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ESSENCE - International Journal for Environmental Rehabilitation and Conservation

Verma, et al./VIII [2] 2017/34 – 42

Volume VIII [2] 2017 [34 – 42] [ISSN 0975 - 6272]

Genotoxic effects of azo dye AR-88 and its microbial degraded products on fry
of Cyprinus Carpio

Verma, Anchal1; Kaur, Arvinder2 and Arya, Shveta1

Received: July 11, 2017  Accepted: August 30, 2017  Online: December 31, 2017


In the present study attempts has been made to and its degradation products at sublethal
examine the peripheral erythrocytes in fish levels will produce genotoxic effects in the
Cyprinus carpio for occurrence of fish significantly affecting their life span and
micronuclei and using the information for reproduction. So micronuclei assay can be
monitoring potential genotoxicity of Azo dye used to know the genotoxicity of Azo dyes and
AR-88 and its degradation product. Azo dye their products in polluted water bodies.
like malachite green causes cancer, mutations, Key words: Cyprinus carpio | Azo dye | AR-
chromosomal fracture and teratogenicity. It 88 | Effluent and Micronuclei
also causes sinusoidal congestion and focal
necrosis in liver, damages mitochondria and Introduction
brings nuclear alterations. The dye and Azo dyes are important colorants having
effluent brought a significant increase in the extensive application in textile, paper, leather,
frequency of micronuclei. The increase in food stuff and cosmetic industries. Azo dyes
frequency was directly proportional to the are characterized by aromatic ring linked
concentration of the dye as well as effluent. together by one or more Azo bonds (-N=N-)
The effluents produced more deleterious and may contain many different substitutes
effects as compared to the dye in the exposed such as sulfonic (-SO3H), chloro (-Cl), methyl
fish with respect to change in the cell shape, (-CH3), nitro (-NO2), amino (-NH2),
clumping and focal necrosis of RBC’s. This hydroxyl (-OH) and carboxyl (-COOH)
clearly indicates that longer exposure to AR88 group. Due to structural stability of Azo dyes,
these are less susceptible to oxidative
For Correspondence:
catabolism and difficult to biodegrade. Azo
1K. L. Mehta Dayanand College for Women, Faridabad,

Haryana dyes are highly soluble in water and

2Department of Zoology, Guru Nanak Dev University,

Amritsar, Punjab
persistent, once discharged in the natural
Email: environment (Bhullar and Sud, 2012). Azo
dyes account for 70% of synthetic dyes used
Verma, et al./VIII [2] 2017/34 – 42

in textile and dyeing industries (Carliell et al., Azo dyes (Rana and Raizada, 1999). This in
1998). turn poses a threat to human beings on
The dye manufacturing, food processing, consumption. Dyes and untreated effluents are
printing, pharmaceutical, leather and textile also mutagenic and carcinogenic (Khanna and
industries produce a large volume of effluents. Das, 1991). In addition, very low
These effluents contain about fifteen percent concentration of dye (less than 1 mg L-1) can
of the total dyes used in the process and other be highly visible in solution and interfere with
products such as starch, surfactants, penetration of light (Sloker and Le Marechal,
dispersants, oil, emulsifiers, caustic soda, 1998). Aniline, toulidine, benzidine,
antifoam etc. (Tan et al., 2000). naphthalene are the cleavage products and
impurities of Azo dyes. Acid Red (AR) 88 dye
Waste water from the textile industry is a
is used for dying wool, nylon, silk, jute fiber,
complex mixture of many polluting
paper and leather. The present study was
substances ranging from organ chlorine-based
undertaken to compare the effect of dye AR-
pesticides to heavy metals associated with
88 and its microbially degraded effluent on fry
dyes and the dyeing process (Barot and
of Cyprinus carpio as no report is available on
Bahadur, 2013). Most of the Azo dyes are
the toxicity of AR-88. The objective of study
water soluble and readily to absorb through
was the determination of minimum lethal
skin contact and inhalation leading to the risk
doses of dye and its effects on the fry which
of cancer and allergic reactions, an irritant for
provides a sensitive measure to know the
the eyes and highly toxic, if inhaled or
toxicity of dye and help in determining the
consumed (Oliveira et al., 2007, Sudha et al.,
levels of dye safe for disposal in natural
2014). Among all the chemical classes of
aquifers. This study will provide a preliminary
dyes, Azo dyes are considered to be
data on toxicity AR-88 for developing a
recalcitrant, nonbiodegradable and persistent
decolourization and detoxification protocol
(Saratale et al., 2011). Moreover, Azo dyes as
for AR-88 before releasing in water bodies.
well as their breakdown products are
cytotoxic or carcinogenic (Khehra et al., Materials and Methods
2006). Dyes may also significantly affect Azo dye AR-88 used for this experiment was
photosynthetic activity in aquatic life by purchased from Punjab Rang Udyog, a dye
reducing light penetration intensity and may manufacturing unit, Amritsar, Punjab. This
also be toxic to some aquatic fauna and flora dye was selected for the present work because
due to the presence of aromatics, metals and it is manufactured and used at large scale in
chlorides (Dhaneshvar et al., 2007). Certain textile industries. Cyprinus carpio were used
acid and basic Azo dyes are acutely toxic to as test organism to estimate the toxicity of dye
aquatic organisms particularly fish, because these are esteemed food fishes and
crustaceans, algae and bacteria (Material widely found in India. The fry of this fish were
Safety Data Sheet, Methyl Red, 2010). collected from Govt. Fish Farm Rajasansi,
Fish being at the highest trophic level of Amritsar. These were transported to
aquatic food chain is maximally afflicted with laboratory in oxygen filled polythene bags.
The fry were kept in the pool of 500ml
Verma, et al./VIII [2] 2017/34 – 42

capacity for 15 days for acclimation to G. Trace element solution per

laboratory conditions. Fishes were fed litre(0.01).The trace element solution
adlibitum with commercial fish feed during used was of following composition
acclimation period. Decolourisation of the dye (mg/l)
was brought in a plug flow bioreactor divided i. ZnSO4.7H2O(10.0mg)
into two compartments anaerobic and aerobic ii. Na2MOO4.2H2O(10.0mg)
with the help of consortium HM-4 which iii. MnCl2.2H2O (3.0mg)
includes Bacillus cere (BN -7), Pseudomonas iv. COCl2.6H2O (1.0mg)
putida (BN-4), Pseudomonas fluorescence v. NiCl2.6H2O (2.0mg)
(BN-5) and Stenotraphomonas vi. H3BO3 (3.0mg)
acidaminiphila (BN-3) collected and purified vii. Cucl2.2H2O (1.0mg)
from industrial sites.
The pH was adjusted to 7.0. The mineral salt
Mineral salt medium was used for growth of medium was supplemented with 25% of yeast
consortium with following compositions and 50% of glucose. At the time of activation
A. Na2 HPO4 (3.6g) the dye was added at a rate of 20mg/l. After
B. (NH4)SO4 (1.0g) sterilization this solution was added at a rate
C. KH2PO4 (1.0g ) of 20mg/l. After sterilization this solution was
run through the plug flow bioreactor. The
D. MgSO4 (1.0g)
effluent that came out of the bioreactor was
E. Fe(NH4) citrate (0.01g) collected and its absorption was recorded at
F. CaCl2.2H2O(10ml) 505nm to observe degradation of dye.



Fig. (A): Spectroscopic analysis for AR-88 and its treated effluent

I. Acute Toxicity Test from these tests. Ten fishes were exposed
According to Standard method 96 hours to control and two concentrations each of
static bioassay tests were performed. Two dye AR-88 as well as its effluent. The
least toxic concentrations were selected concentrations for the dye were 0.5g/L

Verma, et al./VIII [2] 2017/34 – 42

and 1g/L and for the effluents 0.015g/L b. Acute Toxicity Test
and 0.018g/L respectively. Fry were 96 hours static bioassay test were
starved for 24 hour before exposure. The performed to select two concentrations i.e.
behaviour of the exposed fish was 0.5g/L, 1g/L, 0.015g/L and 0.018g/L of
observed during the experiment. The fish dye and effluent respectively.
were prodded with glass rod, if it did not c. Micronuclei Assay
move, it was considered dead. Dead fish The frequency of micronuclei was
were removed immediately to avoid observed from the RBC’s of the exposed
asphyxiation of other test fish. fish fry.
II. Micronuclei Assay i. Control
The micronucleus assay was performed The frequency of micronuclei was 1.6% and
according to the Al–Sabti (1986) with remained constant till 48 hours of exposure
slight modifications. Blood sample was then there was a continuous increase till 96
taken with the heparinised sterile 1ml hours of exposure and the value came to be
plastic syringe. Blood smear was 4.25%. There was no change in shape and size
prepared, air dried and fixed with 99.8% of the nucleus as well as cell. No clumping of
methanol for 5 minutes it was then stained the cell as well as necrosis was observed in the
with 98% Giemsa stain for 20 minutes. slides. The frequency for micronuclei
The slides were observed under occurrence is shown in Table 1) and Fig .1) a.
microscope at 100X. At least 1000 RBC’s
ii. Dye
were observed for the presence of
The frequency for micronuclei occurrence is
micronuclei. The shape of the cells and
shown in Table 2) and Fig. 2) a. Exposure to
nucleus along with necrosis in the cell was
dye brought marked increase in the frequency
also recorded from the slides.
of micronuclei of the blood cell of the fish.
Results The increase was proportional to the
The bacterial degradations of Azo dyes concentration of the dye as well as duration of
initiated by reductive cleavage of Azo bond exposure. The frequency of micronuclei was
under aerobic and anaerobic conditions leads minimum 4.7% after 24 hours but maximum
to decolourization of dye and generation of 18% after 96 hours of exposure. 2-4 cells
aromatic amine that are known to be more remained attached to each other. The
toxic than the parent dye (Shenai, 1996). erythrocytes showed clumping but it was less
a. Degradation of Dye prominent as compared to effluent exposed
The UV-visible spectrum of bioreactor fish. This tendency for clumping increases
feed containing AR-88 shows maximum after 72hours of exposure. Many of the cells
absorptions peak at 503 nm. Treatment of became hypertrophic and this tendency
dye in plug flow bioreactor resulted in increased with the increasing time of
disappearance of peak at 300nm. exposure. There was no prominent change in
Rendering the effluent colourless and shape and size of nucleus. All the values are
indicating complete degradation of the highly significant i.e. (p<0.01 and p<0.001).
Verma, et al./VIII [2] 2017/34 – 42

iii. Effluent frequency was minimum 10.25% after 24

The frequency for micronuclei occurrence is hours and maximum 27% after 96 hours of
shown in Table 3) and Fig. 3) a. All the values exposures. The frequency of occurrence of
are highly significant i.e. (p<0.01 and micronuclei increase with increase in
p<0.001). The frequency of micronuclei in the concentration and all the duration of
erythrocytes increases in concentration of exposure.
effluent as well as duration of exposure. The

Concentration Control Dye

Time (Mean + SD) 0.5g/l 1 g/l
Interval (Mean + SD) (Mean + SD)
24 hours 1.6  0.56NS 4.75  0.35* 7.3  1.55*
48 hours 1.6  0.56 NS
6.35  1.20** 9.6  0.84
72 hours 2.75  0.35* 9.2  1.97 13.8  2.82*
96 hours 4.25  1.06** 12.8  3.11** 18  2.82**
SD- Standard deviation Table 1: Percent occurrence of micronuclei in the RBC of fry
NS -Non Significant Values exposed to (Control and Dye)
*Values Significant at 5% i.e. (p<0.01)
**Values Significant at 1% i.e. (p<0.001)






24 hours 48 hours 72 hours 96 hours
Time Interval

Control Dye (0.5 g/l) Dye (1 g/l)

Figure 1 a: Percent occurrence of micronuclei in the RBC of fry exposed to (Control and Dye)

Concentration Control Effluent

(Mean + SD) (Mean + SD)
0.015g/l 0.018g/l
Time Interval (Mean + SD) (Mean + SD)
24 hours 1.6  0.56NS 10.25  0.35* 11.25  0.35*
48 hours 1.6  0.56 NS 12.5  0.42** 14.25  1.76*
72 hours 2.75  0.35* 16.5  4.38* 18.9  2.96**
96 hours 4.25  1.06** 25.25  6.71 27  7.07*
SD- Standard Deviation Table 2: Percent occurrence of micronuclei in the RBC of fry
NS- Non Significant Values exposed to (Control and Effluent)
*Values Significant at 5% i.e.(p<0.01)
**Values Significant at 1% i.e. (p<0.001)

Verma, et al./VIII [2] 2017/34 – 42






24 hours 48 hours 72 hours 96 hours
Time Interval

Control Effluent (75%) Effluent (95%)

Figure 2 a: Percent occurrence of micronuclei in RBC of fry exposed to (Control and Effluent)

Concentration Time Interval

(mean + SD) 0 hr 24 hrs 48 hrs 72 hrs 96 hrs
CONTROL 0.750.35** 1.6  0.56NS 1.6  0.56NS 2.75 0.35** 4.25  1.06**
DYE (0.5g/l) 2.75 .06* 4.75  0.35* 6.35 1.20* 9.2  1.97* 12.8  3.11*
DYE (1g/l) 4.5 0.70** 7.3  1.55 ** 9.6 0.84** 13.8 2.82** 18  2.82**
EFFLUENT 6  1.41** 10.250.35** 12.50.42** 16.5  4.38** 25.256.71**
EFFLUENT 7.5  1.41* 11.25  0.35* 14.25  1.76* 18.9  2.96* 27  7.07*
SD- Standard Deviation Table 3: Percent occurrence of micronuclei in RBC of fry exposed to (Control,
NS Non Significant Values Dye and Effluent)
*Values Significant at 5% i.e.(p<0.01)
**Values Significant at 1% i.e. (p<0.001)





0 hr 24 hrs 48 hrs 72 hrs 96 hrs
Time Interval
Control Dye (0.5g/l) Dye (1g/l) Effluent (0.015g/l) Effluent (0.018g/l)

Figure 3 a: Percent occurrence of micronuclei in the RBC of fry exposed to (Control, dye and effluent)

Discussion increase in the number of micronuclei in

The mutagenicity of chemicals can be verified control occurred after 24 hrs of exposure
by chromosomal aberrations and micronuclei which then remained constant till 48 hrs again
studies, in turn reflecting potential hazards to showing an increase till 96 hrs of exposure.
individual genetic system (Kar and Das, The increase in micronuclei in the RBC’s of
1987). control fish may be due to a stress of
In the present study there was an increase in starvation as the fry were not fed during the
the frequency of micronuclei in the RBC’s of exposure period Heddle et al. (1991), have
fish in control as well as both the reported that micronuclei are caused by
concentrations of the dye and effluent. In condensation or fragmentation of
control the increase was less prominent as chromosome that are not incorporated into
compared to dye and effluent. A significant

Verma, et al./VIII [2] 2017/34 – 42

daughter nuclei at mitosis due to aneugenic or exposure of genotoxic substance and

clastogenic effects. frequency of micronuclei in fish has been
A highly significant increase was reported under laboratory as well as field
continuously observed with the increase in conditions (Al-Sabti et al., 1995,
concentration as well as duration of exposure Nepomuceno et al., 1997, Ergene et al., 1997
in both dye and its effluent. The increase was and Bombaili et al., 2001). The insolubility of
more prominent in the effluent. The increased AR88 may be another reason for less
frequency of occurrence of micronuclei in the genotoxicity of parent dye ( Shenai et al.,
dyes compared to control may be due to 1996). In our study we observed an increase
genotoxicity of Azo dye to fish, as these dyes in micronuclei frequency with time in all the
have been established to be genotoxic and concentrations. Whereas Cavas et al., (2005).
mutagenic in various organism (Khanna et al., observed an increased with dose and decrease
1991 and Gerundo et al., 1991). Azo dyes like with time in the frequency of micronuclei.
AR 88 and Malachite green are reported to Time dependent effect has also been reported
cause carcinogenesis, mutagenesis, by Campana et al., (1999) and Neponuceno et
chromosomal fracture and teratogenicity al., (1997). Along with micronuclei there was
(Khanna et al., 1991). More number of an increase in the number of RBCs showing
micronuclei in the effluent exposed fish as abnormal cell-shape, bi-nucleated cells and
compared to dye may be because of the necrotic cells. The dye and its effluents also
smaller molecular weight and size of the induced clumping of RBC’s. All these clearly
degraded products. These can easily enter the indicate the cytotoxic and genotoxic potential
cell through the plasma membrane causing of the Azo dyes AR-88 and its degradation
more genotoxic stress to the fish. products.
Intermediates formed during degradation of Conclusion
some Azo dyes are reported to be more A highly significant increase in micronuclei
mutagenic (Faryal, 2007). Genotoxicity of was continuously observed with the increase
effluents of textile industry on fish has been in concentration as well as duration of
reported by several authors (Odeigah et al., exposure in both dye and its effluent. The
1995 and Marlasca et al., 1998). The increase was more prominent in the effluent.
increased frequency may also be due to More number of micronuclei in the effluent
formation off free radicals in the exposed exposed fish as compared to dye may be
organism which interact with the DNA because of the smaller molecular weight and
leading to disruptive changes of and size of the degraded products. These can
producing micronuclei. This gets corroborated easily enter the cell through the plasma
by the findings of Hartwigs (1994) that most membrane causing more genotoxic stress to
of the toxic chemical producing genotoxic the fish.
effect produce reactive oxygen species as well Acknowledgement
electrophillic free radicals and radical Authors are thankful to Dr. S. K. Dhillon,
metabolites that interact with DNA and cause (Head) and Prof. Pushpinder Kaur,
disruptive changes. The correlation between Department of Zoology, GNDU, Amritsar, for
Verma, et al./VIII [2] 2017/34 – 42

providing facilities and guidance during the decolorization of a reactive Azo dye
work. Special thanks to Dr. B.S. Chadha, DR. Environ. Technol. 19 (11): 1133-1137.
H.S. Saini and Dr. Manjinder singh, Cavas, T. and Ergene-Gozukara, S. (2005):
Department of Microbiology GNDU, Micronucleus test in fish cells: A
Amritsar for encouraging help during the bioassay for in situ monitoring of
genotoxic pollution in marine
course of investigation.
environment. Environ. Mol. Mutagen.
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