Toxic effects of chlorpyrifos on morphology and acetylcholinesterase activity in the earthworm, Eisenia foetida

Toxic effects of chlorpyrifos on morphology and acetylcholinesterase activity in the earthworm, Eisenia foetida

Ecotoxicology and Environmental Safety 54 (2003) 296–301 Toxic effects of chlorpyrifos on morphology and acetylcholinesterase activity in the earthwo...

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Ecotoxicology and Environmental Safety 54 (2003) 296–301

Toxic effects of chlorpyrifos on morphology and acetylcholinesterase activity in the earthworm, Eisenia foetida J. Venkateswara Rao,a, Y. Surya Pavan,a and S.S. Madhavendrab a

Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, India b Electron Microscopy Center, Indian Institute of Chemical Technology, Hyderabad 500 007, India Received 6 February 2001; accepted 15 April 2002

Abstract The acute toxicity of chlorpyrifos (O, O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) was determined in the earthworm, Eisenia foetida. A 48-h contact test as described by the Organization for Economic Cooperation and Development (OECD) guideline 207 was carried out. The LC50 of chlorpyrifos was 0.063 mg/cm2. Inhibition of acetylcholinesterase (AChE: EC activity indicated by in vitro neurotoxic potentiality revealed competitive inhibition and altered Km values widely in a dosedependent manner. The Ki value of chlorpyrifos was 4.20  10 6 M. AChE activity of LC50-exposed worms was 62%, 79%, 85%, and 91% inhibited at 12, 24, 36, and 48 h, respectively. Scanning electron microscopic studies revealed the morphological abnormalities in the worms. The present study demonstrates a dose- and time-dependent exposure of chlorpyrifos through skin results, morphological abnormalities, and inhibition of AChE in the earthworm, E. foetida. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Earthworm; Eisenia; Chlorpyrifos; Organization for Economic Cooperation and Development; Acetylcholinesterase; Scanning electron microscopy; Behavior morphology

1. Introduction Organophosphorus insecticides are increasingly used in agriculture as a substitute for organochlorine and carbamate insecticides because of their high efficiency and lower persistence in the environment. Extensive use of organophosphorus compounds for agriculture has resulted in their widespread distribution in the environment. Pesticides either are directly applied to soil to control soilborne pests or are deposited on soil as runoff from foliar applications. Earthworms are one of the most important groups of organisms, considered not only as a biofertilizer and composting agent but at the same time nature’s plough, aerator, moisture retainer, crusher, and biological agent (Eguchi et al., 1995). Earthworms play such an important role that vermicastings have led to significant increases in the yields of several crops, with a significant reduction in pesticide use and almost zero chemical 

Corresponding author. Fax: +91-40-717-3387. E-mail address: [email protected] (J. Venkateswara Rao).

fertilizer input (Dash and Senapathi, 1986). Soil status, nutrients, temperature, moisture, season, and the presence of mixed fertilizers and pesticides influence the population of earthworms (Bhaskaram, 1986; Morgan, 1993; Vishwanathan, 1997). Pesticide and heavy metal poisoning and other disturbances in the natural habitat of earthworms can lead to an ecological imbalance (Laird and Kroger, 1981) and whatever the source, earthworms are exposed to pesticides either through skin contact or by feeding on contaminated litter in soil. Although many toxicity studies have been conducted the fact remains that only a few pesticides and heavy metals in use have been tested against relatively few earthworm species both in laboratory tests and under field conditions (Lebrun et al., 1981; Cathay, 1982; Karnak and Hamelink, 1982; Lofs-Holmin, 1982; Kalaiselvan et al., 1996; Chang et al., 1997; Vishwanathan, 1997; Reinecke Sophie and Reinecke, 1997; Helling et al., 2000). Chlorpyrifos, O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate insecticide, was commercially used in India for more than a decade, particularly to control foliar insects on cotton, paddy fields, pasture

0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. PII: S 0 1 4 7 - 6 5 1 3 ( 0 2 ) 0 0 0 1 3 - 1

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and vegetable crops. Hence, a study was undertaken to evaluate the toxicity of chlorpyrifos to earthworms. For the present work the authors studied the effect of chlorpyrifos on the kinetics of acetylcholinesterase (AChE) and on the morphology by electron microscopy, of the earthworm, Eisenia foetida.

2. Materials and methods All reagents used in the present study were of analytical grade and used without further purification. Chlorpyrifos (purity 498%) was obtained from the Organic Chemistry Division of our laboratory. The earthworms, Eisinea foetida, were purchased from the Vermiculture Project, Kothapet fruit Market, Dilshuknagar, Hyderabad 500 060, India. They were carefully brought to the laboratory within an hour along with the moist soil. Before testing, these worms were acclimated for 7 days under laboratory conditions in feed boxes (36  18  24 in.) containing different 4-in. layers of uncontaminated red soil at the bottom (base soil), a thin layer of leaves, 16 in. of meshed cow dung plus soft soil in a 1:1 ratio, and a thin layer of dried grass on top (growth medium). Wet gunny bags were placed as a cover on the feed boxes. 2.1. Determination of median lethal concentration (LC50) The experiments were conducted by the paper contact toxicity method (OECD Guideline 207). Briefly, the sides of flat-bottom glass vials 8 cm in length and 3 cm in diameter were lined with Whatman filter paper No. 1 without overlapping. The test chemical was dissolved in acetone solvent, the predetermined concentrations were loaded on filter paper with 1 mL of solution, and vial was rotated for uniform distribution of the toxicant. Treated paper was allowed to dry by using a slow stream of compressed air. Controls were also run in parallel with the carrier solvent alone. After drying, 1 mL of deionized water was added to each vial. The acclimated worms (fasted 3 h, on moist filter paper) were washed, dried, and randomly divided into groups of 20 each. They were exposed (one earthworm per vial) to different concentrations, i.e., 0.0158, 0.0317, 0.0476, 0.0634, and 0.0793 mg/cm2, which are equivalent to 1–5 ppm. A record was maintained of the mortality of the worms exposed for 24 and 48 h to each concentration of pesticide and the data were used to estimate the median lethal concentration (LC50) of the test substance using a computer program developed by Reddy et al. (1992), based on the method of Finney (1953). In a separate set of experiments, 3-h fasted test worms were divided into four replicates of 20 each and exposed to LC50’s (OECD Guideline 207). For analysis and an


understanding of the nature of the action of this compound, the worms surviving exposure to LC50’s were selected for biochemical estimations and electron microscopic studies. 2.2. AChE activity Four or five worms from each replicate were chosen and the first six to seven segments separated to estimate AChE activity (AChE, EC They were homogenized (10% w/v) in 0.1 M, phosphate buffer, pH 7.5, using a Potter–Elvehjam homogenizer fitted with a Teflon pestle. The homogenates were centrifuged at 10,000g for 10 min and the supernatant was further centrifuged at 10,000g for 10 min in Beckman tabletop ultra centrifuge. All enzyme preparations were carried out at 41C. The resultant supernatants stored on ice were used as the enzyme source for estimation of AChE activity. Protein was estimated by the method of Lowry et al. (1951). AChE was assayed as described by Ellman et al. (1961). Inhibition of AChE in the LC50-treated worms was monitored after 24 and 48 h of exposure. Similarly, the normal earthworms were used to study the in vitro evaluations of AChE activity. Enzyme activity was determined graphically using double-reciprocal plots of Lineweaver and Burk (1934) transformations. The experiments were repeated three times and the data were analyzed by analysis of variance. The individual means were compared using Duncan’s test for multiple comparisons. Po0:05 was selected as the criterion for statistical significance. 2.3. Electron microscopy studies Samples are fixed in 4% glutaraldehyde in phosphate buffer (pH 6.9, 0.02 M) for 1 h at 41C, washed with phosphate buffer followed by distilled water, passed through an alcohol series (upgrade, 10–100%), air-dried, and mounted on alumina stubs using double-adhesive tape. Samples were coated with gold in Hitachi HUS-5Gb vacuum evaporator and observed in a Hitachi S-520 scanning electron microscope.

3. Results The effect of chlorpyrifos was concentration dependent and the percentage survival decreased with increasing concentration of pesticide. Mortality was recorded at all test concentrations after 24 and 48 h of exposure by the skin contact method. The LC50’s were 0.04770.006 and 0.03770.006 mg/cm2 for 24 and 48 h, respectively (Table 1), which are equivalent to 2.9670.39 and 2.3370.39 ppm.


J. Venkateswara Rao et al. / Ecotoxicology and Environmental Safety 54 (2003) 296–301

Table 1 LC50 values of chlorpyrifos for 24 and 48 h exposure of earthworms determined by skin contact method Exposure period (h)

Acute toxicity range 98% confidence limit 2

24 48

Median LC50 (mg/cm2)

Fold difference

0.04770.006 0.03770.006

1.00 0.79


Upper (mg/cm )

Lower (mg/cm )

0.059 0.047

0.035 0.026

Fig. 1. Effect of 48 h of exposure to chlorpyrifos by paper contact toxicity method to the morphology of earthworms. (A) Control earthworm. (B) Coiling, curling, and mucous secretion at 0.032 mg/cm2 (2.00 ppm). (C) Rupture of the cuticle and bloody lesions at 0.048 mg/cm2 (3.00 ppm). (D) Body constrictions and swellings and degeneration occurred on posterior end at 0.063 mg/cm2 (4.00 ppm). (E) Fragmentation of posterior parts at 0.079 mg/cm2 (5.00 ppm).



Percent inhibition

Morphological changes such as constriction and swelling started to appear from the anterior region within 12 h of exposure. The earthworms exhibited progressive signs, and symptoms like coiling, curling, and excessive mucus secretion (Fig. 1B) with sluggish movements were observed at lower concentrations (0.0158 and 0.0317 mg/cm2). Bulging of the clitellum and extrusion of coelomic fluid resulted in bloody lesions at higher concentrations, e.g., 0.0634 and 0.0793 mg/cm2 (Fig. 1D). Seventy-five percent of the worms exposed to higher concentrations detached their posterior parts either once or twice in 48 h of exposure (Fig. 1E). However, the symptoms at lower concentrations, 0.0158 and 0.0317 mg/cm2, coupled with starvation resulted in degenerative changes commencing from the posterior end (Fig. 1D). AChE activity of earthworms exposed to the LC50 was significantly inhibited and affected by exposure time. It is evident from Fig. 2 that AChE activity was inhibited 60% at 12 h and this inhibition increased further to 79%, 85%, and 91% at 24, 36, and 48 h of exposure, respectively. Most of the worms at 48 h exposures were sluggish, and a lack of response to stimuli was noted. The 50% inhibition (I50 ) studies revealed that a 4.88  10 6 M concentration is required to inhibit 50% of the enzyme activity in vitro (Fig. 3). Inhibition studies in vivo indicated that an amount greater than the I50 concentration was absorbed through the skin and reached the active site in the body of the organism. The kinetics of AChE in relation to chlorpyrifos was also studied further by in vitro evaluation. The




0 12




Time in hours Fig. 2. AChE activity of LC50-exposed earthworms at various intervals. Each value is the mean7SE of three individual observations. Control value is 0.0142 mg/min/mg protein. Values are statistically significant at Po0:05:

dissociation constant of the enzyme–substrate complex, defined as Km (Michaelis constant), was graphically determined by using Lineweavers’ Burk (LB) plots of the reciprocal substrate concentration (1=s) against reciprocal velocity (1=v). The control Km value of the enzyme is 0.228 mM and the Vmax is 0.025. A common intersection (1=Vmax ) on the velocity scale of LB plots of the experimental values indicates the inhibition by chlorpyrifos is competitive in nature (Fig. 4). A relative

J. Venkateswara Rao et al. / Ecotoxicology and Environmental Safety 54 (2003) 296–301

4. Discussion It is evident from earlier reports that the behavioral and morphological abnormalities associated with chlorpyrifos were more abundant than those with other pesticides. Similar symptoms, such as bulging of the clitellum and extrusion of coelomic fluid, were observed when earthworms were exposed to 10–50 ppm of Aldrin, Endosulfan, Heptachlor, and Lindane (Rajendra et al., 1990). The worms became agitated and restless to overcome the toxic affects of chlorpyrifos and required huge amounts of energy that were obtained by autolysis of their own tissue from the posterior region. Similar kinds of autolysis from the same region were observed in

1100 1000


percentage increase in Km was observed with an increase in toxicant concentration (Table 2). The inhibition constant (Ki ), estimated by plotting individual slope values (Km =Vmax ) against pesticide concentration, is presented in the inset of Fig. 4. The Ki value is 4.2  10 6 M. The effect of chlorpyrifos on the morphology and anatomy of earthworms was studied using scanning electron microscopy. It is apparent from Fig. 5 that the soft tissues of the most prominent part, the prostomium, was ruined by continuous contact with the toxicant (Fig. 5B). The transverse section of the body wall clearly indicated necrosis and damage in the structure of circular and longitudinal muscles in protruding areas (Fig. 5D). Most of the body surface affected by the action of pesticide through contact developed cracks on the cuticular membrane and extrusion of coelomic fluid resulted in bloody lesions (Figs. 5E and F). At higher concentrations the rectal area (last 25–30 segments) was detached from the body (Figs. 5G and H).

900 800



100 -6

I 50 value is 4.88 x 10 M

Percent Inhibition




50 45 40 35 30 25 20 15 10 5 0

K i = 4.2x10-6 M

-5 0 5 10 15 20 Concentration x 10-6 M

600 500








100 0












1.78x10 -6 M




0 1E-3





µg Concentration in Log

Fig. 3. Median inhibition (I50 ) of AChE activity calculated by the addition of different concentrations of toxicant added to the reaction mixture consisting 2.8 mL of 0.1 M phosphate buffer, pH 8, 50 mL (0.16 mM) DTNB, 50 mL (1 mg) protein, and 100 mL (0.2 mM) of the substrate acetylthiocholine iodide. Control value is 0.0142 mg/min/mg protein. Each value is the mean7SE of three individual observations. Values are statistically significant at Po0:05:



3.56x10 -6 M 14.25x10



Fig. 4. LB plots of AChE activity in the presence of chlorpyrifos. AChE kinetics and the type of inhibition by chlorpyrifos were assessed by simultaneously allowing 3 mL of each inhibitor concentration in (1.78, 3.56, 7.12, and 14.2  10 6 M) along with various substrate concentrations (0.04, 0.05, 0.066, 0.1, 0.2, and 0.4 mM) to react with the enzyme. Km and Vmax were computed from detailed substrate concentration curves using Lineweaver–Burk transformations. Inset: 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 concentrations.

Table 2 Relative percentage increase in Km with increase in the toxicant concentration Sample no.

Toxicant concentration



Vmax (1/intercept)

Apparent and relative Km ; 1/intercept  slope

1 2 3 4 5

Control 1.78  10 3.56  10 7.12  10 14.2  10

39.42 38.36 39.60 38.00 39.00

9.16 14.01 16.90 23.02 40.96

0.025 0.026 0.025 0.026 0.026

0.232 0.365 0.423 0.599 1.065

6 6 6 6

Note: Figures in parentheses indicate percentage increase in Km :

(57.32) (82.33) (158.19) (359.05)


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Fig. 5. Morphological changes in earthworm after 48 h exposure to chlorpyrifos (LC50) by the paper contact toxicity method. (A) Prostomium of earthworm, control (200  ). (B) Ruined tissue of prostomium (200  ). (C) Transverse section of body wall, control (400  ). (D) Necrosis in circular and longitudinal muscles (400  ). (E) Cuticular breakage and bloody lesions on anular segment (300  ). (F) Rupture of the protrusion on the surface of the body (200  ). (G) Facial surface view of surviving portion after fragmentation (220  ). (H) Facial surface view of anterior fragmented portion (220  ).

Polypheretima elongata due to the toxic effects of textile dyes (Ramaswami and Subbram, 1992). The lack of response to the stimuli could be due to the inhibition of AChE, which interrupted coordination between the nervous and muscular systems. In earthworms, as in most other cases, muscle’s contract in response to acetylcholine-mediated nerve impulses. The concentration of acetylcholine in nerve synaptic terminals is regulated by AChE activity. The observed inhibition of AChE activity is consistent with previous reports on the anticholinergic effect of carbamates, which reduced the efficiency of the burrowing capacity of worms, when combined with the neurotoxic effect (AChE inhibition) and muscular discoordination (Strenersen, 1979; Gupta and Sunderaraman, 1991).

5. Conclusions In the present study E. foetida demonstrated that toxicity increased with the length of exposure to chlorpyrifos. This suggests that the toxicity is associated with accumulation of chlorpyrifos in excess amounts and inhibition of AChE, which proves to be injurious to the earthworms.

Acknowledgments The authors are thankful to the Director, Indian Institute of Chemical Technology, Hyderabad for his

keen interest and encouragement during the course of work.

References Bhaskaram, P.S., 1986. Relation between soil moisture and body weight in tropical earthworms. J. Environ. Biol. 7 (4), 231–238. Cathay, B., 1982. Comparative toxicities of five insecticides to the earthworm, Lumbricus terrestris. Agric. Environ. 7, 73–81. Chang, L.W., Soderberg, L.S., Meir, J.R., Smith, M.K.S., 1997. Application of plant and earthworm bioassays to evaluate remediation of a lead contaminated soil. Arch. Environ. Contam. Toxicol. 32, 166–171. Dash, M.C., Senapathi, B.K., 1986. National Seminar on Organic Waste Utilize, Vermi Comp. Part-13, Proceedings, pp. 157–177. Eguchi, S., Hatano, R., Sakuma, T., 1995. Toshio effect of earthworms on the decomposition of soil organic matter. Nippon DojoHiryogaku 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 acetylcholinesterase activity. Biochem. Phamocol. 7, 88–95. Finney, D.J., 1953. Probit Analysis, 2nd Edition. Cambridge University Press, Cambridge, England. Gupta, S.K., Sunderaraman, V., 1991. Correlation between burrowing capability and AchE activity in the earthworm, Pheretima posthuma, on exposure to Carbyl. J. Res. Ad. Appl. Sci. 2, 350–356. Helling, B., Reinecke, S.A., Reinecke, A.J., 2000. Effects of the fungicide copper oxychloride on the growth and reproduction of Eisenia foetida (Oligochaeta). Ecotoxicol. Environ. Saf. 46 (1), 108–116. Kalaiselvan, K., Spm. Prince, W., Subburam, V., 1996. Toxicity of earthworm to Drawida ramnadana (Michaelsen). Pollut. Res. 15 (1), 15–18.

J. Venkateswara Rao et al. / Ecotoxicology and Environmental Safety 54 (2003) 296–301 Karnak, R.E., Hamelink, J.L., 1982. A standardized method for determining the acute toxicity of chemicals to earthworms. Ecotoxicol. Environ. Saf. 6, 216–222. Laird, J.M., Kroger, M., 1981. Earthworms. CRC Crit. Rev. Environ. Control 11, 189–218. Lebrun, P., Demadts, A., Wauthy, G., 1981. Eco-toxicologie compareeet bioactivite de trois insecticides Carbamates sur une population experimentale de vers de terre, Lumbricus herculeus. Pedobiologia 21, 225–235. Lineweaver, H., Burk, D., 1934. Determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658–666. Lofs-Holmin, A., 1982. Influence of routine pesticide spraying on earthworm Lumbricus in field experiments with winter wheat. Swed. J. Agric. Res. 12, 121–124. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265–275. Morgan, J.E., 1993. Seasonal changes in the tissue metals (Cd, Zn & Pb) concentrations in two ecophysiological dissimilar earthworm species. Environ. Pollut. 82 (1), 1–7.


Rajendra, K., Rajesh, C.G., Mirza, U.B., 1990. Toxicity assesment of four insecticides to earthworm, Pheretima postuma. Bull. Environ. Contam. Toxol. 45, 358–364. Ramaswami, V., Subbram, V., 1992. Effect of selected textile dye on the survival, morphology, and burrowing behavior of the earthworm Polypheretima elongata. Bull. Environ. Contam. Toxol. 48, 253–258. Reddy, P.J., Krishna, D., Suryanarayana Murty, U., Kaiser, J., 1992. A microcomputer FORTRAN programme for rapid determination of lethal concentration in mosquito control. Computer Applications in the Biosciences 8, 209–213. Reinecke Sophie, A., Reinecke, A.J., 1997. The influence of lead and manganese on spermatozoa Eisenia foetida. Soil Biol. Biochem. 29 (3/4), 737–742. Strenersen, J., 1979. 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. Vishwanathan, R., 1997. Physiological basis of assesment of ecotoxicology of pesticides to soil organisms. Chemosphere 35 (1/2), 323–334.