chapter -iv toxicological impact of dyes on anabas...
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Chapter -IV
Toxicological impact of dyes on Anabas testudineus (Bloch)
4.1 Introduction
Alleppy (Alapuzha), the Venice of the East, is also well known for
backwater tourism. The main attraction is the availability of different fish dishes
harvested from lakes and kayals around here. The Kuttanad basin, the rice bowl
of Kerala, is a unique wetland which permits one good crop of rice and one
harvest of fish and an area of thriving water tourism. The area is also popular
for its coconut cultivation, duck rearing & coir industry. The land and water
ecosystem located in the Alleppy region is very much polluted by the pumping
of coir dyeing effluents from small and large-scale coir dyeing industries. Kurup
et al. (1990) observed retardation in the natural propagation of fishes in this
region from the very low fish yield.
The effluent released from coir dyeing industries contains a lot of
hazardous chemicals such as colouring agents (dyes), caustic soda, sodium
chloride, hydrochloric acid, sodium hypochlorite, heavy metals etc. and exert
severe impacts on ecosystems (Bala Subramanian and Manishankar, 1987). Of
the 700,000 tons of dyes produced annually worldwide, approximately 10 to 15
percent of the dye is disposed off during dyeing processes through effluents
(Hussain et al, 2004). The released effluent and sludge contaminate the streams,
rivers and underground water system as well as soil ecosystem nearby, making
it unsuitable for crop production.
Chemical diversity of the organic pollutants in dye effluents causes
variety of toxic effects on aquatic organisms in recipient water bodies. Most of
these substances have been classified as carcinogenic, mutagenic (Ericson and
Larsson, 2000) and endocrinic (Zacharewski et al., 1995). Azo dyes and
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benzidine based dyes are widely used in coir and textile industries. These dyes
cause tissue damage, haematological responses and histopathological
degradations in aquatic organisms.
Fish are at the higher levels of food chain and accumulation of
contaminants in fish biomagnifies the toxicants from water and hence widely
used to evaluate the health of aquatic ecosystem (Kock et al., 1996). Biological
changes in fish due to the exposure of toxicants are called biomarkers and are
used for environmental risk assessment (Van der Oost et al., 2003). Behavioural
changes may be the first response of an organism to environmental change
(Slobodkin, 1968) and are the most sensitive measures of neurotoxicity
(Doving, 1991).The use of haematological and biochemical parameters as
indicators of water quality has been attributed to detect sub-lethal impacts of
pollutants.
Histopathological biomarkers allows examining specific target organs
that is responsible for vital functions, such as respiration, coordination,
excretion, reproduction and the accumulation and biotransformation of toxicants
(Lauren et al.,1990). Furthermore, alterations found in these organs are
normally easier to identify than the functional ones (Fanta et al., 2003) and
serve as warning signs of damage to animal health (Marlasca et al., 1998;
Tetreault et al., 2011).
Climbing perch, Anabas testudineus (Bloch) is a hardy, partially air
breathing fish which can tolerate both well and poorly oxygenated waters. It is
widely seen in paddy fields, ponds and inland water bodies of Kerala, hence
selected as biological indicators of ecotoxicological studies. This study has been
conducted to investigate the acute and chronic toxicity of two azo dyes used in
coir industries viz. Acid Orange 7 (AO7) and Direct Blue 6 (DB6), on Anabas
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testudineus by observing the biochemical, haematological and histopathological
changes before and after exposure to these dyes.
4.2 Materials and methods
4.2.1 Dyes
Two sulphonated azo dyes, Acid Orange 7 (Fig 4.1) and Direct Blue 6
(Fig 4.2) were used for the study and were procured from a local market in
Alleppy. Both the dyes are used for dyeing coir and their chemical structures are
as follows.
Fig 4.1: Structure of Acid Orange 7 Fig 4.2: Structure of Direct Blue 6
4.2.2 Collection and maintenance of experimental fish:
Climbing perch, Anabas testudineus (Fig 4.3) is a fresh water table fish,
which satisfies the qualities of an experimental fish as suggested by Butler et al.
(1971). Fishes were collected from the same stocking pond (Edathua, Alleppy)
not contaminated with industrial effluents including coir dyes. They were
transported in circular plastic containers to prevent crowding or damaging
themselves by striking the walls as suggested by Cox (1974), thereby decreasing
their susceptibility to disease and were disinfected with 0.1% solution of
potassium permanganate for 5 minutes to avoid dermal infection. Further, the
active and healthy fishes of uniform size (22 ± 2.2 g, 11 ± 1.6 cm, as mean ±
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SD) were selected for acclimatization during which they were kept in
quarantine for 16 days (Fig 4.4). The fishes were fed with pieces of live
earthworms during acclimatization.
Healthy fishes with active movements were only considered for acute
and chronic toxicity tests. Glass tanks of 20 litres capacity were chosen for
maintaining the fish. Thorough scrubbing and cleaning of each tank were
carried out and tap water stored for 12 hrs was used for complete replenishment
in the morning, and is used for bioassay experiment. Each tank was filled with
15 litres of water to avoid the fish jumping out of the tank. For 48 hrs prior to
testing, the fishes were starved, as customary in static tests in order to reduce
the amount of waste materials generated by them. All the tanks were disinfected
once a month with 5% Povidone iodine (Alphadine) diluted to 1 ml per litre of
water.
4.2.3 Physicochemical characterization of water
The quality of water used for experiment was determined periodically
according to Standard methods (APHA, 1998). The physicochemical parameters
of the water such as pH, temperature, dissolved oxygen, ammonia and carbon
dioxide were measured and was tabulated (Table 4.1). The pH was determined
using a digital pH meter (Systronics digital) and dissolved oxygen was
measured using a digital, dissolved oxygen meter (Jenway, 9071). While,
temperature was measured using a mercury in-glass thermometer, which was
placed in the medium inside the test container until reading was taken. The
reading was taken at 10.00 a.m. on each day of the experiment.
4.2.4 Chemicals and Glassware
The chemicals, reagents, substrates and standards used in the present
investigation were of analytical grade purchased from HiMedia Industries,
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Mumbai and Sisco Research Laboratories, India. Distilled and deionised water
was used for all analytical work and for preparing stock solutions. Acid and
alkali resistant borosilicate glass wares manufactured by M/S Borosil, India
were used.
4.2.5 Acute Toxicity Tests: Determination of LC50.
Acute Toxicity tests were done by time dependent (96 hours) static
renewal technique in conformity with guidelines suggested by APHA (1998)
followed by probit analysis (Finney, 1971) using the dyes, AO7 and DB6,
dissolved in tap water. Preliminary screening was carried out to determine the
appropriate concentration range for the selected dyes. Selected concentrations in
narrow range from the results of range finding test were used to determine the
LC50 and mortality was recorded at 24, 48, 72 and 96 h. Ten fishes were used
per concentration and the experiment was conducted in triplicate (Fig 4.5). The
control group was kept in experimental water without adding the dyes, keeping
all other conditions same. All experiments were carried out for a period of 96 h.
The number of dead fish was counted every 24 h and removed immediately
from the aquaria. The LC50 value was computed using Probit Analysis
Programme (Version 1.5, US EPA) from the number of fishes that died during
96 hrs of the study, with confidence limits p = 0.05.
4.2.6 Chronic Toxicity studies with sub lethal concentration and exposure periods
In natural or experimental conditions, a sub lethal concentration of a
toxicant is most likely to produce sub lethal effects to alter the morphological,
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Fig 4.3: Anabas testudineus
Fig 4.4: Acclimatisation of fishes used for study
Fig 4.5: Fishes maintained for LC50 study.
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physiological and histological conditions of the fish though it may not cause
immediate death of the individual. Hence, the study of sub lethal dose effect of
azo dye is comparatively more rational than lethal or fatal dose, as in most of
the polluted natural environment, sub lethal concentration is met which may
cause the alteration in the normal survival of organisms over a prolonged period
of time.
For the chronic toxicity studies, the fishes were divided into three
experimental groups and placed in separate glass aquaria. Ten fishes were used
for each group. Group I was maintained in dye free water to serve as control.
Groups II and III were exposed to sub lethal concentration (1/3rd LC50) of both
dyes. The nominal concentrations of dyes tested were 0.92 gm/L of Acid
Orange 7 (AO7) and 0.51 gm/L of Direct Blue 6 (DB6) and maintained upto 90
days. In order to understand the influence of time of exposure over toxicity
effects, the fishes were selected for experiments after 30 days and 90 days of
exposure. The dyes and water were renewed every 48 hour. The experiments
were done in three replicas.
4.2.7 Behavioural Changes:
Behaviour is the recordable and observable activity of living organisms.
The fish depends on an intact nervous system for mediating relevant behaviour.
The nervous system is most vulnerable and injuries to its elements through
toxicants may drastically change the behaviour and consequently the survival of
the fish. The behavioural changes in air gulping, grouping, swimming and
resting were closely observed.
4.2.8 Biochemical parameters: Enzyme Assays
Fishes were exposed to sub lethal concentrations of both dyes and at the
end of the exposure period (30 and 90 days), five fishes each from the
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experimental and control tanks were selected and sacrificed. The organs such as
gills, liver and muscle were dissected out and weighed. It was then minced,
cleaned in saline and homogenized separately using micro pestle with 2 ml 10
mM Tris-HCl buffer and centrifuged at 12000 rpm for 15 minutes. The
activities of aspartate aminotransferase (AST, E.C 2.6.1.1), alanine
aminotransferase (ALT, E.C 2.6.1.2) and alkaline phosphatase (ALP, E.C
3.1.3.1) and acid phosphatase (ACP, E.C 3.1.3.2) were measured using standard
procedures (Appendix I).
4.2.9 Haematological examination: Impact on RBC
After the exposure period of 30 days, fishes from each group were
subjected to blood sampling from the caudal vein and microscopic slides were
prepared for each fish. Two clean slides were taken and a drop of blood was
placed at the edge of one end of the slide. With another slide, held at 45
degrees, the drop was spread evenly and was allowed to dry. Then the blood
film was covered with 15 drops of Giemsa stain for one minute. Then 30 drops
of distilled water were added, mixed well and allowed to stand for five minutes.
Then the slide was washed with distilled water. The stained film was dried in air
at room temperature. The shape and size of RBC were observed under a
microscope and images were taken.
4.2.10 Histopathological examination of Liver and Gill:
The fishes were exposed to sub lethal concentrations of both dyes and at
the end of 30 day exposure period; the organs such as gills and liver were
dissected out and were fixed in 10% buffered formalin. The tissues were then
processed in automatic tissue processor, embedded in paraffin wax and
sectioned at 5µm on a rotary microtome. Sections were placed on glass slides
and stained with Haematoxylin and Eosin (Roberts, 1978). Approximately 2-4
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stained slides were examined with the help of a compound microscope and
photographs were taken. Histopathological changes in these tissues were
recorded and compared with controls under the guidance of a pathologist.
4.2.11 SDS-PAGE analysis:
The SDS-PAGE was performed to analyze protein profile in liver,
muscle and gill of control and dye exposed tissues of fishes exposed to 1/3rd
LC50 concentration for a period of 30 days. 0.5 ml of the supernatant obtained
by centrifugation after homogenizing was taken in an ependroff tube and 0.5 ml
10% trichloroacetic acid was added and centrifuged. The pellet was washed
with chilled acetone and it was dissolved in sample buffer (0.5M Tris-HCl pH
6.8- 2 ml, 40% glycerol- 1.6 ml, 10% SDS- 3.2 ml, 2-mercaptoethanol- 0.8 ml,
0.1% (w/v) bromophenol blue- 0.4 ml) and heated at 950C for 2 minutes. 20 µl
of each sample was added to the wells and the electrophoresis run was carried at
60V for 2 hrs and at 120V for 1.5 hr by watching the movement of the tracking
dye and the gel was analysed by Coomassie Brilliant blue staining method
(Laemmli et al., 1970).
4.2.12 Statistical Analysis
To determine statistically significant differences between experimental
and control groups, all the mean values of data were analyzed using one-way
ANOVA test. The data are presented as the Mean ± SD (Standard Deviation).
The results were evaluated and the following levels of significance were used
p<0.001, p<0.01 and p<0.05 for significant data.
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4.3 Results
4.3.1 Parameters of sample water
The values of physico-chemical parameters of control and experimental
water samples were observed and recorded in Table 4.1. There was a decrease
in pH in dye mixed water but was not significant. Also there is an increase in
carbon dioxide in dye containing samples.
Table 4.1: Physico-chemical parameters of the sample water
Physico-chemical properties Control AO7 dissolved DB6 dissolved
Temperature(0C) 28.50 ± 0.21 28.50 ± 0.40 28.00 ± 0.70
Dissolved Oxygen (mg/L) 6.12 ± 0.30 6.01 ± 0.68 5.97 ± 0.40
pH 7.05 ± 0.20 6.85 ± 0.24 5.91 ± 0.04
Carbon dioxide (mg/L) 9.50 ± 0.15 10.22 ± 0.17 10.06 ± 0.04
Ammonia (mg/L) 0.152 ± 0.003 0.154 ± 0.007 0.151 ± 0.004
Values are average of three replicates and represented as Mean± SD.
4.3.2 Acute toxicity tests: Determination of LC50
The 96 h LC50 is the basic value in the acute toxicity test. The mortality
of A. testudineus exposed to AO7 with exposure time is given in Table 4.2a.
The percentage of mortality was found to be increased with increase in the
concentration of dye.
The estimated Lethal Concentration (LC) values at different exposure
concentration of AO7 with lower and upper confidence limits were calculated
and the results (as per programme software) are given in Table 4.2b.
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Table 4.2a: Mortality of A. testudineus exposed to Acid Orange 7 (gm/L)
Sl. No
Concentration
of AO7(gm/L)
Exposure Time and cumulative no. of fishes
responded
No. of fishes
exposed
No. of fishes died
% of mortality at 96 hrs 24
hrs 48 hrs
72 hrs
96 hrs
1 Control 0 0 0 0 10 0 0
2 1.25 0 0 0 0 10 0 0
3 2.50 0 0 3 4 10 4 40
4 3.75 2 6 8 8 10 8 80
5 5.00 4 8 10 10 10 10 100
6 6.25 8 10 10 10 10 10 100
Table 4.2b: LC Values and Confidence Limits
Point Exposure Conc.
(gm/L)
95% Confidence Limits
Lower Upper
LC 1.00 1.428 0.525 1.938
LC 5.00 1.733 0.805 2.215
LC 10.00 1.921 1.009 2.385
LC 15.00 2.060 1.173 2.512
LC 50.00 2.765 2.124 3.261
LC 85.00 3.712 3.155 5.162
LC 90.00 3.980 3.361 5.932
LC 95.00 4.412 3.657 7.357
LC 99.00 5.355 4.219 11.185
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The mortality of A. testudineus exposed to DB6 with exposure time is
given in Table 4.3a. The percentage of mortality was found to be increased with
increase in the concentration of dye.
Table 4.3a. Mortality of A. testudineus exposed to Direct Blue 6 (gm/L)
Sl. No
Concentration of DB6(gm/L)
Exposure Time and cumulative no. of fishes
responded
No. of fishes
exposed
No. of fishes died
% of mortality at 96 hrs 24
hrs 48 hrs
72 hrs
96 hrs
1 Control 0 0 0 0 10 0 0
2 0.75 0 0 0 1 10 1 10
3 1.50 0 1 2 5 10 5 50
4 2.25 2 5 6 7 10 7 70
5 3.00 5 8 8 9 10 9 90
6 3.75 10 10 10 10 10 10 100
Table 4.3b: LC Values and Confidence Limits
Point Exposure Conc.
(gm/L)
95% Confidence Limits
Lower Upper
LC 1.00 0.460 0.134 0.742
LC 5.00 0.653 0.254 0.954
LC 10.00 0.788 0.357 1.095
LC 15.00 0.894 0.447 1.205
LC 50.00 1.524 1.097 1.913
LC 85.00 2.598 2.059 3.965
LC 90.00 2.948 2.298 4.901
LC 95.00 3.554 2.672 6.787
LC 99.00 5.048 3.477 12.742
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The estimated Lethal Concentration (LC) values at different exposure
concentration of DB6 with lower and upper confidence limits were calculated
and the results (as per programme software) are given in Table 4.3b.
4.3.3 Behavioural Changes
In the present study, morphological and behavioural changes in the
fishes were observed after they were introduced into the dye dissolved water.
They tried to jump out of the water soon after they were introduced into the
medium. During the initial hours, their movement was very fast with erotic
jumping and they were hyperactive. Air gulping behaviour was increased
rapidly and tends to decrease after a short period. Grouping behaviour was
found to be distorted. Two major behavioural changes such as hypo activity and
lethargy were noticed in fish that were exposed to higher concentrations of dyes
after a short period of time. In higher concentrations, after some time the fishes
lost their equilibrium, remained motionless, excess mucus were spread all over
the body surface and ultimately death occurred with slightly bulged out eyes
(Fig 4.6).
Fig 4.6: Fishes treated with acid Fig 4.7: Intense blue colouration of Orange 7 producing viscous mucous muscles due to Direct Blue 6
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4.3.4 Biochemical parameters: Enzyme assays
The aspartate aminotransferase activity (AST) in the tissues of fish
Anabas testudineus on exposure to sublethal concentration of AO7 and DB6
were estimated and the results are given in Table 4.4. There was significant
increase in the AST activity in gills and liver of fishes treated with dye. Also
there was a correlation with increased AST activity with increase in duration of
exposure. When compared, DB6 exposure for 90 days increased the AST
activity by 83.08% in gills, 31.49% in muscles and 73.78% in liver. AO7
exposure increased the activity by 71.32% in gills. All the above values were
significant at P< 0.001.
Table 4.4: AST activity (µmoles oxaloacetate/mg protein/hr)
Tissues Control
Exposure in days
AO7 DB6
30 90 30 90
Gill 1.36 ± 0.06 1.85± 0.45*** 2.33± 0.19*** 1.99± 0.15*** 2.49 ±0.13***
% change 36.03% 71.32% 46.32% 83.08%
Muscle 2.54 ± 0.12 2.96±0.22*** 3.42±0.11*** 2.86±0.17* 3.34 ±0.13***
% change 16.54% 34.65% 12.60% 31.49%
Liver 2.25 ±0.09 2.65± 0.16*** 3.01 ±0.18*** 2.49± 0.11* 3.91±0. 14***
% change 17.77% 33.77% 10.66% 73.78%
Values are expressed in mean ± SD.
*(P<0.05), ** (P<0.01) and *** (P<0.001) denote significant differences from control fish
The alanine aminotransferase activity (ALT) in the tissues of fish
Anabas testudineus on exposure to sublethal concentration of AO7 and DB6
were estimated and the results are given in Table 4.5.
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Table 4.5. ALT activity in µmoles pyruvate/mg protein/hr
Tissues Control
Exposure in days
AO7 DB6
30 90 30 90
Gill 1.62±0.16 1.84±0.04* 2.26±0.08*** 1.82±0.0* 2.35±0.18***
%change 13.58% 39.51% 12.34% 73%
Muscle 4.09±0.11 4.58±0.16** 5.77±0.07*** 4.55±0.05* 6.69±0.21***
% change 11.98% 41.08% 11.25% 63.57%
Liver 4.93±0.04 6.22±0.11** 7.49±0.09*** 6.23±0.19** 8.35±0.15***
% change 26.17% 51.92% 26.37% 69.37%
Values are expressed in mean ± SD
*(P<0.05), ** (P<0.01) and *** (P<0.001) denote significant differences from control fish
There was significant increase in the ALT activity in the tissues of fishes
treated with dye. The increase in ALT activity varies with tissues, liver being
most affected followed by muscle and then gills for AO7 where as for DB6 gills
was the most affected (73%) compared to liver (69.37%) and muscle (63.57%).
The experimental data showed that activity of these enzymes increases with
exposure time.
Table 4.6. The alkaline phosphatase (ALP) activity (µmoles p-nitrophenol/mg protein/hr) in the tissues of fish Anabas testudineus on
exposure to sublethal concentration of AO7 and DB6
Tissues Control
Exposure in days
AO7 DB6
30 90 30 90
Gill 1.84±0.33 2.41±0.31** 2.82±0.25*** 2.14±0.15ns 3.06±0.21***
% change 30.98% 53.26% 16.30% 66.30%
Muscle 1.02±0.22 1.27±0.09* 1.80±0.13*** 1.31±0.14* 2.05±0.11***
% change 24.51% 76.47% 28.43% 100%
Liver 0.69±0.16 1.06±0.06** 1.33±0.24*** 1.02±0.11** 1.56±0.19***
% change 53.62% 92.75% 47.83% 126.09% Values are expressed in mean ± SD *(P<0.05), ** (P<0.01) and *** (P<0.001) denote significant differences from control fish
106 Chapter 4 Chapter 4 Chapter 4 Chapter 4
The alkaline phosphatase (ALP) activity in the tissues of fish Anabas
testudineus on exposure to sublethal concentration of AO7 and DB6 were
estimated and the results are given in Table 4.6. Significant increase (P<0.001)
was observed in all 90 days exposed fish. The percentage change observed in
liver enzyme activity was 126.09% followed by 100% in muscles and 66.30%
in gills for DB6 exposed fishes. Also there was a correlation with increased
ALP activity with increase in duration of exposure.
The acid phosphatase (ACP) activity in the tissues of fish Anabas
testudineus on exposure to sublethal concentration of AO7 and DB6 were
estimated and the results are given in Table 4.7. There was significant increase
in the ACP activity in gills and liver of fishes treated with dye. Also there was a
correlation with increased ACP activity with increase in duration of exposure.
The percentage of increase in acid phosphatase activity was more pronounced in
liver with a value of 82.14% for DB6 and 73.21% for AO7 treated fish.
Table 4.7. The Acid phoshatase activity (µmoles p-nitrophenol/mg protein/hr) in the tissues of fish Anabas testudineus on exposure to
sublethal concentration of AO7 and DB6.
Tissues Control
Exposure in days
AO7 DB6
30 90 30 90
Gill mean SD 2.52±0.56 3.15±0.24* 3.81±0.22*** 3.43±0.27** 4.29±0.31***
% change 25% 51.19% 36.11% 70.24%
Muscle mean SD
1.45±0.08 1.76±0.09** 1.93±0.19*** 1.68±0.07* 2.24±0.13***
% change 21.38% 33.10% 15.86% 54.48%
Liver mean SD 0.56±0.06 0.68±0.04* 0.97±0.05*** 0.71±0.03** 1.02±0.11***
% change 21.43% 73.21% 26.78% 82.14%
Values are expressed in mean ± SD
*(P<0.05), ** (P<0.01) and *** (P<0.001) denote significant differences from control fish
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4.3.5 Haematological parameter
Blood smear: Impact on Erythrocytes
The results are presented in Fig 4. 8: A, B and C. The control fishes
showed elliptical RBC's with a central nucleus. The blood smear of dye treated
fish showed a number of morphological changes in the erythrocyte. AO7 treated
fish blood smear showed a very slight deviation from the normal elliptical forms
of the control whereas DB6 treated fish blood exhibited more pronounced
departures in the shape of the cells from the elliptical forms of the controls,
vacuolation of the cytoplasm and drastic changes in the shape of the nucleus.
4.3.6 Histopathology of Liver and Gill
The normal structure of the gill of Anabas testudineus is of the
teleostean type described by Hughes and Morgan, (1973). The primary gill
filaments in each arch for two rows are joined at the base by a gill septum.
These filaments are flat and leaf like structures situated alternately on either side
of the septum. Numerous semicircular secondary gill lamellae, vary slightly in
size and shape is lined up along both sides of the primary gill lamellae. The
primary gill lamellae consist of centrally placed rod like supporting axis with
blood vessels on either side. The secondary lamellae also termed as respiratory
lamellae are highly vascularised and covered with a thin layer of epithelial cells.
The epithelial layers of two sides are separated by pillar cells or pilaster cells.
Blood vessels are extended into each of the secondary gill filaments. The blood
cells of the secondary gill lamellae have a single nucleus, which are flattened in
appearance. The region between the two adjacent secondary gill lamellae is
known as inter lamellar region. Histopathology of normal gill is shown in Fig
4.9a.
108 Chapter 4 Chapter 4 Chapter 4 Chapter 4
a) b) c)
Fig 4.8: RBC of (a) normal fish,(b) AO7 treated fish and (c)DB6 treated fish
a) b) c)
Fig 4.9: Histopathology of gill of (a) normal fish, (b) AO7 treated fish and (c) DB6 treated fish
a) b) c)
Fig 4.10: Histopathology of liver of (a) normal fish, (b) AO7 treated fish and (c) DB6 treated fish
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Exposure to sub lethal concentrations of dye has induced marked
pathological changes in fish gills (Fig 4.9 b,c). The changes include the bulging
of tips of primary gill filaments. The secondary gill filaments lost their original
shape and curling of secondary gill filaments was observed. The pillar cells
nucleus showed necrosis and development of vacuoles in the secondary gills
epithelium. In AO7 and DB6 treated samples the arrangement of the pillar cells
was greatly disturbed. Necrosis was seen in both samples and severe in DB6
treated in the lamellar region. At some places a fusion of adjacent gill lamellae
was seen. A number of secondary gill lamellae were damaged in fish exposed to
AO7. The pillar cell system also appeared to collapse. The pilaster columns
were seen to be curled up and their blood spaces were engorged. Pools of
congested blood were also visible within the sub-epithelial spaces. The
respiratory epithelia were slightly swollen in the AO7 treated sample whereas it
was either swollen or lost in the case of DB6 exposed fish. Respiratory
epithelium was ruptured at different points, so that the capillaries were exposed
to water and hemorrhage exudates could be seen at many places over the
lamellar surface and in the branchial cavity. Swollen nuclei were also observed.
Inflammatory alterations were seen in the epithelium. Shrinkage of blood
capillaries and loss of micro ridges were seen in both samples.
Liver:
The liver of control Anabas testudineus was composed of parenchymal
cells (hepatocytes) arranged in a typical tubular sinusoid pattern, the liver cords
were characteristically two cells thick and alternated with sinusoids. The
hepatocytes were morphologically polygonal and vacuolated. The nuclei were
spherical in shape and uniform in size. The blood vessels with red blood cells
were found in good condition (Fig.4.10a)
110 Chapter 4 Chapter 4 Chapter 4 Chapter 4
In contrast, the liver of fish after the dye treatment at sublethal
concentration, exhibited marked pathological changes. Diffuse necrosis in
parenchymal cells with cytoplasmic vacuolation was found. Increased
cytoplasmic granularity was seen in AO7 and DB6 exposed fish. Blood
congestion in the central vein can be easily noticed. Significant loss of radial
orientation and parenchymal shrinkage were readily observed in DB6 exposed
fish than in AO7 exposed fish. Pycnotic nuclei were seen scattered throughout
the liver. Fibrosis was also observed in dye exposed fishes. The tubule sinusoid
arrangement was partly or completely lost in DB6 exposed fishes when
compared to AO7 exposed fishes (Fig 4.10b and 4.10c).
4.3.7 SDS PAGE Analysis
The gills, muscles and liver of the fish was dissected (Fig 4.11) and SDS
PAGE analysis was done. The electrophoretogram (Fig 4.12) represents the
difference in the intensity of proteins in tissues such as gills, muscle and liver.
When compared to control protein pattern, each tissue sample showed
difference in protein intensities. In gill protein, there is an increase in intensities
of proteins with molecular weights 44 kDa, 40 kDa and 20 kDa for AO7 treated
sample and 68 kDa, 40 kDa in case of DB6 treated sample. In case of liver
protein bands, new bands were observed in DB6 treated sample when compared
to control. In muscle protein, AO7 treated sample showed new protein bands
with molecular weights 56 kDa and 42 kDa.
Toxicological impact of dyeToxicological impact of dyeToxicological impact of dyeToxicological impact of dyes on s on s on s on AnabasAnabasAnabasAnabas testudineus (Bloch)testudineus (Bloch)testudineus (Bloch)testudineus (Bloch) 111
Fig 4.11: Gills, muscle and liver dissected for SDS PAGE
Lanes 1,2,3: Normal gill, gill of AO7 treated, gill of DB6 treated Lanes 4,8 : Markers Lanes 5,6,7: Normal liver, liver of AO7 treated, liver of DB6 treated Lanes 9,10,11:Normal muscle, muscle treated with AO7, muscle treated with DB6
Fig 4.12: SDS-PAGE analysis of protein profile in gill, liver and muscle
112 Chapter 4 Chapter 4 Chapter 4 Chapter 4
4.4 Discussion
Fish mortality due to dye exposure mainly depends upon its sensitivity
to the toxicants, concentration and duration of exposure. The evaluation of
LC50 concentration is an important step in ecotoxicological studies. Different
species respond differently to same type of toxicant. Anabas, being a hardy fish,
can tolerate usual disturbances and contaminants in aquatic ecosystem.
However, increased concentration of toxicant will make severe damages to
different systems and finally leads to death of the organism. In the present study
A.testudineus was exposed to different concentrations of two azo dyes used in
coir industries in Kerala.
The LC50 values for 96 hrs were 2.765 gm and 1.524 gm respectively for
AO7 and DB6. Air breathing fish have a higher LC50 value compared to other
water-breathing fish (Dutta et al., 1992a). Anabas, being an obligate air
breathing fish takes only less toxicant through gills from the water (Jacob et al.,
1982) and is highly tolerant to toxicants. Mercy et al. (2000) studied the lethal
toxicity of monocrotophos, a pesticide used in Kuttanad area and found that the
LC50 value for Anabas was 0.102 gm whereas for Etroplus, a sensitive fish, the
value was 0.0034 gm. It was clear that the sensitivity of a particular species
itself varies with internal factors such as sex, age, size (Williams et al., 1984)
and external factors such as period of exposure, pH, hardness of water and
dissolved content of the medium (Mc leese, 1974 and Brungs et al., 1977). The
comparative high value of LC50 of these dyes makes them more dangerous since
they are not potent killers and no signs of extensive damage can be seen when
the water is polluted.
During present study fish, Anabas testudineus showed hyper excitation,
erratic swimming, jerky movements and thick mucous covering over the whole
Toxicological impact of dyeToxicological impact of dyeToxicological impact of dyeToxicological impact of dyes on s on s on s on AnabasAnabasAnabasAnabas testudineus (Bloch)testudineus (Bloch)testudineus (Bloch)testudineus (Bloch) 113
body surface. Similar results were observed by Srivastava et al. (2007) when
Labeo rohita and Channa punctatus exposed to paper mill effluent. These
observations were more profound in fishes exposed to higher concentrations
than lower which reveals the positive correlation between toxicant
concentration and behavioural pattern. An earlier report shows that acute
toxicity primarily damages the central nervous system which leads to breathing
difficulties, instability and sluggishness (Holden, 1973). The lethargic condition
due to dye exposure would affect the fish in several ways. Feeding and food-
capture will be altered and fishes living in streams may be swept downstream. It
has been shown that some fish behaviours (e.g., locomotor activity and
avoidance) are extremely sensitive to pollutants (Heath, 1987). Similar
hypoactive and lethargic conditions were observed also in fish Labeo rohita and
Anabas testudineus exposed to malathion (Dutta et al.,1994), Anabas
testudineus exposed to monocrotophos (Santhakumar.,1998), Anabas
testudineus exposed to biopesticide prepared from Calotropis gigantean
(Bharathi.,2005) and Cirrihnus mrigala exposed to cypermethrin (Prasanth et
al.,2005). The observed mucus accumulation on the gills and skin of the fishes
exposed to dyes were probably due to toxic effects of dyes, because respiratory
epithelium might be the main target site of toxicity during the period of
experiment. The mucus may be an adaptive response which provides an
additional protection against the corrosive nature of dyes. This agrees with
earlier findings of Sadhu (1993) with pesticides using Anabas as the
experimental fish. Out of the two dyes selected, DB6 showed more toxicity in
terms of behaviour changes. Aquatic organisms exhibit a broad range of
responses to pollutants depending on the compound, exposure time, water
condition and species (Coppage and Matthews, 1974).
114 Chapter 4 Chapter 4 Chapter 4 Chapter 4
The enzymes like aspartate and alanine aminotransferase, acid and
alkaline phosphatases etc. also serve as diagnostic tool to evaluate the toxicity
stress of chemicals in the living organisms (Harper, 1991). AST activity of gills
was increased significantly (P<0.001) for 30 days and 90 days exposed fishes
and reveals that gills are the primary target of toxicants. The increase in the
activity of AST and ALT was in the order of gills, liver and muscle for DB6.
Acid phosphatases and Alkaline phosphatases are hydrolytic enzymes released
by lysosomes for the hydrolysis of foreign body. Also these are inducible
enzymes, whose activity increases with the concentration of toxicant. The
activity of these enzymes was significantly (P<0.001) increased in gills, liver
and muscle for 90 days exposure period. Similar observations were reported by
Josephine Paulina (2003) in the sub-lethal concentrations of latex of Calotropis
gigantea and in Anabas testudineus exposed to monocrotophos (Santha
Kumar.,1998).
The results for the toxicity impact on the shape and size of erythrocytes
are presented in Fig.4.8. The control fishes showed ovoid RBC’s, size of 9 x 5
micron, with an oval shaped central nucleus. The blood smear of treated fishes
showed a number of morphological changes in the RBC. In DB6 treated fish,
the RBC appears less ovoid in shape and more towards rounded up shape. The
cells appear mildly enlarged than normal. The cytoplasm remains with well
defined outline. The nucleus was less elliptical and there was mild rounding
with no significant chromatin. In AO7 treated fish, apart from the above
observations, anisocytosis was seen. The nucleus was round, enlarged with open
chromatin. Also the RBC count were lower in treated fish compared to normal
and the decrease was highly significant (P<0.01).
Histopathology provides a rapid method to detect the effects of irritants
in various organs. Histological observation on gills showed that both the dyes
Toxicological impact of dyeToxicological impact of dyeToxicological impact of dyeToxicological impact of dyes on s on s on s on AnabasAnabasAnabasAnabas testudineus (Bloch)testudineus (Bloch)testudineus (Bloch)testudineus (Bloch) 115
caused remarkable pathological changes after 30 days of exposure. Since gills
are the important organ for respiration and osmoregulation, it is more vulnerable
to damage than any other tissue. The pathological conditions include necrosis in
lamellar area, swelling of secondary gill filaments and bulging of tips of
primary gill filaments. Most of these changes were noticed in fishes exposed to
different concentrations of pesticides like malathion (Singh and Sahai.,1984),
with latex (Preethakumari.,2004) and with biopesticide of Calotropis gigantean
(Bharathi., 2005). Hyperplasia of secondary lamellae was reported in the gills of
Puntius exposed to Khan River water with industrial sewage (Chauhan and
Pandey., 1987). Swollen nucleus and inflammatory alterations of lamellar
epithelium are due to accumulation of fluids on account of factors like increased
capillary permeability (Roberts., 1978) or lowered efficiency of the epithelial
cells in maintaining normal water balance (Skidmore and Towell., 1972). The
results are in agreement with the studies of Roy and Datta Munshi (1991) in
which fresh water carp Cirrhinus mrigala exposed to sublethal dose of
Malathion for 48 hrs. The effects like collapse of pillar cell system and rupture
of gill epithelium can cause the stagnation of lamellar blood flow which can
ultimately limit the respiratory capacity of the gills (Skidmore and
Towell.,1972).
The liver is the primary organ of metabolism, detoxification of
pollutants and its excretion. There are mechanisms to eliminate toxic materials
but elevated concentrations of pollutants results in cell damage. The dye water
caused alterations of the liver parenchyma and the histopathological lesions
observed were necrosis, focal heamorrhages and congestion in AO7 treated fish
whereas vascular congestion and focal cellular necrosis were observed in DB6
treated fishes. Similar studies showed that hypertrophy and vacuolisation
followed by necrosis and cirrhosis have been observed in hepatocytes of
116 Chapter 4 Chapter 4 Chapter 4 Chapter 4
Heteropneustes fossilis following treatment with malachite green (Srivastava et
al., 1998a). Exposure to this dye also causes severe damage to gills, resulting in
necrosis of lamellar cells and gill epithelium, as well as leucocyte infiltration in
rainbow trout (Gerundo et al., 1991) and Heteropneustes fossilis (Srivastava et
al., 1998b). Pyconosis of nuclei as reported by King (1962) in DDT exposed
trout and endosulfan treated A. Testudineus (Satheesh Kumar Reddy.,1994) is
noted in the present study. Also observations such as congestion of blood in
central vein, loss of orientation and parenchymal shrinkage are similar to that
described by Boyd (1949).
In the present study, SDS polyacrylamide gel electrophoresis was
performed for organs such as liver, gill and muscle of A. testudineus exposed to
AO7 and DB6 and compared to that of normal. The protein subunits of dye
exposed tissues showed decrease in intensity and some protein sub units were
disappeared. The proteins showed more increase in intensity in DB6 exposed
tissue samples than AO7. This indicates that DB6 alters the protein expression
and induces it and this may due to more toxicity of this dye when compared to
AO7. It may be due to the difference in chemical and structural properties of
these dyes. The variations in protein subunit band patterns may be due to
change in the turn over (synthesis /degradation) of various proteins. Marinovich
et al. (1994) found that Diazinon could induce inhibition of proteins in HL 60
cells at 24 hr exposure. The inhibition of proteins may be due to tissue necrosis
which leads to losses of intracellular enzymes or other proteins. Sherif et al.
(2009) observed slight reduction or decrease in intensity of proteins in Diazinon
treated fish Nile Tilapia, which indicates that these proteins were highly
affected by the stress caused by the pesticides. It is observed that the changes in
the protein banding are more pronounced in DB6 than AO7.
Toxicological impact of dyeToxicological impact of dyeToxicological impact of dyeToxicological impact of dyes on s on s on s on AnabasAnabasAnabasAnabas testudineus (Bloch)testudineus (Bloch)testudineus (Bloch)testudineus (Bloch) 117
The present study clearly revealed the toxic nature of the selected dyes
and the adverse effects on non-target organisms due to deterioration of water
quality. The selected fish Anabas testudineus, is a very hardy fish, which can
tolerate extreme conditions showing severe damage at behavioural,
biochemical, haematological and histopathological levels should be a warning
sign of the extent of pollution.