Research Article NBU J. Anim. Sc.

11: 45-49 (2017)

ISSN 0975-1424

ACUTE TOXICITY OF CHLORPYRIFOS TO ZEBRAFISH,

DANIO RERIO (CYPRINIDAE)

Swati Singh1, Min Bahadur 1* Soumen Bhattacharjee2 and Joydeb Pal3

1
Genetics and Molecular Biology Lab, Department of Zoology, University of North Bengal,
P.O NBU, District Darjeeling, West Bengal
2
Cell and Molecular Biology Lab, Department of Zoology, University of North Bengal,
P.O NBU, District Darjeeling, West Bengal
3
Ecology and Toxicology Lab, Department of Zoology, University of North Bengal,
P.O NBU, District Darjeeling, West Bengal

Received: September 4, 2017 Revised & Accepted: November 17, 2017

ABSTRACT
Chlorpyrifos is a widely used organophosphate insecticide which causes toxic effects to non-target aquatic organisms,
especially fishes. In this study experiments were made in the laboratory to determine median lethal concentration (LC50)
of chlorpyrifos to Zebrafish, Danio rerio (Cyprinidae) and behavioural effects of the insecticide on the fish. The 96 hour
LC50 value of chlorpyrifos, determined from the mortality data using Probit analysis, was found as 289 µg/L for the
Zebrafish, D. rerio. The fish exhibited respiratory distress such as gasping in air, loss of balance and erratic swimming
behaviour prior to death. The results indicate that chlorpyrifos is extremely toxic to fish and should be used with extreme
caution.

Keywords: Zebrafish, toxicity, chlorpyrifos, Probit, behaviour, LC50

INTRODUCTION (AChE) inhibitor, and affects the nervous system

Pesticides are used extensively in
agriculture to control pests. After application, less
than 0.1% of the pesticides reach the specific target
organisms whilst the rest 99.9% enters the
environment (Pimentel, 1995) causing environmental
contamination and toxicity to non-target organisms,
both terrestrial and aquatic. Pesticides cause chronic
and acute fish poisoning and damage the vital organs
(Joshi and Sahu, 2007), reduce reproductive ability
(Sharma and Ansari, 2010), skeletal deformities
(Kumar and Ansari, 1984) and induce various
biochemical changes in fish.
Chlorpyrifos (CPF), an organophosphate
insecticide, is used worldwide for the control of
agricultural and non-agricultural insect pests (Lemus
and Abdelghani, 2000). It is an acetylcholine esterase
*Author for correspondence: Dr. Min Bahadur: bahadurmin@rediffmail.com / min.b@rediffmail.com
(Sun and Chen, 2008). This insecticide is useful for
the control of a wide range of important insects and
other arthropod pests. Chlorpyrifos, being a non-
systemic insecticide, is effective by ingestion,
inhalation and direct contact (Tomlin, 2006).
Chlorpyrifos is activated to its oxon metabolite by
cytochrome P450 to become toxic and also inhibits
the AChE activity (Barata et al., 2004). Half life of
chlorpyrifos is 25.6 days (water pH 7.0) (Shi et al.,
2000), 30 days (soil), 60 days (less alkaline soils)
and weeks or months (indoors) (Arcury et al.,
2007). Chlorpyrifos passes via air drift of surface
run-off into the surrounding waters and gets
accumulated in different aquatic organisms,
particularly fishes, adversely affecting them. The
occurrence of chlorpyrifos in freshwater bodies is
harmful to the fish and other non-target aquatic

45
 Swati Singh, Min Bahadur, Soumen Bhattacharjee and Joydeb Pal
organisms (Starner et al., 2005). Chlorpyrifos has
been reported to be used in the tea plantations of
the Terai and the Dooars region of the Darjeeling
foothills to curb the insect pest menace (Bishnu et
al., 2009, Singh et al., 2015). Water bodies have
been reported to be contaminated with this
insecticide with a residue level of 0.0091 ± 0.0020
ppm (Singh et al., 2015). Fish muscles of Puntius
sp. showed a concentration of chlorpyrifos as high
as 5.0371 ± 1.4236 ppm in river Deomoni of Terai
region of West Bengal (Singh et al., 2015).
Zebrafish is used as model organism in
various researches including toxicological research
(Spitsbergen and Kent, 2003). The International
Organization for Standardization and the
Organization for Economic Co-operation and
Development (OECD) recommends the fish for
toxicological researches (OECD, 1992) because
large clutch sizes, small body sizes reduce the
experimental costs.
Objectives of the present study were to
determine median lethal concentration (LC 50 ) of
chlorpyrifos to Zebrafish D. rerio and changes in
the behaviour of the fish exposed to the insecticide.
MATERIALS AND METHODS
Insecticide: The chlorpyrifos 20% EC was
purchased from the local agrochemicals supplier
(Dursban, Trade Mark of Dow AgroSciences LLC,
USA).
Test animal: Danio rerio having average body
weight of 289 mg and total body length 2.55 cm
was used in the present study. The fish specimens
were collected from local hatchery, stocked at
appropriate density in 20 litre glass aquaria
containing de-chlorinated tap water and were
acclimatized in this condition for at least 10 days
before they were subjected to investigations. The
fish were fed with commercial artificial feed equal
to 1/10th of their body weight. Renewal of water,
clearing of unconsumed feed and fecal wastes were
46
done daily by siphoning out 80% of water and refilling
with fresh water and the aquaria screens were also
cleaned daily (Sreedevi et.al, 2014). The test fishes
were starved for 24 hrs before the start of the
experiment (OECD, 1992).
Experimental procedure: Static bioassays were
conducted in the laboratory in a series of glass
aquaria of 20 litres capacity. Aquaria were filled
with water. A stock solution of the insecticide was
prepared by dissolving Dursban (Chlorpyrifos 20%
EC) in acetone. For the toxicity test, four
concentrations of Chlorpyrifos (100, 200, 300, 400
µg/L) previously diluted in acetone were used. Three
replicates of 8 fishes for each concentration of the
pesticide were used. Eight (8) acclimatized fishes
of nearly uniform size were stocked in each
aquarium as soon as the test material (chlorpyrifos)
was mixed. Each experiment was accompanied with
a control having the same volume of acetone but
without the pesticide. The acute toxicity assay was
performed following the guidelines of the
Organization for Economic Co-operation and
Development (Test no. 203, OECD, 1992) for
testing of chemicals. A fish was considered dead
when its gill movements ceased and it did not respond
to gentle prodding. Dead fish was removed from
the aquarium to avoid deterioration. The behaviour
of each test fish was observed and the mortality of
the Zebrafish was recorded for different exposure
periods, 24, 48, 72 and 96 hr at different
concentrations. The dead fishes were removed as
soon as they were seen.
Probit analysi: Data obtained in each experiment
were evaluated by the use of probit analysis
statistical method (Finney, 1964) in Microsoft Excel
2007.
RESULTS AND DISCUSSION
Behavioural study: Fishes showed abnormal
behaviour when exposed to the various
concentrations of chlorpyrifos. No abnormal
response was observed in the control group. Some
 Acute toxicity of chlorpyrifos to zebrafish
of the pesticide exposed fishes aggregated at the
corner of the aquarium, took rest at the bottom of
the aquarium laterally, frequently came to the surface
to gulp air, showed stronger opercular movements,
and occasionally turned upside down. They also
showed erratic and darting swimming behaviour and
lost their equilibrium compared to the controls. Their
body colour darkened, pectoral and pelvic fins got
expanded with frequent movement of fins and the
fish rolled vertically with erratic swimming prior to
death. Erratic/darting swimming behaviour and loss
of equilibrium after the pesticide exposure was likely
due to inhibition of Acetylcholine Esterase (AChE)
activity. Similar results have also been reported by
Kristen et al. (2009) in hybrid striped bass. Inhibition
of AChE activity is a typical characteristic of
organophosphate pesticides (Timchalk et al. 2002).
The observed behavioural responses in the present
study are in accordance with the previous reports
of Ismail et al. (2014).
LC50 and probit analysis: The 96 hr LC 50 of
Chlorpyrifos for the fish, Danio rerio was found to
be 289 µg/L. Fig. 1 shows the regression line
between the Probit kill of Danio rerio and log
concentration of Chlorpyrifos. Tiwari and Ansari
(2014) have reported the 24, 48, 72 and 96 hour
LC50 values for Chlorpyrifos in Zebrafish as 0.40,
0.29, 0.21 and 0.16 µg/L, respectively, which is very
less as compared to the present study. Reports by
Rao et al. (2005) to chlorpyrifos toxicity in
Oreochromis mossambicus and Gambusia affinis
by semi-static method was found to be 0.0259 mg/
L and 0.297 mg/L, respectively, which is comparable
to results of the present study. Jeon et al., 2016
reported LC50 value of 709.43µg/L for chlorpyrifos
in adult Zebrafish after 96 hr exposure which is much
higher as compared to results of the present study
(289 µg/L).
The LC50 values of chlorpyrifos vary with
species of fish. Johnson and Finley (1980) reported
96 hour LC50 value of chlorpyrifos as 0.280 mg/L
and 2.4 µg/l for channel catfish, Ictalurus punctatus
47
Fig.1. Regression line between the Probit kill of Danio
rerio and log concentration of Chlorpyriphos
and Lepomis michrochirus, respectively. In another
study conducted by Ali et al. (2008) the 96 hr LC50
values of chlorpyrifos for the freshwater fish,
Channa punctatus was found as 811.98 µg/L.
Similarly, Werner et al. (2008) have shown that the
96 hr LC50 value of chlorpyrifos for fathead minnow
(Pimephales promelas) was 311 µg/L. The LC50
value of chlorpyrifos to Cyprinus carpio was
reported as 0.16 mg/L and 0.58 mg/L by Ramesh
and Munniswamy (2009) and Xing et al. (2011)
respectively. The 96 hr LC50 values of chlorpyrifos
in juvenile and adult of Oreochromis niloticus were
determined as 98.67 µg/L and 154.01 µg/L,
respectively, which reveal that chlorpyrifos can be
rated as highly toxic to fish (Oruc, 2010). The 96 hr
LC50 value of chlorpyrifos to Poecila reticulata was
found as 0.176 ppm/L (Sharbidre et al., 2011). In a
study conducted by Ismail et al. (2014) the acute
LC50 (96hr) value of Chlorpyrifos for Rohu (Labeo
rohita) was found as 442.8 µg/L. Such variations in
the LC50 values among different fish species may
be attributed to the different behaviour of the species
due to differences in their genetic makeup that may
have a role in influencing the ability to detoxify certain
chemicals. This may also be due to the resistance
of different fish species to the pesticide and water
quality parameters. It can be concluded that
chlorpyrifos and other organophosphate pesticides
are highly toxic to fish species including Zebrafish.
CONCLUSION
The behavioural changes observed in the
Zebrafish due to exposure to the chlorpyrifos indicate
 Swati Singh, Min Bahadur, Soumen Bhattacharjee and Joydeb Pal
that Zebrafish is highly sensitive to chlorpyrifos. The
96hr LC50 value of chlorpyrifos to zebrafish also
indicates that chlorpyrifos is highly toxic to fish and
its application in the agricultural fields should be
made judiciously so that non-target organisms are
protected from the hazards of the insecticide.
ACKNOWLEDGEMENTS
Authors are thankful to the Head,
Department of Zoology for providing the
Departmental Central Instrument facility which is
supported by the Fund for Improvement of Science
and Technology Infrastructure programme (FIST),
Department of Science and Technology, New Delhi,
India and the Special Assistance Programme (SAP)
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