Chapter -IV Toxicological impact of dyes on Anabas ...shodhganga.inflibnet.ac.in/bitstream/10603/28477/15/15_chapter4.pdf · Then the slide was washed with distilled water. The stained

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

92 Chapter 4 Chapter 4 Chapter 4 Chapter 4

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 ±


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,


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,


Fig 4.3: Anabas testudineus

Fig 4.4: Acclimatisation of fishes used for study

Fig 4.5: Fishes maintained for LC50 study.


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


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


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.


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.


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


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


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


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.


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


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


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



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.


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


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)


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.


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


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


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).


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


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


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.


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.

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