the
number of the germinated seeds and measuring the average root length observed in each
sample compared to control treatments as reported by Komilis et al. (2005). A seed was
considered germinated if its root length exceeded 5 mm. In contrast, a root length below
5 mm was considered equal to 0 and the seed was not considered germinated. The aver-
age sum of root lengths comprised the sum of the lengths of all germinated seeds in a Petri
dish. Results finally were expressed according to the following formula:
GI ¼ (number of germinated seeds in sample/number of germinated seeds in control)
 (average of root lengths in sample/average of root lengths in control)  100.

3. Results and discussions

3.1 Adsorption of methyl orange on titanium oxide
Understanding the theory behind the common reactor operational parameters and their
interactions is one of the key factors for process optimization and modification. Conse-
quently, the kinetics of MO adsorption has been determined for different initial concen-
trations ranging from 5 to 30 mg L1 (Figure 1). It can be clearly noticed that the
adsorption equilibrium is reached within 30 min whatever the tested concentrations.
Therefore, 30 min had been selected as time allocated for the dark adsorption. Several

Figure 1. Kinetics of adsorption of MO in the dark. Experimental conditions: m (TiO2) ¼ 1 g; V ¼

1000 mL; T ¼ 25 C ; natural pH.
 Toxicological & Environmental Chemistry 5

authors have shown that the classical Langmuir adsorption model could be applied to
numerous compounds in aqueous suspensions (Hachem et al. 2001; Saquib and Muneer
2002). At the equilibrium, the adsorbed quantity could be calculated according to the fol-
lowing expression:
Kads Ceq
Qeq ¼ Qmax ;
1 þ Kads Ceq

where Ceq corresponds to the concentration of the compound at the adsorption equilib-
rium and Qeq corresponds to the adsorbed equilibrium quantity. The linear transform of
the above equation can be expressed as Ceq/Qeq ¼ f(Ceq). The linearity of the transform
indicates that the Langmuir isotherm fits well MO dark adsorption on TiO2 catalyst
implying a monolayer adsorption model (Figure 2). The maximum adsorbed quantity
Qmax of the dye, the adsorption constants Kads as well as the areal density of the adsorbed
dye molecules Dmax were determined and were, respectively, Qmax ¼ 39.84 mg/gcat,
Kads ¼ 3.4 102 L mg1 and Dmax ¼ 1.47 molecules nm2. We recall that the word
“areal” refers to a surface area unit as defined by Robert and Burwell (1977) in the
IUPAC report.

3.2 Photocatalytic decolorization of methyl orange

Figure 3 gives the comparative results of the changes of MO (C0 ¼ 9.16  104 mol L1)
in an aqueous solution remaining with reactive time in photolysis and photocatalysis pro-
cess, respectively. In adsorption process, the results show that 29.47% of MO was adsorbed
on TiO2 after 30 min adsorption in dark condition. This means that the TiO2 has obvious
adsorption for MO in the aqueous solution. The experiments performed in the absence of
TiO2 (neat photochemical regime) show that degradation of MO via photolysis could be
neglected with respect to the corresponding photocatalysis. In photocatalytic process the
complete decolorization of MO was observed after 300 min irradiation time. In fact, the

Figure 2. Linear transform of Langmuir isotherm illustrated by plot of Ce/Qe vs. Ce. Experimental
conditions: m (TiO2) ¼ 1 g; V ¼ 1000 mL; T ¼ 25 C; natural pH.
6 H. Trabelsi et al.

Figure 3. Disappearance rate of methyl orange azo dye in 9.16  104 mol L1 solution (~) pho-
tocatalysis () without TiO2.

time-dependent UV–vis spectral changes during photocatalytic degradation of MO aque-

ous solution are shown in Figure 4. The spectra of the raw MO solution showed one peak
at l ¼ 273 nm in the UV region and a main band at l ¼ 465 nm in the visible region. The
absorbance at 273 nm is assigned to the benzene ring in MO as reported by Galindo et al.
(2000) and the absorbance at 465 nm is due to the azo linkage of MO. From Figure 4, it
can be noticed that the intensity of the visible band started to decrease upon solution irradi-
ation. In parallel, a temporary appearance of a peak associated with the aromatic com-
pounds was observed in the near-UV region at about 250 nm. This phenomenon was also
stated by Chen et al. (2008) and was assigned to the formation of some intermediates con-
tributing thus to the elevation of the absorbance in that region. This result corroborates also
with that reported by Ince et al. (1997). It can be assumed that the transient by-products
containing aromatic rings are easy to break down in the present TiO2 suspension, suggest-
ing the high mineralization efficiency of MO as mentioned by Dai et al. (2007).

3.3 COD and TOC removal

In order to confirm the above-mentioned hypothesis and to assess the degree of minerali-
zation reached during the photocatalytic treatment, COD and TOC measurements have
been performed. Figure 5 shows COD and TOC reduction vs. treatment time. It can be
seen that after 180 min of UV irradiation, more than 61% of the initial COD was
removed. However, only 45% of TOC content was reached, while over 92% of the decol-
orization efficiency was achieved. This is due to the fact that the intermediate products
such as carboxyl acids are more difficult to oxidize than their parent compound (azo dye
in this case), and complete oxidation proceeds at a much slower reaction rate (Ince et al.
2004). Augugliaro et al. (2002) have observed a complete decolorization of MO in few
hours but mineralization occurred after a longer time with the formation of CO2, nitrates
 Toxicological & Environmental Chemistry 7

Figure 4. Change in the absorption spectra during the photo-degradation of MO at the initial con-
centration C0 ¼ 9.16  104 mol L1 in an aqueous TiO2 suspension (1000 mg L1) and natural pH.

and sulfates. Sakthivel et al. (2003) have reported similar trend in TOC removal under
similar conditions and demonstrated that the complete decolorization of the acid brown
14 occurs in 300 min and the complete degradation was achieved in 420 min. Therefore,
it could be expected that more aggressive conditions are required to achieve higher TOC
removal than those employed to simply break the chromophore group.

3.4 Effects of inorganic additives on MO photo-degradation

The occurrence of inorganic ions is rather common in dye-containing industrial wastewa-
ter. Often, wastewater contains a mixture of pollutants, organic solvents as well as dis-
solved organic matter and humic substances. If mixed with other waste streams, these
substances may compete for the active sites on the TiO2 surface and deactivate the photo-
catalyst decreasing hence the degradation rate of the target dyes. Alternatively, they may
act as light screens, thus reducing the photon receiving efficiency. For instance, the UV/
TiO2 photocatalytic degradation of different classes of dyes were reported to be delayed
by many commonly used industrial solvents and acids, as well as by many naturally abun-
dant mineral species and dissolved organic matter (Bandara et al. 1997). On the other
hand, it is worth noting that most of the studies related to effluents from textile
manufacturing report that these wastewaters are highly variable in composition with rela-
tively high residual dyes, sodium hydroxide, inorganic salts such as Na2SO4 (5500 mg
L1) and various organic components such as detergents, softening, dispersing and fixing
agents (Pekakis et al. 2006). Keeping this in mind, the effect of the addition of anionic
additives (Cl, SO42, S2O82) at the dose of (1000 mg L1) on the photo-degradation of
8 H. Trabelsi et al.

Figure 5. COD (a) and TOC removal (b). Experimental conditions: [MO]0 ¼ C0 ¼ 9.16  104
mol L1; m (TiO2) ¼ 1000 mg; T ¼ 25 C; natural pH.

MO solution was studied. The obtained results are shown in Figure 6. Logarithmic linear-
ization of results reported in Figure 3 is also illustrated in the insert of Figure 3. The val-
ues of the apparent first-order rate constant kapp deduced from the slope of the linear
regression and corresponding to different solution compositions are regrouped in Table 2.
From the insert of the Figure 6, it can be seen that the photocatalytic degradation of MO
is a pseudo-first-order reaction. The confirmation of the first-order rate kinetics was
derived from the linearity of the plot. Among the anionic species studied, Na2SO4 exhib-
ited the strongest inhibition effect followed by NaCl as also found by Daneshvar et al.
(2003) and Epling and Lin (2002). Inhibition effects of anions can be explained as the
reaction of positive holes and hydroxyl radical with anions that behaved as hþ and OH
scavengers (Equations (1)–(5)) resulting from prolonged color removal. According to
Bahnemann et al. (1994) and S€ €
okmen and Ozkan (2002), the adsorbed anions compete
with the dye for the photo-oxidizing species on the surface and prevent the photocatalytic
degradation of the dyes. Also, the formation of inorganic radical anions (e.g. Cl, SO4)
under these circumstances is possible to occur (Abdullah et al. 1990;Chen et al. 2007):

Cl þ hþ  
VB !Cl  or Cl þ OH!ClOH ; ð1Þ
þ 
4 þ h !SO4 ;
SO2 ð2Þ
 
4 þ OH!SO4 þ OH :
SO2 ð3Þ

The literature reports that although the reactivity of these radicals may be considered,
they are not as reactive as hþ and OH (Hu et al. 2003), and thus the observed retardation
 Toxicological & Environmental Chemistry 9

Figure 6. (a) Kinetics of the photocatalytic degradation of MO in the presence of different anionic
additives. (b) first-order linear transform lnC0/C ¼ f (t). Experimental conditions: an aqueous TiO2
suspension (1000 mg L1); [MO]0 ¼ C0 ¼ 9.16  104 mol L1; T ¼ 25 C; natural pH.

effect is still thought to be related to the strong adsorption of the anions on the TiO2 sur-
face. Indeed, the addition of sodium peroxydisulfate Na2S2O8 was beneficial for the
photo-oxidation of MO. The disappearance profile of MO confirmed this behavior, clearly
indicating a neat mineralization rate increase when S2O82 is added. The reactive radical
intermediate (SO4) formed from this oxidant by reactions with the photogenerated elec-
trons can exert a dual function: as a strong oxidant itself and as an electron scavenger,
thus inhibiting the electron–hole recombination at the semiconductor surface (Gr€atzel
et al. 1990). Other fact, it was also noticed that in the presence of S2O82 the pH
decreases in the course of reaction up to the value of 2.4 for all the runs due to formation
of hydrogen ions according to the following reactions:

 
8 þ eCB ! SO4 þ SO4 ;
S2 O2 ð4Þ
2

SO þ
4 þ H2 O ! SO4 þ OH þ H :
2
ð5Þ

Table 2. Pseudo-first-order apparent constant values for MO degradation.

Composition kapp (min1) R2

MO 0.004 0.0003 0.990

MO þ Na2S2O8 0.006 0.0004 0.991
MO þ NaCl 0.002 0.0002 0.988
MO þ Na2SO4 0.001 0.0001 0.990
10 H. Trabelsi et al.

The positive influence of S2O82 on the mineralization rate of MO is also due to the
strong decrease of pH during photocatalysis which probably enhances the photoreactivity
of titanium dioxide. Guettaı and Ait Amar (2005) have demonstrated that best results are
obtained in the oxidation of MO in acidic medium.

3.5 Determination of the photo-degradation products

After having studied the reactivity in heterogeneous photocatalysis of MO in an aqueous
solution, we tried to identify the products of photocatalytic degradation of this azo dye.
The identification carried out by using GC–MS analysis has mainly been oriented towards
the aromatic derivatives formed during the first 30 min of MO degradation process due to
the probability of their higher toxicity. With the aim of identifying the present soluble
compounds in the irradiated dye solution, the ethylacetate extract of the aqueous MO
solution has been analyzed. The main fragments detected in the solution of MO irradiated
for 30 min. The degradation products were identified according to their molecular ion
and MS fragmentation peaks are shown in Table 3.
This table presents thus the photo-degradation products classified by their characteris-
tic fragments and their relative intensities. In the present work, there are at least five types

Table 3. GC–MS data for intermediates obtained after 30 min of photocatalytic degradation of
methyl orange.

No. Intermediates Retention Main ions (m/z) (%

time (min) abundance)

1
306 ([M–H] þ,14);
8.282 189 (18);
147 (100); 73 (54)

2 9.373 292(Mþ,10);
191 (100);
147 (56); 73 ( 68)

3 11.035 304 (Mþ,); 289 (26);

231 (100);
190 (8); 147 (44)

4 14.74 320 (Mþ,weak);

304 (100); 214 (18);
147 (55); 73 (60 )

5 31.735 322 (Mþ,70);

264 (9) 129 (70);
73 (100)
 Toxicological & Environmental Chemistry 11

of intermediates detected; some of these intermediates were also found by other research-
ers (Augugliaro et al. 2002; Baiocchi et al. 2002; Bianco Prevot et al. 2004; Comparelli
et al. 2005; Coutinho et Gupta 2009; Ruan et al. 2001). The degradation products formed
by the cleavage of the azo bond of the dye molecule are not the primary reaction inter-
mediates. The sample after 30 min of radiation showed the peak at tR ¼ 11.035 min (m/z
304 of medium intensity) attributed to the ionization of the dye molecule and another
peak at tR ¼ 14.74 min (m/z 320), which corresponds to the monohydroxylated product
of MO. There is a numerical agreement between some molecular weights and the hypoth-
esis of the formation of monohydroxylated derivatives that appear, respectively, at m/z
320, 306, and 292 and the dihydroxylated product with m/z 322. Considering that the
methyl group can be easily separated from the dimethylamino group in the monohydroxy-
lated compound, we suggest that the compound of m/z 306 is produced by heterolytic
breaking of the nitrogen–carbon bond resulting in the substitution of the methyl group by
the hydrogen atom as also stated by Dai et al. (2007). Further loss of one methyl group
and the introduction of two hydroxyl groups to benzene ring of the quinonoid species in
the ionized dye molecule with m/z ¼ 304 may lead to form a compound with m/z ¼ 322.
All the intermediate compounds detected by GC–MS still hold the chromophore moiety
so that the photo-bleaching cannot be linked to their formation, but could reasonably be
due to the formation of smaller molecules. After 90 min of UV irradiation, the degrada-
tion products of MO subsequently decrease without visible formation of new products by
GC–MS. Further oxidation gives rise to the opening of the aromatic ring leading to the
formation of alcohols, aldehydes, and carboxylic acids. Eventually, the mineralization of
MO leads to produce CO2, H2O, SO42, NH4þ and NO3 (Lachheb et al. 2002).

3.6 Evaluation of acute toxicity of MO

To evaluate the toxicities of the solutions before and after the photocatalytic reaction,
avoidance tests with the earthworms were performed. No mortality was observed during
the whole runs. Significant statistical differences were found between Organisation for
Economic Co-operation and Development (OECD) soil and OECD modified soils by add-
ing raw and treated MO solutions. When induction of the avoidance behavior occurred, it
followed apparent dose–response relationship (Figure 7). It can be noted that in the first
30 min of irradiation, there was a significant fluctuation of the avoidance of earthworms.
This behavior could be correlated to intermediates formed during the degradation. It is
believed that this phenomenon can be attributed to substances, generated in very low con-
centrations, and presenting a high level of toxicity. No intermediates, in the concentra-
tions in which they were generated in the reaction mixture, presented a higher toxicity
than the parent compound MO. Photocatalysis was quite efficient in removing toxicity. It
is clear that the avoidance of earthworms before the reaction were much higher than those
after the reaction. These results indicate that the toxicity of the solution containing MO is
decreased as a result of oxidation with TiO2/UV photocatalysis. In contrast, Rismayani
et al. (2004) have reported that the degradation of orange II by catalytic oxidation using
iron (III) phthalocyanine-tetrasulfonic acid leads to the increase of toxicity of the final
oxidized solution. In earlier work, we have shown that by means of photocatalytic degra-
dation significant removal of the overall toxicity of 4-chlorophenol was accomplished
(Elghniji et al. 2012). In this study, OECD soil spiked with the original MO solution was
generally the most toxic to E. andrei, while OECD soil spiked with the treated solutions
was least toxic. The avoidance bioassay was demonstrated to be sensitive indicator of soil
contamination and may be used to evaluate the quality of remediated colored solution.
12 H. Trabelsi et al.

Figure 7. Avoidance responses (average þ SD) of Eisenia andrei to OECD artificial soil contami-
nated by residual MO solutions irradiated during 0, 30, 60 and 180 min. An
indicates statistical
differences (p < 0.0001) by the Fischer’s exact test, i.e. a significant avoidance response of the test
soil. Data points above the dotted line indicate limited habitat function of the respective soil,
according to the Hund–Rinkecriteria (Hund-Rinke and Simon 2005).

3.7 Evaluation of phytotoxicity of MO

Phytotoxicity tests were conducted to assess the impact of the release of the treated solu-
tion to the environment as well as to evaluate the possible reuse of the pre-treated aqueous
solution in the irrigation field. Indeed, this practice can alleviate the burden on under-
ground water overexploitation and promote the practice of using treated water to irrigate
golf courses, parks and gardens. Phytotoxicity of raw and treated MO solution was tested
using seed germination assays and is depicted in Figure 8. As clearly shown, the seed

Figure 8. Germination index of Bromus ramosus in raw and treated MO solution as a function of
reaction time under UV illumination, using TiO2 catalyst.
 Toxicological & Environmental Chemistry 13

germination of B. ramosus was strongly inhibited by the raw as well as by the 30 min a
photocatalysed solution reflecting the recalcitrant nature of MO. However, as photocatal-
ysis progressed, a fast increase of GI was observed probably linked to a decrease of by-
products phytotoxicity. At 180 min, the GI reaches a maximum of 91% and the toxicity
of the treated solutions falls within the non-toxic range as classified by Wang et al.
(2001) and Tamer et al. (2006). Hence, our data indicated that after a definite time, no
germination inhibition of B. ramosus by MO metabolites at (350 mg L1) concentration
was noticed. In this way, it is worth noting that Parshetti et al. (2006) showed that the ger-
mination of T. aestivum using a raw malachite green solution is less compared to an irri-
gation test using a photocatalysed solution and the control test. Anyhow, we can suggest
that the germination of seeds using any photo-degraded dye solution could help promot-
ing the reuse of the treated water in the irrigating field under certain circumstances. In the
light of our findings, it is suggested that methyl-orange photo-treated solution should be
used precautiously for irrigation of agricultural lands.

4. Conclusions
Decolorization of an aqueous solution contaminating a stable azo dye MO was achieved
using UV/TiO2 photocatalytic process. Almost 92% of the decolorisation of relatively
high concentrated MO solution (C0 ¼ 9.16 104mol L1) was obtained after 180 min of
irradiation using HPK 125 UV immerged lamp reactor. The high concentration of dye
solution is used to simulate real colored wastewater. The disappearance of color together
with the abatement of COD and TOC contents were monitored. It was found that the reac-
tion of MO degradation follows the first-order kinetic model. The presence of inorganic
ions such as SO42 and Cl, which are usually present in real wastewater produced by
textile industry, has an inhibitory effect on MO degradation in a sequence of SO42>Cl.
The experiments also demonstrated that the addition of the peroxydisulfate ion, S2O82
in MO/TiO2 suspension leads to a higher degradation rate, which is beneficial to the treat-
ment of azo dye wastewater. The determination of the nature of the principal organic
intermediates and the evolution of the mineralization will help further understand the
mechanistic studies of the photocatalytic reaction. The structures of the transient inter-
mediates formed during the photocatalytic process are consistent with a degradation route
mainly based on demethylation, methylation and hydroxylation processes. The demethyl-
ation seems to take precedence over the hydroxylation process, but the hydroxylation
mechanism results in the most intermediates in the present system. Further oxidation
gives rise to the opening of the aromatic ring leading consequently to the complete miner-
alization. Finally and according to invertebrate and plant bioassays, it was confirmed that
photocatalytic reaction destroys the toxicity of the MO mother molecule.

Acknowledgements
The authors are indebted to Ms. Hajer Rebai for her kind help in brushing the English of the
manuscript.

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