Namata 1st ppr
Published on: Mar 3, 2016
Transcripts - Namata 1st ppr
Decolorization of Reactive Blue 171 Dye Using
Ozonation and UV/H2O2 and Elucidation of the
Namata N. Patil and Sanjeev R. Shukla
Department of Fibres and Textile Processing Technology, Institute of Chemical Technology, (University under the Section-3 of
UGC Act 1956), Nathalal Parekh Marg, Matunga, Mumbai 400019, India; email@example.com or firstname.lastname@example.org (for
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ep.12171
In this report, the degradation of a commercially impor-
tant dye C. I. Reactive Blue 171 (RB 171) has been investi-
gated using peroxidation under UV light and ozone. The
effect of operational conditions such as the dye concentra-
tion, operating pH and use of the oxidants such as ozone
and UV/H2O2 was studied. Complete decolorization of RB
171 (50 mg/L) was obtained using ozonation, while UV/
H2O2 showed poor decolorization efﬁciency. The COD
removal by ozone was fair enough at 33%, whereas with UV/
H2O2 it was lower than 2%. Characterization using TLC,
FTIR and HPLC indicated degradation of the organic bonds
of the dye. To the best of our knowledge, for the ﬁrst time a
possible fate of metabolism of intact dye molecule by ozone
has been proposed using GC/MS analysis, which showed the
production of benzene and aliphatic/aromatic sulfonate
derivatives. Phytotoxicity studies revealed extensive reduction
in toxicity of RB 171 with ozone. It may be concluded that
the ozonation is a promising way of rapid decolorization
along with effective mineralization. VC 2015 American Institute
of Chemical Engineers Environ Prog, 00: 000–000, 2015
Keywords: ozonation, UV/H2O2, HPLC, GC/MS, toxicity
Annual worldwide production of dyes used for textile col-
oration is over 10,000 tons, out of which the azo
chromophore-based reactive dyes constitute a major share
. The presence of even ppm level concentration of dyes in
the efﬂuent streams creates aesthetic problems, hinders the
photosynthesis and aquatic life . The dyeing of reactive
dyes produces color on cotton through strong covalent
bonding and hence is in large demand. However, their pick
up is poor. As a result, over 20% of unutilized dye gets dis-
charged into wastewater during dyeing. Intense washing
of dyed cotton further adds up due to removal of unﬁxed
The conventional technique for the treatment of waste-
water in the textile industry mainly includes coagulation-
ﬂocculation, which produces large amount of sludge merely
transferring the contaminants from wastewater to secondary
waste. The biological treatment that follows has the draw-
back of system maintenance as well as treatment time .
Advanced oxidation processes (AOPs) have gained attention
as a clean and efﬁcient technology. The key AOPs, UV/
H2O2, and ozonation are based on the generation of OH•
which act as a powerful oxidizing agent (2.8 V), destroying
various organic and inorganic compounds nonselectively .
The decolorization of reactive dyes is marked by the cleav-
age of the chromophoric system; however, the generated
intermediates/by-products may or may not be toxic. Ideally,
the decolorized water used for recycling should not
adversely affect the dyeing process and should have reduced
toxicity. Considering these requirements, the study of inter-
mediate compounds formed during degradation process
seems to be crucial. Recently, the identiﬁcation of intermedi-
ates formed during the degradation process of reactive dyes
has been reported [6,7]. Degradation mechanism involving
ZnO-mediated photocatalytic treatment of Reactive Blue 160
was revealed by using GC/MS and IC techniques . Agrawal
et al.  have studied the degradation of Acid Black 210 by
using the enzyme Providencia sp. SRS82 with different char-
acterization techniques such as FTIR, HPTLC, HPLC, GC/MS,
C. I. Reactive Blue 171 (RB 171) dye, based on azo chro-
mophore, is largely used for dyeing of cotton textiles. How-
ever, only a few studies on its degradation have been
reported [8,9]. Sun et al. adsorbed RB 171 on ﬂy ash .
Khan and Husain have decolorized the same dye by using
potato (Solanum tuberosum) soluble and immobilized poly-
phenol oxidase . This work explores the degradation of
RB 171 by UV/H2O2 and O3. Operating parameters like pH,
reagent dosage and time of treatment of dye solution were
optimized. Characterization of degradation products through
FTIR, TLC and HPLC conﬁrmed the fragmentation of dye
structure into smaller components, which were further sub-
jected to the toxicity studies followed by GC/MS analysis for
their identiﬁcation so as to predict the probable mechanistic
pathway of the dye degradation.
C. I. Reactive Blue 171 (RB 171) dye, procured from Atul
Ltd., India, was used without further puriﬁcation. The struc-
tural details of the selected dye are shown in Figure 1. All
the chemicals were of LR grade and purchased from SigmaVC 2015 American Institute of Chemical Engineers
Environmental Progress & Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep Month 2015 1
Aldrich. All solutions were prepared with distilled water.
Except in concentration optimization study, the dye concen-
tration was kept at 50 mg/L. pH of the solution was adjusted
by using 0.1 M HCl/0.1 M Na2CO3/NaOH.
Ozonation of 50 mL dye solution was carried out at alka-
line pH in a semi-batch lab scale 100 mL glass reactor with
ozone dose of 72 mg/L. Ozone was generated from pure
oxygen at an input current of 0.3A with a ﬂow rate of 3 L/
min by ozone generator unit (A. M. Ozonics Pvt. Ltd., India)
having an ozone production capacity of 10 g/h. The excess
ozone gas coming out from the reactor outlet was passed
into a column of activated carbon to quench it. Most of the
test runs lasted for 3 min.
The photolytic oxidation setup consists of a closed
wooden chamber with a single low pressure UV tube
(UVC) with a power rating of 36 W, which was procured
from Philips, India. The power output was 15.3 W, which
was 35–40% of the input power. The UV tube was placed
above the 100 mL reactor, at a distance of 13 cm. The reac-
tor was ﬁlled with 50 mL dye solution of known concentra-
tions. Runs were carried out at pH 4, 7, 9 and 10.5 with
H2O2 dosing varied up to 8% (v/v). The treatment time was
ﬁxed at 30 min and the sample was collected after every 5
min. The dye solution was mechanically agitated using a
magnetic stirrer at a speed of 400 rpm, so that H2O2 is uni-
The studies were performed at room temperature
(30 6 28
C), with pH varying between 4 and 10.5, repeated
twice to ensure data reproducibility. The experimental errors
were within 1.5% of the reported values of dye degradation.
Analysis of Aqueous and Extracted Dye Solutions and
Their Degradation Products
The concentration of dissolved ozone was determined
using iodometric procedure . Concentration of RB 171
was measured by using UV-VIS Spectrophotometer (Model
8500, TECHCOMP, Hong Kong) at the characteristic kmax of
605 nm of the dye. Some of the samples were also analyzed
for the extent of chemical oxygen demand (COD) removal.
The samples were digested using closed reﬂux micro method
and analyzed on Hach colorimeter (model DR/850) at a ﬁlter
value 610 nm .
After these measurements, initial and ozone treated aque-
ous dye solutions were ﬁltered. The supernatant obtained
was used to extract metabolites with an equal volume of
ethyl acetate; dried over anhydrous Na2SO4, concentrated in
a rotary vacuum evaporator followed by its dissolution in the
HPLC grade methanol and was further used for characteriza-
tion by FTIR, TLC, HPLC and GC/MS.
The changes in functional groups were investigated using
FTIR spectrum 2000 Perkin-Elmer spectrophotometer in the
mid IR region of 750 2 4000 cm21
with 16 scan speed at a
resolution of 4/cm. The dye was analyzed in powder form,
whereas the dye degradation products were analyzed in the
form of their methanol extracts .
TLC is considered as an easy and conﬁrmatory test before
moving on to the HPLC analysis. The extracts mounted on
silica gel TLC plates (Merck) were run in the trough chamber
previously saturated with mobile phase consisting of hex-
ane/ethyl acetate/methanol (5:3:2 v/v). The resolved chroma-
tograms were observed under UV light (254 nm) and were
developed using iodine chamber.
HPLC was performed on Agilent 1100 Series model,
equipped with an auto sampler. Water Hypersil C18, 5 lm
(4.6 m 3 250 mm) reverse phase column was used to sepa-
rate individual components that were detected using Diode
Array Detector. The mobile phase consisted of HPLC grade
methanol. The column was run at a ﬂow rate 1 mL/min for
10 min without controlling the temperature and the eluate
was monitored at wavelength 254 nm using isocratic elution.
The metabolites formed after degradation were subjected
to identiﬁcation using GC/MS (Shimadzu 2010 MS). The ioni-
zation voltage was 70 eV. Gas chromatography was con-
ducted in the temperature programming mode with a Restek
column (0.25 mm id, 60 m long, nonpolar; XTI-5). The initial
column temperature was 808C for 2 min, raised linearly at
108C/min to 2808C, and held for 7 min. The temperature of
the injection port was 2808C and the GC/MS interface was
maintained at 2908C. Helium was used as a carrier gas with a
ﬂow rate of 1.0 mL/min. Degradation products were identi-
ﬁed by comparison of retention time and fragmentation pat-
tern, as well as with the mass spectra in the NIST spectral
library support stored in the GC/MS solution software (ver-
sion 1.10 beta, Shimadzu) .
Establishing the toxicity levels of the dye and its degrada-
tion products is important to decide the mode of recycling of
decolorized solution. Two types of crops, Phaseolus mungo
(dicot) and Triticum aestivum (monocot) were selected for
phytotoxicity analysis as they are regularly consumed in
India. Solutions of the dye RB 171 (500 mg/L) and methanol
extracts of the treated solutions (obtained by using each
AOP), all were adjusted to pH 7, were prepared in distilled
water. Ten healthy seeds of each crop were separately
sowed into pots containing sand. The toxicity study was car-
ried out at room temperature i.e., 328C by daily watering
5 mL for each using distilled water (as control)/mineralized
wastewater . Germination and the lengths of plumule
(shoot) and radicle (root) were recorded after 8 days of
growth. Germination (%) was estimated using the formula
Germination %ð Þ 5 ðNo: of seeds germinated
3100=No: of seeds sowedÞ
Analysis of variance (ANOVA) is a collection of statistical
models used in order to analyze the differences between
Figure 1. C. I. Reactive Blue 171 (RB 171).
Environmental Progress & Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep2 Month 2015
group means and their associated procedures to test the null
hypothesis against an alternative hypothesis. In its simplest
form, ANOVA provides a statistical test of whether or not the
means of several groups are equal. The effect of ozonation
and UV/H2O2 treatments was studied on two types of seeds
and the null hypothesis would be that all treatments have
the same effect. Rejecting the null hypothesis (F FCritical)
would imply that the two treatments result in different
effects. Statistical summary reviews the values of variance,
average, sum and count obtained by applying ANOVA to
phytotoxicity study and gives the sum of squares of residuals
(SS) together with the corresponding degrees of freedom
(df), F-values, P-values. F-value is the ratio of variation asso-
ciated with the model and variation associated with the
experimental error about its mean. Greater F-value indicates
that the components explain the variation . The critical
value of F is a function of the degrees of freedom (df) in the
numerator and the signiﬁcance level (a) in the denominator.
Adjusted mean square (MS) is the ratio of sum of squares
(SS) to degree of freedom (df). P-values assist in understand-
ing the pattern of the mutual interactions between the test
variables. The smaller the P-value, the more signiﬁcant the
corresponding coefﬁcient is.
RESULTS AND DISCUSSION
Effect of Ozonation Time and Various Dye Concentrations on Extent
RB 171 dye solutions of various concentrations (50–
200 mg/L) were ozonated for 3 min. As can be seen from
Figure 2, the extent of decolorization decreased as the con-
centration of dye was increased to 200 mg/L. This may be
attributed to the insufﬁciency of OH•
with respect to the
higher amount of dye molecules present in solution and the
competition of dye degradation products with the virgin dye
molecules for the available OH•
to get further oxidized
[15,16]. It can be seen from UV-Vis spectra of 50 mg/L aque-
ous RB 171 solution (Figure 3) that maximum absorbance for
RB 171 at k 5 605 nm decreases as a function of ozonation
time, which could be correlated to more population of OH•
that becomes available for dye degradation with longer time
In order to compare the degradation efﬁciency, the con-
centration of dye solution was kept constant at 50 mg/L with
a volume of 50 mL.
Effect of pH on Decolorization
For optimization, pH of the dye solutions was adjusted to
4, 7, 9, and 10.5 and each one was subjected to ozonation
for 1 min. Decolorization was found to be the maximum at
the alkaline pH of 10.5. Ozonation experiments were con-
ducted under alkaline pH using NaOH/Na2CO3 for pH
adjustment. From Figure 4, it may be observed that using
NaOH, decolorization enhanced by 7% (for 1 min ozonation)
at pH 10.5. However, reactive dyeing on cotton is carried out
under alkaline conditions using Na2CO3 and not NaOH. For
this reason, we have not chosen NaOH to maintain the pH,
although carbonate ions are known to scavenge OH•
At alkaline pH, ozone undergoes self-decomposition to
generate much powerful OH•
5 2.8 V) that attack the
dye pollutant nonselectively . Mechanism for enhanced
decolorization of RB 171 solution could be explained by
higher concentration of hydroxide ions at alkaline pH that
Figure 2. Effect of ozonation time on decolorization and COD removal at different dye concentration.
Figure 3. UV-Vis spectra of RB 171 solution at different ozo-
Environmental Progress Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep Month 2015 3
initiates decomposition of ozone thereby generating hydro-
peroxyl anion (HO2
) which further reacts with ozone yield-
ing super-oxide anion radical (O2
) as follows:
O3 1 OH- ! HO-
O3 1 HO-
2 ! OH•
2 1 O2
In the propagation step, ozone further reacts with gener-
ated super-oxide anion radical to generate ozonide anion
), which is immediately decomposed to OH•
2 ! O-•
3 1 O2
3 $ O-•
1 H2O ! OH•
The termination stage comprises of radicals which react
among themselves to form another molecule as described by
Cremaso and Mochi . The rate of the attack by OH•
times faster than the corresponding reac-
tion rate for molecular ozone. Thus, the concentration of
and molecular ozone can be controlled by adjusting the
solution pH to 10.5 and 4, respectively.
It is worth mentioning here that the pH of the spent reac-
tive dye bath is essentially alkaline favoring alkaline ozona-
tion and eliminating the pH adjustment step. This is an
additional advantage of the ozone treatment for decoloriza-
tion of reactive dye baths, which dominates the market of
various types of dye classes applicable on cotton fabric.
Effect of Various Concentrations of Chloride and Sulfate Ions on
Dyeing with reactive dyes requires the addition of consid-
erably large quantities of salts (sodium chloride or sodium
sulfate) to the dye bath for enhanced pick up of dye on cot-
ton. However, the solubility of ozone is readily affected by
pH and presence of radical scavengers in the liquor. Hence,
this parameter was studied under optimized conditions (RB
171 dye concentration 50 mg/L, solution pH 10.5 and volume
50 mL) at various salt concentrations (60, 500, and 1000 mg/
L) to evaluate the efﬁciency of ozone in decolorizing reactive
dye bath (Figure 5). Decolorization efﬁciency was found to
decrease marginally after 1 min of ozonation, more so with
the higher concentration (1000 mg/L) of NaCl/Na2SO4. This
negative effect of salt on RB 171 decolorization was possibly
due to the depletion of OH•
at alkaline pH as the salt anions
and dye compete for OH•
 as shown below:
Cl- 1 OH•
Cl- 1 Cl•
! Cl2 1 e-
Addition of Na2SO4 also had similar effect, which could
be explained by the reaction mentioned below:
4 1 OH•
4 1 OH-
Muthukumar and Selvakumar  reported that higher the
chloride and sulfate ion content, longer is the time required
for complete decolorization. This is in agreement with our
Figure 4. Effect of pH on the decolorization of RB 171 in
the ozonation treatment.
Figure 5. Effect of chloride ions on the extent of decoloriza-
tion by ozonation.
Table 1. Decolorization of RB 171 using UV/H2O2.
In absence of UV
In presence of UV
15 min at pH 30 min at pH
(%, v/v) 4 7 9 10.5 4 7 9 10.5
0 0 1.4 0.5 0.4 0 1.9 0.6 0 0
1 0.6 9.3 3.7 2 2.8 22.1 6.1 4.3 3.9
2 1.4 12.4 5.4 3.5 5.2 22.1 6.9 4.9 5.7
4 1.9 14.4 9.5 5.9 8.0 23.5 10.8 6.9 9.6
6 2.3 16.9 10.3 6.4 7.4 24.4 10.8 5.9 6.5
8 3.1 17 12.3 7.9 6.9 23.9 12.8 8.3 8.7
Environmental Progress Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep4 Month 2015
Effect of Ozonation Time on COD Removal
As can be seen in Figure 4, under the optimized pH con-
ditions, within ﬁrst 1 min of ozonation, more than 80% dye
solution was decolorized, but COD removal (Figure 2) was
not even 10%. At the end of 3 min ozone treatment, the
COD removal increased, but only to 33%. Thus a longer
duration of treatment is essential to achieve better minerali-
zation of the dye. Perhaps, in the ﬁrst 1 min, the OH•
ated by ozone in alkaline medium attack the azo
chromophore of dye thereby loosing the color. Mineraliza-
tion was found to be poor with respect to decolorization, as
decolorization was rapid than COD reduction, which
depends essentially on the smaller size products of dye deg-
Effect of UV Exposure on Decolorization
The data given in Table 1 indicates insigniﬁcant decolor-
ization by UV or H2O2 alone. Li et al. also observed the same
. When the treatment under UV was combined with 6%
H2O2, the extent of decolorization increased with UV expo-
sure time, perhaps due to increased population of active
, by extended duration of photolysis .
H2O21UV ! 2 OH•
Effect of pH on Decolorization
Maximum decolorization was obtained at pH 4 for each
H2O2 dosage studied (Table 1). Decreased decolorization at
alkaline pH might be a consequence of the following possi-
ble reasons. The concentration of the conjugate base of
) increases at alkaline pH which reacts with a
nondissociated molecule of H2O2 resulting in oxygen and
water, in place of producing OH•
under UV radiation as
shown below :
H2O2 ! HO-
pKa ¼ 11:6ð Þ
21 H2O2 ! H2O1 O21 OH-
Hence, the instantaneous concentration of OH•
lower than expected. Secondly, the reaction of OH•
is approximately 100 times faster than its reaction with
H2O2 that has been very well explained by Mitrovic et al.
. Furthermore, the self-decomposition rate of hydrogen
peroxide has been observed to increase in alkaline
Figure 6. FTIR spectra of the RB 171 (a) and its degradation products by ozonataion (b).
Environmental Progress Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep Month 2015 5
conditions leading to the decrease of OH•
et al.  also indicated that best results on reduction of
COD and color were obtained when the treatment was per-
formed under acidic pH rather than an alkaline pH. Reduc-
tion in pH after the treatment from initial 4 to nearly 2.5
can be attributed to the formation of organic and inorganic
Effect of H2O2 Dosage on Decolorization and Extent of COD Removal
The extent of RB 171 decolorization increased to 24.4%
(Table 1) till an optimum H2O2 dosage of 6% (v/v). How-
ever, higher dosage of H2O2 [8% (v/v)] resulted in the inhibi-
tion of decolorization limiting it to 23.9%, which may be
attributed to further increase in OH•
, scavenging H2O2 itself
 as follows:
H2O2 1 OH•
2 1 H2O
2 1 HO•
! H2O1 O2
Even after 30 min of the reaction, the COD removal was
very poor (less than 2%). UV/H2O2 treated samples were not
considered for elucidation of degradation due to poor decol-
orization and COD removal, however, those were considered
for the toxicity studies.
Analysis of methanol extracts to study degradation by FTIR, TLC,
HPLC, and GC/MS
Figure 6a shows the appearance of hump in the region of
3330 2 3550 cm21
conﬁrming the presence of primary, sec-
ondary and tertiary amines (aliphatic as well as aromatic) in
Figure 7. GC/MS spectra obtained after the degradation of RB 171 by ozonation.
Environmental Progress Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep6 Month 2015
Table 2. GC spectral details of intermediates obtained by ozonation of RB 171.
GC/MS fragments by Ozonation Details of the intermediate Structure
a Chemical Formula: C9H7ClN5NaO3S sodium
b Chemical Formula: C10H9N4NaO4S sodium
c Chemical Formula: C9H7N4NaO2S sodium
(1) Chemical Formula: C31H18ClN10Na5O16S5sodium
(2) Chemical Formula: C15H10ClN5Na2O6S2 sodium 3,
(3) Other fragments like sodium hydroxide, sodium
benzenesulfonateand SO3 ions
(4) Chemical Formula: C6H6NNaO3S sodium
Environmental Progress Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep Month 2015 7
the dye structure which was found to diminish in the treated
samples. In addition, band at 1610 cm21
conﬁrmed the pres-
ence of AN@NA linkage while the bands between 1530 and
indicated aromatic C@C stretching , followed
by the band between 550 and 850 cm21
thus marking CACl
stretching. All the above mentioned bands were found to be
absent in the spectra of the ozone treated sample Figure 6b,
conﬁrming the degradation of dye to different species. The
FTIR spectra of the treated samples showed two peaks
between each 2800 2 2900 cm21
, 1400 2 1450 cm21
1000 2 1120 cm21
regions representing CAH stretching,
CAH bending, and CAN stretching vibrations of aliphatic
amines, respectively, thus indicating the production of ali-
phatic saturated compounds due to oxidation of RB 171 by
In the case of TLC analysis, the Rf value for the parent
dye RB 171 was much higher (0.87) in comparison with that
for ozone treated sample (0.45). The same has been con-
ﬁrmed by Sahasrabudhe and Pathade  and Zope et al.
Table 3. Phytotoxicity study of the dye RB 171 and the metabolites obtained after AOP treatments for the Phaseolus mungo
and Triticum aestivum.
(Dicot) Phaseolus mungo (Monocot) Triticum aestivum
Dist. Water 100 20.3 6 0.8 7 6 0.6 100 16.4 6 0.8 4 6 0.5
500 ppm RB 171 70 14.4 6 0.9 3 6 0.7 60 11.3 6 0.6 1.8 6 0.2
UV/H2O2 70 15.2 6 0.8 3.1 6 0.4 70 13.3 6 0.4 2.7 6 0.4
Ozonated 90 17.8 6 0.4 4.2 6 0.4 90 14.8 6 0.4 3.1 6 0.3
Data was analyzed by one-way analysis of variance (ANOVA) with Tukey-Kramer Multiple Comparison Test and mentioned
values are the mean of ten germinated seeds of four sets SEM (6).
Figure 8. Proposed degradation pathway for the dye RB 171 by ozonation.
Environmental Progress Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep8 Month 2015
HPLC spectrum of RB 171 showed one major peak at
retention time of 6.467 min and a minor peak at 6.360 min.
The spectrum for ozone treated sample showed the peaks at
2.413 and 3.393 min. This difference in the retention times of
the original dye and the fragments formed by ozone treat-
ment conﬁrms the dye degradation into different small
organic fragments by ozonation, further to cleavage of azo
linkage that is responsible for decolorization.
The GC/MS spectrum of ozone treated sample is shown in
Figure 7. The details of intermediates detected by GC/MS
were labeled alphabetically and those undetected-but-
reorganized as necessary intermediates were labelled numeri-
cally and are given in Table 2. Inferring Figure 8, it could be
assumed that the dye structure was attacked by OH•
to asymmetric azo bond cleavage and yielded intermediate
(a); (m/z 5 322) followed by the formation of a unidentiﬁed
reactive intermediate (1). It might have further cleaved at sec-
ond azo position by OH•
giving two intermediates, one of
them was labeled as Intermediate (b); (m/z) 5 304) and sec-
ond as Intermediate (2). Oxidative cleavage of this intermedi-
ate (2) between N and C of the triazine ring led to the
formation of intermediate (c); (m/z) 5 258 and intermediate
(3 and 4). Thus, probable degradation mechanism showed the
production of benzene and aliphatic/aromatic sulfonate deriv-
atives. Similar were the observations by Bansal and Sud .
Toxicity of 500 mg/L of the dye RB 171 solution before
and after the treatments was conﬁrmed by inhibition in ger-
mination for Phaseolus mungo (dicot) and Triticum aestivum
(monocot), as against distilled water. This untreated solution
adversely affected both shoot and root lengths (Table 3).
However UV/H2O2 and ozone treated set showed excellent
results comparable to distilled water, thus indicating exten-
sive reduction in toxicity by the treatment order ozone UV/
H2O2. This study paved the route for the treated water
towards its reuse for irrigation purpose as well. Kurade et al.
 also acknowledged similar reduction in toxicity of Scarlet
RR dye after Consortium BL-GG treatment.
Higher difference in variance values (Table 4) between
ozone and UV/H2O2 treated samples with respect to that
between ozone and control (distilled water) samples conﬁrms
that ANOVA result is in agreement with the experimental data
stating higher reduction in toxicity by ozonation than UV/
H2O2 treatment. In all cases, P value 0.05 (Table 5) along
with F FCritical for a certain number of degrees of freedom at
level of 95% signiﬁcance a (a 5 0.05) proves that null hypoth-
esis is rejected (Both treatments have different efﬁciency in
toxicity removal). Much lower P value for Phaseolus mungo
with respect to Triticum aestivum indicate that among both
the prior resulted in good germination and better growth.
From this study the following conclusions may be drawn:
1. The fastest decolorization (100% within 2 min) and high-
est COD removal (33% within 3 min) was achieved by
ozonation at pH 10.5. However, UV/H2O2 was found to
be time consuming (30 min) leading to only 24.4% decol-
orization at acidic pH 4. COD removal was poor with
both the AOPs.
2. FTIR, TLC, and HPLC analysis of ozone-treated samples
conﬁrmed the structural changes in the large dye mole-
cule in terms of shift/disappearance of IR peaks, retention
time and elution peaks, respectively.
3. Probable degradation illustrated the production of ben-
zene and aliphatic/aromatic sulfonate derivatives. This
might be attributed to lower COD removal values.
4. Toxicity studies revealed possible reuse of treated dye
solution for irrigation purpose.
5. It may be concluded therefore that ozonation is most
promising technique for abatement of the pollution
caused by the presence of color in the wastewater.
Table 4. Statistical summary indicating the values corresponding to “Sum,” obtained by summation of shoot and root length
values (from Table 3), for Phaseolus mungo and Triticum aestivum.
Phaseolus mungo (Dicot) Triticum aestivum (Monocot)
Variance Average Sum Count Summary Count Sum Average Variance
89.2448 13.63 27.26 2 D.W 2 20.37 10.185 76.26125
64.65469 8.671429 17.34286 2 RB 171 2 13.06667 6.533333 45.125
72.68735 9.171429 18.34286 2 UV/H2O2 2 16.07143 8.035714 56.02867
92.54477 10.96905 21.9381 2 Ozonated 2 17.96667 8.983333 68.18525
7.247746 16.90964 67.63857 4 Shoot length 4 55.79413 13.94853 4.688716
3.368698 4.31131 17.24524 4 Root length 4 11.68063 2.920159 0.853955
Average values are function of “Sum” in numerator to “Count” in denominator. Variance calculated by ANOVA measures the
variability from an average or mean.
Table 5. ANOVA analysis conﬁrms that seeds germinated in RB 171 solution are signiﬁcantly different from those germinated
in metabolites at P 0.05. Better germination and growth was obtained for Phaseolus mungo at P 0.0001 than Triticum
Phaseolus mungo (Dicot) Triticum aestivum (Monocot)
F crit P-value F MS df SS
Variation SS df MS F P-value F crit
9.276628 0.020518 17.7834 10.0512 3 30.1537 Treatments 14.278 3 4.75929 16.0753 0.00863 9.2766
10.12796 0.000165 561.632 317.436 1 317.436 Shoot root
243.25 1 243.25 310.51 0.0004 10.128
Environmental Progress Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep Month 2015 9
Authors would like to express their gratitude to UGC for
funding of this project under Major Research Project grant.
Thanks are also due to Mr. A. Moolji (Director, M/S A. M.
Ozonics Pvt. Ltd., Mumbai) for his timely and valuable
inputs. Ms. Namata Patil is also grateful for UGC-SAP fellow-
ship under CAS.
1. Forgacs, E., Cserhati, T., Oros, G. (2004). Removal of
synthetic dyes from wastewaters: A review, Environment
International, 30, 953–971.
2. Allegre, C., Moulin, P., Maisseu, M., Charbit, F. (2006).
Treatment and reuse of reactive dyeing efﬂuents, Journal
of Membrane Science, 269, 15–34.
3. Solıs, M., Solıs, A., Manjarrez, N., Flores, M. (2012).
Microbial decolouration of azo dyes: A review, Process
Biochemistry, 47, 1723–1748.
4. Beyerbach, A., Rothman, N., Bhatnagar, V., Kashyap, R.,
Sabbioni, G. (2006). Hemoglobin adducts in workers
exposed to benzidine and azo dyes, Carcinogensis, 27,
5. Glaze, W., Kang, J. D.H. (1987). The chemistry of
water treatment processes involving ozone, hydrogen
peroxide and ultraviolet radiation, Ozone: Science
Engineering: The Journal of the International Ozone
Association, 9, 335–352.
6. Bansal, P., Sud, D. (2012). Photodegradation of
commercial dye, CI Reactive Blue 160 using ZnO
nanopowder: Degradation pathway and identiﬁcation of
intermediates by GC/MS. Separation and Puriﬁcation
Technology, 85, 112–119.
7. Agrawal, S., Tipre, D., Patel, B., Dave, S. (2014).
Optimization of triazo Acid Black 210 dye degradation by
Providencia sp. SRS82 and elucidation of degradation
pathway., Process Biochemistry, 49, 110–119.
8. Sun, D., Zhang, X., Wu, Y., Liu, X. (2010). Adsorption
of anionic dyes from aqueous solution on ﬂy ash, Journal
of Hazardous Materials, 181, 335–342.
9. Khan, A., Husain, Q. (2007). Decolorization and
removal of textile and non-textile dyes from polluted
wastewater and dyeing efﬂuent by using potato (Solanum
tuberosum) soluble and immobilized polyphenol oxidase,
Bioresource Technology, 98, 1012–1019.
10. Wang, Y., Zhang, H., Chen, L., Wang, S., Zhang, D.
(2012). Ozonation combined with ultrasound for the
degradation of tetracycline in a rectangular air-lift reactor,
Separation and Puriﬁcation Technology, 84, 138–146.
11. Is¸ık, M., Sponza, D. (2008). Anaerobic/aerobic
treatment of a simulated textile wastewater, Separation
and Puriﬁcation Technology, 60, 64–72.
12. Lade, H., Waghmode, T., Kadam, A., Govindwar, S.
(2012). Enhanced biodegradation and detoxiﬁcation of
disperse azo dye Rubine GFL and textile industry efﬂuent
by deﬁned fungal-bacterial consortium, International Bio-
deterioration Biodegradation, 72, 94–107.
13. Kulkarni, A., Kadam, A., Kachole, M., Govindwar, S.
(2014). Lichen Permelia perlata: A novel system for
biodegradation and detoxiﬁcation of disperse dye Solvent
Red 24, Journal of Hazardous Materials, 276, 461–468.
14. Kurade, M., Waghmode, T., Kagalkar, A., Govindwar, S.
(2012). Decolorization of textile industry efﬂuent
containing disperse dye Scarlet RR by a newly developed
bacterial-yeast consortium BL-GG, Chemistry Engineering
Journal, 184, 33–41.
15. Li, G., Zhao, X., Ray, M. (2007). Advanced oxidation of
orange II using TiO2 supported on porous adsorbents:
The role of pH, H2O2 and O3, Separation and Puriﬁcation
Technology, 55, 91–97.
16. M.S.G. De Souza, D., Bonilla, K.S.A.A. (2010). Removal
of COD and color from hydrolyzed textile azo dye by
combined ozonation and biological treatment, Journal of
Hazardous Materials, 179, 35–42.
17. Gul, S., Yildirim, O. (2009). Degradation of reactive
red 194 and reactive yellow 145 azo dyes by O3 and
H2O2/UV-C processes, Chemistry Engineering Journal,
18. Santana, M., Da Silva, L., Freitas, A., Boodts, J.,
Fernandes, K., De Faria, L. (2009). Application of
electrochemically generated ozone to the discoloration
and degradation of solutions containing the dye Reactive
Orange 122, Journal of Hazardous Materials, 164, 10–17.
19. Cremasco, M., Mochi, V. (2013). Reaction of dissolved
ozone in hydrogen peroxide produced during
ozonization of an alkaline medium in a bubble column,
Acta Scientiarum Technology, 36, 81–85.
20. Muthukumar, M., Selvakumar, N. (2004). Studies on the
effect of inorganic salts on decolouration of acid dye
efﬂuents by ozonation, Dyes and Pigments, 62, 221–228.
21. Alvares, A., Diaper, C., Parsons, S. (2001). Partial
oxidation of hydrolysed and unhydrolysed textile azo dyes
by ozone and the effect on biodegradability, Process
Safety and Environmental Protection, 79, 103–108.
22. Galindo, C Kalt, A. (1998). UV-H2O2 oxidation of
monoazo dyes in aqueous media: a kinetic study, Dyes
and Pigments, 40, 27–35.
23. Ghodbane, H., Hamdaoui, O. (2010). Decolorization of
antraquinonic dye, C.I. Acid Blue 25, in aqueous solution
by direct UV irradiation, UV/H2O2 and UV/Fe(II)
processes, Chemistry Engineering Journal, 160, 226–231.
24. Mitrovic´, J., Radovic´, M., Bojic´, D., Anbelkovic´, T.,
Purenovic´, M., Bojic´, A. (2012). Decolorization of the
textile azo dye reactive orange 16 by the UV/H2O2 process,
Journal of the Serbian Chemical Society, 77, 465–481.
25. Azbar, N., Yonar, T., Kestioglu, K. (2004). Comparison
of various advanced oxidation processes and chemical
treatment methods for COD and color removal from a
polyester and acetate ﬁber dyeing efﬂuent,
Chemosphere, 55, 35–43.
26. Basiri Parsa, J., Hagh Negahdar, S. (2012). Treatment of
wastewater containing Acid Blue 92 dye by advanced
ozone-based oxidation methods, Separation and Puriﬁca-
tion Technology, 98, 315–320.
27. Hu, Q., Zhang, C., Wang, Z., Chen, Y., Mao, K., Zhang,
X., Xiong, Y., Zhu, M. (2008). Photodegradation of
methyl tert-butyl ether (MTBE) by UV/H2O2 and UV/
TiO2, Journal of Hazardous Materials, 154, 795–803.
28. Arjunan, V., Subramanian, S., Mohan, S. (2004). FTIR
and FTR spectral studies of 2-amin-6-bromo-3 formyl-
chromone, Spectrochimica Acta Part A Molecular and
Biomolecular Spectroscopy, 60, 995–1000.
29. Sahasrabudhe, M., Pathade, G. (2012). Decolourization
and degradation of C.I.Reactive Red 195 by Georgenia
sp.CC-NMPT-T3, Indian Journal of Experiamental
Biology, 50, 290–299.
30. Zope, V., Kulkarni, M., Chavan, M. (2007).
Biodegradation of synthetic textile dyes reactive red 195
and reactive green 11 by Aspergillus niger grp: An
alternative approach, Journal of Scientiﬁc and Industrial
Research, 66, 411–414.
Environmental Progress Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep10 Month 2015