Toxicity of azodrin on the morphology and acetylcholinesterase
activity of the earthworm Eisenia foetida
J. Venkateswara Rao* and P. Kavitha
Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad 500-007, India
The acute toxicity of azodrin (monocrotophos, an organophosphorus insecticide) was determined on a soil organism, Eisenia
foetida. The median lethal concentrations (LC50) were derived from a 48-h paper contact test and from artificial soil tests. The LC50
of azodrin in the paper contact test was 0.4670.1mgcm?2(2376mgL?1) and those in the 7- and 14-day artificial soil tests were
171721 and 132720mgkg?1, respectively. The neurotoxic potentiality of azodrin was assessed by using a marker enzyme,
acetylcholinesterase (AChE; EC 184.108.40.206) in both in vitro and in vivo experiments. The progressive signs of morphological destruction
are correlated with percentage inhibition of AChE in the in vivo experiments. The kinetics of AChE activity in the presence and
absence of azodrin indicated that the toxicant is competitive in nature. This study demonstrated that azodrin causes concentration-
dependent changes in the morphology and AChE activity of the earthworm E. foetida.
Keywords: AChE; Eisenia foetida; Azodrin; Morphology; Earthworm
Extensive usage of organophosphorus (OP) com-
pounds in agriculture has resulted in a widespread
distribution in the environment. Earthworms play
important roles in agriculture. They are considered not
only biofertilizers and composting agents but also
nature’s ploughs, aerators, moisture retainers, crushers,
and biological agents (Eguchi et al., 1995). Vermicast-
ings have led to significant increases in the yields of
several crops, with significant reductions in pesticide use
and almost zero chemical fertilizer inputs (Dash and
Insecticide residues reach the soil in a variety of ways,
causing toxicity to earthworms (Paoletti et al., 1988;
Pizl, 1989). These residues enter the environment
through industrial and agricultural activities (Edwards
et al., 1992), reaching the earthworms from soil and
water (Connell and Markwell, 1990). Earthworms can
be used as bioindicators to detect pesticide contamina-
tion in agricultural soils (Stenersen, 1979a,b). OP
insecticides as neurotoxic agents are known to cause
acute toxic effects in earthworms (Scott-Fordsmand and
Weeks, 2000; Venkateswara Rao et al., 2003a) and in
other organisms (Venkateswara Rao et al., 2003b,c).
Signs and symptoms include excess of acetylcholine due
to inhibition of acetylcholinesterase (AChE; EC 220.127.116.11)
enzyme. AChE inhibition of different animals by many
OP insecticides is well established (Rao et al., 1991;
Yamin Hussian Quadri et al., 1994; Jain-Rang et al.,
1998). Certain cholinesterase-inhibiting insecticides have
been tested against earthworms under both laboratory
and field conditions (Stenersen et al., 1992; Vishwa-
nathan, 1997). Further study on the influence of OPs on
the kinetic properties of earthworm AChE is still
Azodrin is an OP insecticide extensively used in India
for agriculture purposes (Ray et al., 1985; Swamy,
1995). It reduced the survival rate of earthworms in
agricultural soils (Takaiyosus, 1977; Rajendra et al.,
1990). The present study assessed the acute toxic effects
of azodrin using paper contact and artificial soil test
methods under laboratory conditions with special
reference to the morphology and AChE activity of
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*Corresponding author. Fax: +91-40-2717-3387.
E-mail address: email@example.com (J.V. Rao).
2. Materials and methods
All reagents used in the present study were of
analytical grade and were used without any further
purification. Acetylthiocholine (ATC) iodide and 5,50-
dithiobis(2-nitrobenzoic acid) (DTNB) were purchased
from Sigma–Aldrich Chemical Co. (USA). Technical-
grade azodrin (purity o95%) was a gift from NOCIL,
The earthworms, Eisinea foetida, were purchased
from the Vermiculture Project, Kothapet Fruit Market,
Dilsukhnagar, Hyderabad, India. They were carefully
brought to the laboratory along with the moist soil
within 1h. Before testing, these worms were acclima-
tized for 7 days under laboratory conditions in feed
boxes (36?18?24 inches) containing a 4-inch layer of
uncontaminated red soil at the bottom (base soil), a thin
layer of leaves, 16 inches of meshed cow dung plus soft
soil (1:1), and a thin layer of dried grass on top (growth
medium). Wet gunny bags were placed as a cover on the
2.1. Determination of median lethal concentration
The acute toxicity experiments were conducted in two
ways, i.e., direct contact test through a filter paper
method (48h) and artificial soil test for 14-day exposure
(OECD, 1984). For the filter paper contact test, the sides
of flat-bottomed glass vials (8cm in length and 3cm in
diameter) were lined with Whatman filter paper No. 1
without overlapping (the circumference of the paper is
63cm2). The test chemical, azodrin, was dissolved in
water and predetermined amounts, 0.1, 0.3, 0.6, 1.2, and
2.5mgcm?2, were loaded onto the filter paper (1mL of
6.25, 18.75, 37.5, 75, and 125mgL?1solution, respec-
tively). The vial was rotated horizontally to ensure
uniform distribution of the toxicant. Controls were also
run in parallel with water alone. Prior to exposure,
earthworms were placed on moist filter paper for 3h to
adjust to the test environment under starvation. They
were then randomly divided into groups of 20 earth-
worms per treatment and were exposed (1 adult earth-
worm per vial; 9.5270.25cm in length and 0.7270.03g
in weight) to different concentrations of azodrin
The artificial soil test (using an evenly blended dry-
weight mixture of 68% No. 70-mesh silica sand, 20%
kaolin clay, and 10% sphagnum peat) was conducted
according to the OECD (1984). Different concentrations
of azodrin (100, 150, 200, and 250mgkg?1) were
homogeneously mixed with artificial soil. Briefly, 400,
600, 800, or 1000mg of azodrin was dissolved in
1400mL of distilled water and thoroughly mixed with
2.6kg of artificial soil (dry weight) to obtain 35%
moisture; pH was maintained at 6.070.5 by addition of
calcium carbonate (total weight of mixture=4kg). Each
test mixture (concentration) was divided into four equal
quantities of 1kg each (as determined by weight
including 35% moisture) which were placed into
separate 1.5-L earthen pots to make four replicates.
The test containers were covered with perforated plastic
film to prevent the test organisms from escaping the
earthen pots and to retain the moisture content in the
media for 14 days. The control media was the same
quantity of water without any additive agent. Testing
was done in continuous light at 2072?C.
Batches of 40 adult earthworms of approximately
equal length (9.5270.25cm) and weight (0.7270.03g)
were divided into four replicates of 10 earthworms. Each
batch was exposed to each concentration of azodrin,
plus a control. The behavioral and morphological
abnormalities and the percentage mortalities were
recorded after days 7 and 14 of exposure. The
concentration verses percentage mortality along with
the sample size were subjected to probit analysis
(Finney, 1953) for calculating the median lethal
concentration (LC50) of the test substance in both the
2.2. Acetylcholinesterase activity
Earthworms exposed in the artificial soil experiments
were used to estimate the AChE activity. The anterior
parts (six to seven segments) of four or five earthworms
exposed in the artificial soil experiments were dissected
and then homogenized (10% w/v) in 0.1M, pH 7.5,
phosphate buffer using a Potter–Elvehjam homogenizer
fitted with a Teflon pestle. The homogenates were
centrifuged at 10,000g for 10min and the resultant
supernatant was recentrifuged at 10,000g for 10min in a
Beckman table-top ultracentrifuge (TLX-361544). All
enzyme preparations were carried out at 4?C. The
supernatants stored on ice were used as the enzyme
sources for the estimation of AChE activity. Protein was
estimated by the method of Lowry et al. (1951). AChE
assays were performed spectrophotometrically utilizing
the method of Ellman et al. (1961).
The assay consists of 2.8mL of 0.1M phosphate
buffer, pH 7.2, 50mL of 0.16mM DTNB, 50mL of
protein, and 100mL of 0.2mM ACT iodide as sub-
strate. The reactions were performed at 37?C and were
initiated by adding the substrate (ACT iodide). The
measurement of the rate of production of thiocho-
line was accomplished by measurement of the contin-
uous reaction of the thiol with DTNB to produce
the yellow anion of 5-thio-2-nitrobenzoic acid. The
rate of color production was continuously recorded
for 6min at 412nm in a spectrophotometer (Spectra
MAX Plus; Molecular Devices; supported by SOFTmax
PRO-3.0). The activity was calculated as mmol/mg
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Similarly, the supernatants derived from unexposed
earthworms were used to study the in vitro evaluations
of AChE activity. The maximum velocity of the
substrate hydrolysis (Vmax) and the Michaelis–Menten
constant (Km) were estimated by the double-reciprocal
method of Lineweaver and Burk (1934) transforma-
tions, using various substrate concentrations (0.02,
0.023, 0.025, 0.030, 0.035, 0.040, 0.050, 0.065, 0.1, and
0.2mM), 0.16mM DTNB, and known amount of
protein in 3-mL assay volume. The effect of azodrin
and its mode of inhibition were assessed by adding
3.74?10?5, 7.47?10?5, 1.49?10?4, and 2.24?10?4M
azodrin along with the various substrate concentrations
to react with the enzyme. The Ki was determined
graphically from reciprocal plots made at different
inhibitor concentrations. The slopes of intercepts of
these lines were plotted against the inhibitor concentra-
tions (Dixon and Webb, 1965). Data are expressed as
mean7SE of three separate experiments, each assayed
The experiments were repeated three times in tripli-
cate and the data were analyzed by analysis of variance.
The individual means were compared using Duncan’s
test for multiple comparisons. A probability of?Po0:05
was selected as the criterion for statistical significance.
3. Results and discussion
The toxic effects of azodrin against earthworms were
recorded at 48h for the paper contact test and at days 7
and 14 of exposure for the artificial soil test. The median
lethal concentrations (LC50) were 0.4670.2mgcm?2
(2376mgL?1) for the paper contact test and 171721
and 132720mgkg?1respectively, for days 7 and 14 of
the artificial soil test (Table 1). The present study reveals
that lower concentrations are enough to cause 50%
mortality and similar morphological symptoms when
the length of exposure is increased from 7 to 14 days.
The earthworms showed progressive signs and symp-
toms of toxicity such as coiling, curling, and excessive
mucus secretion with sluggish movements and swelling
of clitellum at lower concentrations (0.1–0.6mgcm?2).
Extrusion of coelomic fluid resulting in bloody lesions
occurred atthe higher
2.5mgcm?2). Morphological changes such as constric-
tion and swelling started appearing in the anterior
regions of exposed worms (0.46mgcm?2) within 12h of
exposure and degenerative changes appeared at the
posterior end of the exposed earthworms after 48h of
exposure (Fig. 1B). This type of degeneration may
indicate a complete drain of utilizable levels of energy
reserves and subsequent autolysis of its own tissues to
meet its energy requirements. A similar kind of auto-
lysis from the posterior region was observed in earth-
worms, Polypheretima elongata, exposed to textile dyes
(Ramaswami and Subbram, 1992). Fifty percent of the
worms detached one or two of their posterior parts
during 48h of exposure (Fig. 1C).
Similar symptoms were also observed when earth-
worms were exposed to azodrin in the artificial soil test.
Numerous protrusions were observed on the anterior
parts of the earthworms exposed to X150mgkg?1
azodrin. The worms exposed to 150–250mgkg?1
azodrin remained at the bottom of the test container
(from day 1 onward), which could be due to the bulging
of clitellar regions and to protrusions that might have
restricted their free movement. Disappearances of
metameric segmentation and loss of pigmentation were
observed at high concentrations (200 and 250mgkg?1)
at day 7 that lead to fragmentation by day 14 of
exposure, whereas the control earthworms exhibited
excellent borrowing movements in the lower two-thirds
of the container and exhibited no other extraordinary
The in vivo AChE activity was estimated in earth-
worms treated with azodrin in artificial soil after 24h, 1
week, and 2 weeks of exposure. It is evident from Fig. 2
that Vmax (indicative of total enzyme activity) of
earthworm AChE was inhibited significantly in all the
test concentrations in a concentration-dependent man-
ner. The inhibition after 24h of treatment was 11% in
100mgkg?1, 17% in 150mgkg?1, 25% in 200mgkg?1,
and 31% in 250mgkg?1. The percentage inhibition of
AChE was further increased by day 7 of exposure, and
after day 14 was inhibited by 90% at the highest
concentration of azodrin.
In vitro AChE response to azodrin, the dissociation
constant of the enzyme–substrate complex defined as Km
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Fig. 1. (A–C) Morphological abnormalities in earthworms after 48h
of exposure (LC50concentration) to azodrin using the paper contact
Median lethal concentration of azodrin to the earthworm E. foetida in
the paper contact and artificial soil tests
Median lethal concentration
Paper contact method Artificial soil method
(Michaelis–Menten constant), was graphically deter-
mined by applying the Lineweaver–Burk plot of the
reciprocal substrate concentration (1=s) against the
reciprocal velocity (1=v) (Fig. 3). The Vmax and Km
values of AChE enzyme in control and each concentra-
tion of the toxicant were derived from their regression
equations, in which the 1/intercept value is the Vmaxand
the 1=intercept ? slope value is the Michaelis–Menten
The estimated Vmaxand apparent Kmvalues of AChE
of the control earthworms were 0.415 and 0.162mM,
respectively. The kinetic constants (Vmaxand apparent
Km) describing the hydrolysis of the ATC iodide
substrate by AChE of earthworms are presented in
Table 2. Regression of reciprocal plots yielded lines with
slopes corresponding to increasing inhibitor concentra-
tion. A common intersect of all the slopes at ordinate
and an increase in the Km indicates close structural
resemblance of inhibitor to substrate, thus enabling it to
compete for the active site of the enzyme, which
indicates that the inhibition by azodrin is competitive
The inhibition constant (Ki) was derived from the
double-reciprocal plots of Km=Vmax regressed against
inhibitor concentration (Dixon and Webb, 1965). The Ki
value of azodrin in moles is 4.74?10?5in earthworms
(Fig. 4). The potency of azodrin is probably due to the
effect of its high affinity to enzyme and the high
phosphorylation process during interactions between
AChE and inhibitor.
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-100 10 2030
1/ AChE Activity
(µ moles/min/mg protein]
Fig. 3. Lineweaver–Burk plots of AChE activity of earthworms in the
absence and presence of azodrin as a function of substrate concentra-
tion. —&—, control; —J—, 3.74?10?5; —n—, 7.47?10?5; —,—,
1.49?10?4; —B—, 2.24?10?4. Each point indicates the activity7SE
expressed in different concentrations of substrate used in the assay
Azodrin concentration x10-5
-10-505 1015 2025303540
Fig. 4. Slopes of Lineweaver–Burk plots at different concentrations of
azodrin (to determine inhibitory constant (Ki)).
Mean Percent AChE
Inhibition ± SE
100 mg kg-1
150 mg kg-1
200 mg kg-1
250 mg kg-1
Fig. 2. Percentage inhibition of AchE after exposure to azodrinat 1, 7,
and 14 days.
Kinetic constants of earthworm AChE activity in the presence of azodrin
Toxicant concentration (mol) InterceptSlopeApparent Km(1/intercept)?(slope)
Values in parentheses indicate % increase.
In this study E. foetida demonstrated that toxicity
increased with the length of exposure to azodrin. This
suggests that the toxicity is associated with accumula-
tion of azodrin in excess amounts and with inhibition of
AChE, which is injurious to the earthworms. The 48-h
paper contact test is more suitable for observing the
pathological changes that were apparent and reprodu-
cible than was the artificial soil test. The minute and
specific observations such as cuticular breakage, extru-
sion of coelomic fluid, and fragmentation are clearly
visible only with the paper contact test, although the
artificial soil test is close to reality but specific symptoms
are promptly visible in the 48-h paper contact test. The
kinetic plots for the inhibition of earthworm AChE in
the presence of increasing azodrin concentrations
indicate that the azodrin is a competitive inhibitor.
Based on the in vitro kinetic data it is proposed to do
similar evaluations on other OP and carbamate pesti-
cides, which are currently being used in the agricultural
system. The generated data will be helpful to assess the
impact of such chemicals on the population and toxicity
of earthworms. It is evident from the results that the
rapid and reliable paper contact method can be
employed initially (prior to the artificial soil test) to
assess the toxicity of the agricultural chemicals.
We are thankful to the Director of IICT for his
interest and encouragement throughout the study. The
junior author P. Kavitha is grateful to the Department
of Ocean Development for providing a fellowship.
Connell, D.W., Markwell, R.D., 1990. Bio-accumulation in the soil to
earthworm. Chemosphere 20, 91–100.
Dash, M.C., Senapathi, B.K., 1986. National Seminar on Organic
Waste Utilise, Vermi comp. Part-13 Proceedings, pp. 157–177.
Dixon, M., Webb, C.E., 1965. ENZYMES, 3rd Edition. Longman
Group Limited, New York.
Edwards, C.A., Edwards, W.M., Shipitalo, M.J., 1992. Earthworm
populations under conservation tillage and their effects on
transport of pesticides into groundwater. Proc. British Crop
Protection Conf. Pests and Diseases 7C (16), 859–864.
Eguchi, S., Hatano, R., Sakuma, T., 1995. Toshio Effect of earth-
worms on the decomposition of soil organic matter. Nippon Dojo-
Hiryogaku Zasshi 66 (2), 165–167.
Ellman, G.L., Courtney, K.D., Andres Jr., V.V., Featherstone, R.M.,
1961. A new and rapid colorimetric determination of acetylcholi-
nesterase activity. Biochem. Pharmacol. 7, 88–95.
Finney, D.J., 1953. Probit Analysis 2nd Edition. Cambridge University
Press, Cambridge, UK.
Jain-Rang, G., Rao, J.V., Gerald, E.W., Kun Yan Zhu, 1998.
Purification and kinetic analysis of acetylcholinesterase from
western corn rootworm, Diabrotica virgifera (Coleoptera: Chy-
somelidae). Arch. Insect. Biochem. Physiol. 39, 118–125.
Lineweaver, H., Burk, D., 1934. Determination of enzyme dissociation
constants. J. Ann. Chem. Soc. 56, 658–666.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951.
Protein measurement with folin phenol reagent. J. Biol. Chem. 193,
Organisation for Economic Co-operation and Development (OECD).
1984. OECD Guidelines for Testing of Chemicals, Earthworm,
Acute Toxicity Tests (Filter paper test and Artificial soil test),
No. 207, pp. 1–9.
Paoletti, M.G., Iovane, E., Cortese, M., 1988. Bioindicators and heavy
metal. Rev. Ecol. Biol. Sol. 25 (1), 33–58 (Dip. Biol., Univ. Padova
I-35131 Padua, Italy).
Pizl, V., 1989. Earthworms as bioindicators for soil contamination by
pesticide. Ekologiya 5, 86–88.
Rajendra, K., Rajesh, C., Gupta, Mirza, U.B., 1990. Toxicity
assessment of four insecticides to earthworm Pheretima postuma.
Bull. Environ. Contam. Toxicol. 45, 358–364.
Ramaswami, V., Subbram, V., 1992. Effect of selected textile dye on
the survival, morphology, and burrowing behavior of the earth-
worm Polypheretima elongata. Bull. Environ. Contam. Toxicol. 48,
Rao, J.V., Swamy, A.N., Yamin, S., 1991. In vitro brain acetylcho-
linesterase response among three inbred strains of mice to
monocrotophos. J. Environ. Sci. Health B 26 (4), 449–458.
Ray, P.K., Prasad, A.K., Nandan, R., 1985. Pesticides-major
environmental problems. Sci. Cult. 51, 363–370.
Scott-Fordsmand, J.J., Weeks, J.M., 2000. Biomarkers in earthworms.
Rev. Environ. Contam. Toxicol. 165, 117–159.
Stenersen, J., 1979a. Action of pesticides on earthworms. Part I: The
toxicity of cholinesterase-inhibiting insecticides to earthworms as
evaluated by laboratory tests. Pestic. Sci. 10, 66–74.
Stenersen, J., 1979b. Action of pesticides on earthworms. Part 3:
Inhibition and reactivation of cholinesterases in Eisenia foetida
(Savigny) after treatment with cholinesterase-inhibiting insecti-
cides. Pestic. Sci. 10, 113–122.
Stenersen, J., Brekke, E., Engelstad, F., 1992. Earthworms for toxicity
testing, species differences in response towards cholinesterase
inhibiting insecticides. Soil Biol. Biochem. 24 (12), 1295–1307.
Swamy, A., 1995. Modulation of haemotalogical and biochemical
parameters by new organophosphorus pesticides in fish. Thesis,
Osmania University, Hyderabad.
Takaiyosus, T., 1977. Effect of pesticides on soil organisms. Nippon
Dojo-Hiryogaku Zasshi 48 (3), 74–80.
Venkateswara Rao, J., Surya Pavan, Y., Madhavendra, S.S., 2003a.
Toxic effects of chlorpyrifos on survival, morphology and
acetylcholinesterase activity of the earthworm Eisenia foetida.
Ecotoxicol. Environ. Saf. 54, 296–301.
Venkateswara Rao, J., Shoba Rani, D., Kavitha, P., 2003b. Toxicity of
chlorpyrifos to the fish Oreochromis mossambicus. Bull. Environ.
Contam. Toxicol. 70, 985–992.
Venkateswara Rao, J., Shilpanjali, D., Kavitha, P., Madhavendra,
S.S., 2003c. Toxic effects of profenofos on tissue acetylcholinester-
ase and gill morphology in a euryhaline fish, Oreochromis
mossambicus. Arch. Toxicol. 77, 227–232.
Vishwanathan, R., 1997. Physiological basis of assessment of
ecotoxicology of pesticides to soil organisms. Chemosphere
35 (1–2), 323–334.
Yamin Hussian Quadri, Swamy, A.N., Rao, J.V., 1994. Species
difference in brain acetylcholinesterase response to monocrotophos
in vitro. Ecotoxicol. Environ. Saf. 28, 91–98.
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