Anabas testudineus, Channa punctatus and Barbodes gonionotus were exposed to 5.62, 6.25, 6.87, 7.50, 8.12 and 8.75 ppm; 1.13, 2.26, 3.39, 4.52, 5.65 and 6.78 ppm; and 2.00, 2.50, 3.00, 3.50, 4.00 and 4.50 ppm of Diazinon 60 EC, respectively. The median lethal concentration (LC50) values of Diazinon 60 EC on A. testudineus, C. punctatus and B. gonionotus were 6.55, 3.09 and 2.72 ppm for 96 hrs of exposure. The fish species showed several abnormal behaviors which included restlessness, arena movements, loss of equilibrium, increased opercular activities, strong spasm, paralysis and sudden quick movements during the exposure. For histopathological studies, A. testudineus, C. punctatus and B. gonionotus were exposed for 7 days to sublethal concentrations of 1.13 and 3.75 ppm; 1.13 and 2.26 ppm; and 1.13 and 2.26 ppm of Diazinon 60 EC, respectively. Hypertrophy, necrosis and pyknosis of hepatocytes, pyknosis and degenerative changes such as necrosis of tubular and haematopoietic cells of kidney were the major histopathological effects

Ahmed, G.U.

Hossain, Z.

Mollah, M.F.A.

Rahman, M.Z.

English

Aquatic Commons

Naga, The ICLARM Quarterly (Vol. 25, No. 2) April-June 20028
Effect of Diazinon 60 EC on
Anabas testudineus, Channa punctatus and
Barbodes gonionotus
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Anabas testudineus, Channa punctatus and Barbodes gonionotus were exposed to 5.62, 6.25, 6.87, 7.50,
8.12 and 8.75 ppm; 1.13, 2.26, 3.39, 4.52, 5.65 and 6.78 ppm;  and 2.00, 2.50, 3.00, 3.50, 4.00 and 4.50 ppm of
Diazinon 60 EC, respectively. The median lethal concentration (LC50) values of Diazinon 60 EC on A. testudineus,
C. punctatus and B. gonionotus were 6.55, 3.09 and 2.72 ppm for 96 hrs of exposure. The fish species showed
several abnormal behaviors which included restlessness, arena movements, loss of equilibrium, increased
opercular activities, strong spasm, paralysis and sudden quick movements during the exposure. For
histopathological studies, A. testudineus, C. punctatus and B. gonionotus were exposed for 7 days to sublethal
concentrations of 1.13 and 3.75 ppm; 1.13 and 2.26 ppm; and 1.13 and 2.26 ppm of Diazinon 60 EC, respectively.
Hypertrophy, necrosis and pyknosis of hepatocytes, pyknosis and degenerative changes such as necrosis of
tubular and haematopoietic cells of kidney were the major histopathological effects.
Aquaculture Section
of the Network of Tropical Aquaculture
and Fisheries Professionals (NTAFP)Editor: M.V. Gupta
AQUABYTE
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Pesticides are useful tools in
agriculture and forestry, but their
contribution to the gradual
degradation of the aquatic
ecosystem cannot be ignored
(Konar 1975, Basak and Konar
1976, 1977). The aquatic ecosystem
as a greater part of the natural
environment is also faced with the
threat  of a shrinking genetic base
and biodiversity. Anabas
testudineus, Channa punctatus
and other indigenous small fishes use
paddy fields as breeding and nursery
grounds. Barbodes gonionotus is
an important species for integrated
rice-fish farming.  Pesticides at high
concentrations are known to reduce
the survival, growth and reproduction
of fish (Mckim et al. 1975) and
produce many visible effects on fish
(Johnson 1968).
Due to the residual effects of
pesticides, important organs like the
kidney, liver, gills, stomach, brain,
muscles and genital organs are
damaged. Until the use of pesticides
in crop farming is replaced by other
means of pest control such as
integrated pest management, less
toxic pesticides at lowest possible
doses need to be recommended.
Organophosphorus pesticides vary
in their toxicity to different species.
Very limited work has been done on
the histo-pathological effects of
pesticides on fishes. This study
determines the median lethal
concentration (LC50) values and
behavioral impacts of the pesticide
Diazinon 60 EC on the three fish
species mentioned. The histo-
pathological state of the liver and
kidney of tested fish species at
sublethal concentration for 7 days
exposure time was also studied.
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A. testudineus, averaging 6.12 ±
0.258 cm and 4.369 ± 0.452 g; C.
punctatus, averaging 10.20 ± 0.506
cm and 11.722 ± 1.208 g  and B.
gonionotus averaging 2.82 ± 0.166
cm and 0.240 ± 0.046 g were used
in the study.
Twenty-one glass aquaria of size
60 cm x 30 cm x 30 cm with 50 liters
of tap water were used in the
experiments. Initially, range finding
tests were performed. It was
observed that none of the A.
testudineus, C. punctatus and B.
gonionotus died when exposed to
concentrations below 5.625, 1.13
and 2.00 ppm and complete mortality
was observed at concentrations of
8.750, 6.78 and 4.50 ppm,
respectively. Based on this, six
concentrations of the pesticide were
tested for each of the species. The
Naga, The ICLARM Quarterly (Vol. 25, No. 2) April-June 2002 9
different concentrations of Diazinon
60 EC for A. testudineus,
C. punctatus and B. gonionotus
were 5.62, 6.25, 6.87, 7.50, 8.12 and
8.75 ppm; 1.13, 2.26, 3.39, 4.52, 5.65
and 6.78 ppm; and 2.00, 2.50, 3.00,
3.50, 4.00 and 4.50 ppm,
respectively, with three replications
including a control. Ten acclimated
fish were released in each aquarium
containing different concentrations
of pesticide as well as in the control.
All tests were done at room
temperature. The behavior and
other external changes in the body
of fishes were observed. Dead
fishes were removed and mortality
was recorded at 6, 12, 24, 48, 72 and
96 hrs of exposure time.
Temperature, dissolved oxygen and
pH were recorded daily. The LC
50
values for different fish species were
calculated for 96 hours of exposure
time by probit analysis.
For histopathological investiga-
tions, the fish were exposed to
various sublethal concentrations: 1.13
and 3.75 ppm for A. testudineus, 1.13
and 2.26 ppm for C. punctatus and
1.13 and 2.26 ppm for
B. gonionotus for a period of 7 days
with three replications for each.
At the end of 7-day exposure
period, liver and kidney samples (1
cm3) were collected from the three
fish species from each
concentration and preserved in small
plastic vials with ten times 10%
neutral buffer formalin fixatives. The
numbers at section of samples was
prepared using a microtome, stained
and studied under a microscope.
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General effects
The effects of different concen-
trations and exposure time of
Diazinon 60 EC on the three species
are presented in Tables 1, 2 and 3.
The LC
50
 values based on  probit
analysis were found to be 6.55, 3.09
and 2.72 ppm for A. testudineus, C.
punctatus and B.  gonionotus for
96 hrs of exposure, respectively.
Abnormal behavior such as
restlessness, sudden quick
movement, rolling movements,
swimming on the back (at higher
doses) were observed when the
media started to act on test species.
The affected fish became very
weak, settled at the bottom and died
in increasing numbers at the higher
doses. Normal color and behavior
were observed in the control groups.
However, the color became pale
progressively with higher doses at
the end of 96 hours of exposure time.
The average dissolved oxygen
was higher at lower concentrations.
However, the water quality
parameters of temperature,
dissolved oxygen and pH varied little
in different treatments.
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Liver
The hepatocytes and other cells
of the liver in control groups were
normal and systematically arranged.
Hypertrophy of hepatocytes,
mild necrosis and minor vacuolation
were found at the dose of 1.13 ppm
for A. testudineus. At the dose of
3.75 ppm, severe necrotic
hepatocytes, pyknosis, hypertrophy,
haemorrhage and vacuolation were
observed for the same fish (Fig. 1).
Hypertrophy, minor vacuolation
and haemorrhage were recorded
at the dose of 1.13 ppm for
C. punctatus while at a dose of 2.26
ppm severe necrosis and
hypertrophy occurred resulting in
disappearance of liver cells and
vacuolation. Haemorrhagic areas
were also noticed (Fig. 2).
Liver tissue from B. gonionotus
Concentration Cumulative mortality  %
(ppm) 6 h 12 h 24 h 48 h 72 h 96 h
Control 00 00 00 00 00 00
1.13 00 00 00 00 00 00
2.26 00 00 00 00 17 27
3.39 00 00 00 03 23 57
4.52 00 03 03 20 53 80
5.65 00 10 13 33 67 93
6.78 07 20 23 50 70 97
Table 2. Cumulative mortality of Channa punctatus at Diazinon 60 EC treatments during the
experimental period.
Table 1. Cumulative mortality of Anabas testudineus at Diazinon 60 EC treatments during the
experimental period.
Concentration Cumulative mortality  %
(ppm)
6 h 12 h 24 h 48 h 72 h 96 h
Control 00 00 00 00 00 00
5.625 00 00 00 03 13 17
6.250 00 03 17 23 33 37
6.875 00 13 30 40 53 63
7.500 00 13 23 43 63 77
8.125 10 17 27 47 73 93
8.750 43 53 60 70 83 100
Concentration Cumulative mortality  %
(ppm) 6 h 12 h 24 h 48 h 72 h 96 h
Control 00 00 00 00 00 00
2.00 00 00 00 00 03 17
2.50 00 00 03 10 23 37
3.00 00 03 07 23 43 63
3.50 00 03 10 17 47 77
4.00 03 10 27 50 70 90
4.50 10 10 40 73 80 100
Table 3.  Cumulative mortality of Barbodes gonionotus to Diazinon 60 EC treatments during
the experimental period.
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Naga, The ICLARM Quarterly (Vol. 25, No. 2) April-June 200210
treated with 1.13 ppm showed
necrosis, hypertrophy and
vacuolation. Marked hypertrophy,
necrotic hepatocytes and pyknotic
cells resulting in empty spaces at the
dose of 2.26 ppm were observed for
the mentioned fish species (Fig. 3).
Kidney
Kidney tubules and haemato-
poietic cells were normal and
systematically arranged in the control
treatment for all the three tested fish
species.
Kidney tissue from A.
testudineus exposed to 1.13 ppm
showed necrosis, degenerated
kidney tubules and pyknosis having
haemorrhagic areas. Degeneration
of kidney tubules and haemato-
poietic cells, necrosis, pyknosis and
haemorrhage were also recorded at
the dose of 3.75 ppm for the fish
species (Fig. 4).
Kidney tissue from C. punctatus
exposed to 1.13 ppm  showed mild
necrosis, pyknosis and tubular
degeneration (Fig. 5). Severe
necrosis, vacuolation and tubular
degeneration were recorded at the
dose of 2.26 ppm.
At the dose of 1.13 ppm, mild
necrosis, pyknosis and melano-
genesis having inflammatory cells
were recorded for B. gonionotus.
Severe degenerative changes such
as necrosis of tubular and
haematopoietic cells, pyknosis and
vacuolation were observed at the
dose of 2.26 ppm (Fig. 6).
The mean water temperature,
dissolved oxygen and  pH of
different treatments including
controls during the experimental
period for histological study were
27.85 ± 0.76ºC, 4.98 ± 0.88 ppm and
7.82 ± 0.68, respectively. Fig. 6. Photomicrography of kidney of B.
gonionotus after 7 days exposure to 2.26
ppm Diazinon 60EC. Severe necrosis (N),
pyknosis (P) and vacuolation were marked.
Fig. 3. Photomicrography of liver of B.
gonionotus  after 7 days exposure to 2.26
ppm Diazinon 60EC. Marked hypertrophy
(Hy), necrotic hepatocytes and pyknotic cells
(P) resulted some empty spaces.
Fig. 2. Photomicrography of liver of C.
punctatus after 7 days exposure to 2.26 ppm
Diazinon 60EC. Hypertrophy (Hy), severe
necrosis (N) of hepatocytes, pyknosis (P),
hypertrophy (Hy) and haemorrhagic (H)
areas were observed.
Fig. 5. Photomicrography of kidney of C.
punctatus after 7 days exposure to 1.13 ppm
Diazinon 60EC. Mild necrosis (N), pyknosis
(P) and tubular degeneration (Kt) were
observed.
Fig. 4. Photomicrography of kidney of A.
testudineus after 7 days exposure to 3.75
ppm Diazinon 60EC. Degenerated kidney
tubules (Kt), necrosis (N), pyknosis (P) and
haemorrhage (H) were observed.
Fig. 1. Photomicrography of liver of A.
testudineus after 7 days exposure to 3.75
ppm Diazinon 60EC. Severe necrosis (N) of
hepatocytes, pyknosis (P), hypertrophy (Hy),
haemorrhage (H) and vacuolation (V) were
observed.
Naga, The ICLARM Quarterly (Vol. 25, No. 2) April-June 2002 11
C,'0%'',/1
The LC
50
 values for 96 hours
were 6.55, 3.09 and 2.72 ppm for A.
testudineus, C. punctatus and B.
gonionotus, respectively. The LC
50
value in the present study for C.
punctatus is similar to the findings
of Haider and Inbaraj (1986), who
used formulations of Malathion. Rao
et al. (1985) reported the LC50 value
of Elson for C. punctatus to be 0.43
ppm for 48 hrs of exposure. Hoque
et al. (1993) observed the LC50
value to be 3.67 ppm at 96 hours
when B. gonionotus of 12.7 cm in
length were exposed to Diazinon.
By taking into account the size of
the present experimental fish (2.82
± 0.166 cm), the LC50 value is close
to the result obtained by Hoque et
al. (1993). Al-Arabi et al. (1992)
reported the LC50 value for 96 hours
of exposure to Diazinon for the
fingerlings of Labeo calbasu to be
1.54 ppm which is almost close to
present findings, while the LC
50
value of 1.002 ppm reported by Alam
et al. (1995) was not comparable
with that of the present work. In both
cases, however the test fishes were
of different sizes.
Several abnormal behaviors such
as restlessness, loss of equilibrium,
increased opercular activities,
surface to bottom movement,
sudden quick movement, resting at
the bottom, etc. were similar to the
observations of Hoque et al. (1993)
and Lovely (1998). However,
swelling in the abdominal region and
gas-filled stomach were not
observed, which is contrary to the
findings of Kabir and Begum (1978)
and Lovely (1998). It is an indication
that the effect of pesticides is
species-specific.
Temperature, hardness, pH,
alkalinity, sex, age and other
physiological status of the test
animals have profound effects on
the toxicity of agro-chemicals (Boyd
1979, Rand and Petrocelli 1985).
Meletev et al. (1971) reported that
pesticides affect the gas exchange
of fish and other aquatic organisms.
This might be one of the probable
reasons for the decline of oxygen
concentrations in the lower to higher
concentration test media during the
present work. As the recorded
parameters had little variation, it is
evident that physical and chemical
properties of aquarium water were
within the desirable range of fish
culture (Boyd 1979).
At the agricultural dose of 1.13
ppm, mild pyknotic and necrotic
hepatocytes, hypertophy and in a
few cases vacuolation were
recorded for all the fish species. At
the highest doses (3.75 ppm  for A.
testudineus, 2.26 ppm for both C.
punctatus and B. gonionotus),
degenerative changes of
hepatocytes and in some samples
severe degenerative changes like
necrosis, pyknosis, vacuolation,
rupture of blood vessel causing
haemorrhage were recorded for all
the fish species. Kabir and Begum
(1978) reported cytoplasmic
degeneration, pyknotic nuclei in liver
tissues; vacuolation in hepatic cells
and rupture of blood vessels;
degenerative hepatic cells and
necrotic nuclei when Heteroneutes
fossilis was exposed for 25 days to
5, 10 and 20 ppm Diazinon,
respectively. Shastry and Sharma
(1979) exposed C. punctatus to a
sublethal concentration (0.01 mg/l)
of Endrin and observed hypertrophy
of hepatic cells and liver cord
disarray, vacuolation of cytoplasm
and necrosis, rupture of hepatic cell
membrane and necrotic centrolo-
bular area.
At the agricultural dose of 1.13
ppm, mild tubular degeneration,
necrosis, pyknosis and haemorrhage
were observed in cells of kidney. At
the highest dose (3.75 ppm for A.
testudineus and 2.26 for both
C. punctatus and B. gonionotus),
degeneration of kidney tubules and
haematopoietic cells, necrosis,
pyknosis and haemorrhage were
recorded. Rand and Petrocelli (1985)
reported necrosis of tubular and
surrounding haematopoietic cells,
nuclear changes of pyknosis and
karyorrhexis of kidney tissue when
coho salmon was exposed to 100
ppm Amitrole for 144 hours.
>"9"&"10"'
Al-Arabi, S.A.M., M.A. Mazid and
M.G.M. Alam. 1992. Toxicity of
Diazinon to three species of Indian major
carps.  Bangladesh J. Tran. Dev. 5(1):
79-86.
Alam, M.G.M., S.A.M Al-Arabi, G. C.
Haldar and M.A. Mazid. 1995. Toxicity
of  Diazinon to the fry of Indian major
carp (Cirrhinus mrigala, Hamilton).
Bangladesh J. Zool. 23(2): 183-186.
Basak, P.K. and S.K. Konar. 1976. Pollution
of water by pesticides and protection
of  fishes: parathion. Proc. Nat. Acad.
Sci. 46B: 382-392.
Basak, P.K. and S.K. Konar. 1977.
Estimation of safe concentration of
insecticides, a new method tested on
DDT and BHC.  J. Inl. Fish. Soc., India
9: 9-29.
Boyd, C.E. 1979. Water quality in warm
water fish ponds. Alabama Agricultural
Research Station, Alabama. 362 p.
Haider, S. and R.M. Inbaraj. 1986. Relative
toxicity of technical material and
commercial formulation of Malathion
and Endosulfan to a fresh water fish,
Channa punctatus (Bloch).  Ecotoxicol.
Environ. Saf. 11(3): 347-351.
Hoque, M.M., M.J.A. Mirja and M.S.
Miah. 1993. Toxicity of Diazinon and
Sumithion to Puntius gonionotus.
Bangladesh J. Tran. Dev. 6(1): 19-26.
Johnson, D.W. 1968. Pesticides and Fishes
a review of selected literature.  Trans.
Am. Fish. Soc. 97: 398-424.
Kabir, S.M.H. and R. Begum. 1978. Toxicity
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Konar, S.K. 1975. Pesticides and aquatic
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80-85.
Lovely, F. 1998. Toxicity of three commonly
used organophosphorus insecticides to
Thai sharputi (Barbodes gonionotus)
and African catfish (Clarias gariepinus)
fry. Department of Fisheries Biology
and Genetics, Bangladesh Agricultural
University, Mymensingh, Bangladesh.
83 p. M.S. thesis.
Mckim, J.M., D.A. Benoit, K.K. Biesinger,
W.A. Brungs and R.E. Siefert. 1975.
Effects of pollution on fresh water fish.
J. Wat. Pollut. Contr. Fed. 47: 1711-
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Meletev, V.V., A.S. Kanaev and N.G.
Dzasokhova. 1971. Water toxicology.
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Fundamentals of aquatic toxicology.
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Rao, K.R., S. Sumbasiva, K.S. Basu and K.V.
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the Indian snakehead Channa punctatus.
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M.Z. Rahman, Z. Hossain and M.F.A.
Mollah are from the Department of
Fisheries Biology and Genetics, Bangladesh
Agricultural University, Mymensingh-2202,
Bangladesh. G.U. Ahmed is from the
Department of Aquaculture, Bangladesh
Agricultural University, Mymensingh-2202,
Bangladesh.


Effect of diazinon 60 EC on Anabas testudineus, Channa punctatus and Barbodes gonionotus

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Effect of diazinon 60 EC on Anabas testudineus, Channa punctatus and Barbodes gonionotus

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