ESSENCE - International Journal for Environmental Rehabilitation and Conservation
Verma, et al./VIII [2] 2017/34 – 42
Volume VIII [2] 2017 [34 – 42]
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
Abstract
In the present study attempts has been made to
examine the peripheral erythrocytes in fish
Cyprinus carpio for occurrence of
micronuclei and using the information for
monitoring potential genotoxicity of Azo dye
AR-88 and its degradation product. Azo dye
like malachite green causes cancer, mutations,
chromosomal fracture and teratogenicity. It
also causes sinusoidal congestion and focal
necrosis in liver, damages mitochondria and
brings nuclear alterations. The dye and
effluent brought a significant increase in the
frequency of micronuclei. The increase in
frequency was directly proportional to the
concentration of the dye as well as effluent.
The effluents produced more deleterious
effects as compared to the dye in the exposed
fish with respect to change in the cell shape,
clumping and focal necrosis of RBC’s. This
clearly indicates that longer exposure to AR88
For Correspondence:
1K. L. Mehta Dayanand College for Women, Faridabad,
Haryana
2Department of Zoology, Guru Nanak Dev University,
Amritsar, Punjab
Email: aanchal1verma@gmail.com
[ISSN 0975 - 6272]
[www.essence-journal.com]
and its degradation products at sublethal
levels will produce genotoxic effects in the
fish significantly affecting their life span and
reproduction. So micronuclei assay can be
used to know the genotoxicity of Azo dyes and
their products in polluted water bodies.
Key words: Cyprinus carpio | Azo dye | AR-
88 | Effluent and Micronuclei
Introduction
Azo dyes are important colorants having
extensive application in textile, paper, leather,
food stuff and cosmetic industries. Azo dyes
are characterized by aromatic ring linked
together by one or more Azo bonds (-N=N-)
and may contain many different substitutes
such as sulfonic (-SO3H), chloro (-Cl), methyl
(-CH3), nitro (-NO2), amino (-NH2),
hydroxyl (-OH) and carboxyl (-COOH)
group. Due to structural stability of Azo dyes,
these are less susceptible to oxidative
catabolism and difficult to biodegrade. Azo
dyes are highly soluble in water and
persistent, once discharged in the natural
environment (Bhullar and Sud, 2012). Azo
dyes account for 70% of synthetic dyes used
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 Verma, et al./VIII [2] 2017/34 – 42
in textile and dyeing industries (Carliell et al.,
1998).
The dye manufacturing, food processing,
printing, pharmaceutical, leather and textile
industries produce a large volume of effluents.
These effluents contain about fifteen percent
of the total dyes used in the process and other
products such as starch, surfactants,
dispersants, oil, emulsifiers, caustic soda,
antifoam etc. (Tan et al., 2000).
Waste water from the textile industry is a
complex mixture of many polluting
substances ranging from organ chlorine-based
pesticides to heavy metals associated with
dyes and the dyeing process (Barot and
Bahadur, 2013). Most of the Azo dyes are
water soluble and readily to absorb through
skin contact and inhalation leading to the risk
of cancer and allergic reactions, an irritant for
the eyes and highly toxic, if inhaled or
consumed (Oliveira et al., 2007, Sudha et al.,
2014). Among all the chemical classes of
dyes, Azo dyes are considered to be
recalcitrant, nonbiodegradable and persistent
(Saratale et al., 2011). Moreover, Azo dyes as
well as their breakdown products are
cytotoxic or carcinogenic (Khehra et al.,
2006). Dyes may also significantly affect
photosynthetic activity in aquatic life by
reducing light penetration intensity and may
also be toxic to some aquatic fauna and flora
due to the presence of aromatics, metals and
chlorides (Dhaneshvar et al., 2007). Certain
acid and basic Azo dyes are acutely toxic to
aquatic organisms particularly fish,
crustaceans, algae and bacteria (Material
Safety Data Sheet, Methyl Red, 2010).
Fish being at the highest trophic level of
aquatic food chain is maximally afflicted with
Azo dyes (Rana and Raizada, 1999). This in
turn poses a threat to human beings on
consumption. Dyes and untreated effluents are
also mutagenic and carcinogenic (Khanna and
Das, 1991). In addition, very low
concentration of dye (less than 1 mg L-1) can
be highly visible in solution and interfere with
penetration of light (Sloker and Le Marechal,
1998). Aniline, toulidine, benzidine,
naphthalene are the cleavage products and
impurities of Azo dyes. Acid Red (AR) 88 dye
is used for dying wool, nylon, silk, jute fiber,
paper and leather. The present study was
undertaken to compare the effect of dye AR-
88 and its microbially degraded effluent on fry
of Cyprinus carpio as no report is available on
the toxicity of AR-88. The objective of study
was the determination of minimum lethal
doses of dye and its effects on the fry which
provides a sensitive measure to know the
toxicity of dye and help in determining the
levels of dye safe for disposal in natural
aquifers. This study will provide a preliminary
data on toxicity AR-88 for developing a
decolourization and detoxification protocol
for AR-88 before releasing in water bodies.
Materials and Methods
Azo dye AR-88 used for this experiment was
purchased from Punjab Rang Udyog, a dye
manufacturing unit, Amritsar, Punjab. This
dye was selected for the present work because
it is manufactured and used at large scale in
textile industries. Cyprinus carpio were used
as test organism to estimate the toxicity of dye
because these are esteemed food fishes and
widely found in India. The fry of this fish were
collected from Govt. Fish Farm Rajasansi,
Amritsar. These were transported to
laboratory in oxygen filled polythene bags.
The fry were kept in the pool of 500ml
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 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.

Dye 

Effluent 

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

36 
 Verma, et al./VIII [2] 2017/34 – 42
and 1g/L and for the effluents 0.015g/L
and 0.018g/L respectively. Fry were
starved for 24 hour before exposure. The
behaviour of the exposed fish was
observed during the experiment. The fish
were prodded with glass rod, if it did not
move, it was considered dead. Dead fish
were removed immediately to avoid
asphyxiation of other test fish.
II. Micronuclei Assay
The micronucleus assay was performed
according to the Al–Sabti (1986) with
slight modifications. Blood sample was
taken with the heparinised sterile 1ml
plastic syringe. Blood smear was
prepared, air dried and fixed with 99.8%
methanol for 5 minutes it was then stained
with 98% Giemsa stain for 20 minutes.
The slides were observed under
microscope at 100X. At least 1000 RBC’s
were observed for the presence of
micronuclei. The shape of the cells and
nucleus along with necrosis in the cell was
also recorded from the slides.
Results
The bacterial degradations of Azo dyes
initiated by reductive cleavage of Azo bond
under aerobic and anaerobic conditions leads
to decolourization of dye and generation of
aromatic amine that are known to be more
toxic than the parent dye (Shenai, 1996).
a. Degradation of Dye
The UV-visible spectrum of bioreactor
feed containing AR-88 shows maximum
absorptions peak at 503 nm. Treatment of
dye in plug flow bioreactor resulted in
disappearance of peak at 300nm.
Rendering the effluent colourless and
indicating complete degradation of the
dye.
b. Acute Toxicity Test
96 hours static bioassay test were
performed to select two concentrations i.e.
0.5g/L, 1g/L, 0.015g/L and 0.018g/L of
dye and effluent respectively.
c. Micronuclei Assay
The frequency of micronuclei was
observed from the RBC’s of the exposed
fish fry.
i. Control
The frequency of micronuclei was 1.6% and
remained constant till 48 hours of exposure
then there was a continuous increase till 96
hours of exposure and the value came to be
4.25%. There was no change in shape and size
of the nucleus as well as cell. No clumping of
the cell as well as necrosis was observed in the
slides. The frequency for micronuclei
occurrence is shown in Table 1) and Fig .1) a.
ii. Dye
The frequency for micronuclei occurrence is
shown in Table 2) and Fig. 2) a. Exposure to
dye brought marked increase in the frequency
of micronuclei of the blood cell of the fish.
The increase was proportional to the
concentration of the dye as well as duration of
exposure. The frequency of micronuclei was
minimum 4.7% after 24 hours but maximum
18% after 96 hours of exposure. 2-4 cells
remained attached to each other. The
erythrocytes showed clumping but it was less
prominent as compared to effluent exposed
fish. This tendency for clumping increases
after 72hours of exposure. Many of the cells
became hypertrophic and this tendency
increased with the increasing time of
exposure. There was no prominent change in
shape and size of nucleus. All the values are
highly significant i.e. (p<0.01 and p<0.001).
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 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)
20

18

16

14
Concentration

12

10

0
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)

38 
 Verma, et al./VIII [2] 2017/34 – 42

30

25

20

Concentration
15

10

0
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**
(0.015g/l)
EFFLUENT 7.5  1.41* 11.25  0.35* 14.25  1.76* 18.9  2.96* 27  7.07*
(0.018g/l)
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)
30

25

20
Concentration

15

10

0
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

39 
 Verma, et al./VIII [2] 2017/34 – 42
daughter nuclei at mitosis due to aneugenic or
clastogenic effects.
A highly significant increase was
continuously observed with the increase in
concentration as well as duration of exposure
in both dye and its effluent. The increase was
more prominent in the effluent. The increased
frequency of occurrence of micronuclei in the
dyes compared to control may be due to
genotoxicity of Azo dye to fish, as these dyes
have been established to be genotoxic and
mutagenic in various organism (Khanna et al.,
1991 and Gerundo et al., 1991). Azo dyes like
AR 88 and Malachite green are reported to
cause carcinogenesis, mutagenesis,
chromosomal fracture and teratogenicity
(Khanna et al., 1991). More number of
micronuclei in the effluent exposed fish as
compared to dye may be because of the
smaller molecular weight and size of the
degraded products. These can easily enter the
cell through the plasma membrane causing
more genotoxic stress to the fish.
Intermediates formed during degradation of
some Azo dyes are reported to be more
mutagenic (Faryal, 2007). Genotoxicity of
effluents of textile industry on fish has been
reported by several authors (Odeigah et al.,
1995 and Marlasca et al., 1998). The
increased frequency may also be due to
formation off free radicals in the exposed
organism which interact with the DNA
leading to disruptive changes of and
producing micronuclei. This gets corroborated
by the findings of Hartwigs (1994) that most
of the toxic chemical producing genotoxic
effect produce reactive oxygen species as well
electrophillic free radicals and radical
metabolites that interact with DNA and cause
disruptive changes. The correlation between
exposure of genotoxic substance and
frequency of micronuclei in fish has been
reported under laboratory as well as field
conditions (Al-Sabti et al., 1995,
Nepomuceno et al., 1997, Ergene et al., 1997
and Bombaili et al., 2001). The insolubility of
AR88 may be another reason for less
genotoxicity of parent dye ( Shenai et al.,
1996). In our study we observed an increase
in micronuclei frequency with time in all the
concentrations. Whereas Cavas et al., (2005).
observed an increased with dose and decrease
with time in the frequency of micronuclei.
Time dependent effect has also been reported
by Campana et al., (1999) and Neponuceno et
al., (1997). Along with micronuclei there was
an increase in the number of RBCs showing
abnormal cell-shape, bi-nucleated cells and
necrotic cells. The dye and its effluents also
induced clumping of RBC’s. All these clearly
indicate the cytotoxic and genotoxic potential
of the Azo dyes AR-88 and its degradation
products.
Conclusion
A highly significant increase in micronuclei
was continuously observed with the increase
in concentration as well as duration of
exposure in both dye and its effluent. The
increase was more prominent in the effluent.
More number of micronuclei in the effluent
exposed fish as compared to dye may be
because of the smaller molecular weight and
size of the degraded products. These can
easily enter the cell through the plasma
membrane causing more genotoxic stress to
the fish.
Acknowledgement
Authors are thankful to Dr. S. K. Dhillon,
(Head) and Prof. Pushpinder Kaur,
Department of Zoology, GNDU, Amritsar, for
40 
 Verma, et al./VIII [2] 2017/34 – 42
providing facilities and guidance during the
work. Special thanks to Dr. B.S. Chadha, DR.
H.S. Saini and Dr. Manjinder singh,
Department of Microbiology GNDU,
Amritsar for encouraging help during the
course of investigation.
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