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Toxicity of azodrin on the morphology and acetylcholinesterase activity of the earthworm Eisenia foetida
ARTICLE IN PRESS
Environmental Research 96 (2004) 323–327
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 Received 20 August 2003; received in revised form 17 February 2004; accepted 27 February 2004
Abstract 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 0.4670.1 mg cm2 (2376 mg L1) and those in the 7- and 14-day artificial soil tests were 171721 and 132720 mg kg1, respectively. The neurotoxic potentiality of azodrin was assessed by using a marker enzyme, acetylcholinesterase (AChE; EC 3.1.1.7) 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 concentrationdependent changes in the morphology and AChE activity of the earthworm E. foetida. r 2004 Elsevier Inc. All rights reserved. Keywords: AChE; Eisenia foetida; Azodrin; Morphology; Earthworm
1. Introduction Extensive usage of organophosphorus (OP) compounds 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). Vermicastings 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 Senapathi, 1986). 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 contamination in agricultural soils (Stenersen, 1979a, b). OP *Corresponding author. Fax: +91-40-2717-3387. E-mail address: [email protected] (J.V. Rao). 0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.02.014
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 3.1.1.7) 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; Vishwanathan, 1997). Further study on the influence of OPs on the kinetic properties of earthworm AChE is still warranted. 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 earthworms.
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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). Technicalgrade azodrin (purity o95%) was a gift from NOCIL, Mumbai, India. 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 1 h. Before testing, these worms were acclimatized 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 feed boxes. 2.1. Determination of median lethal concentration (LC50)
calcium carbonate (total weight of mixture=4 kg). Each test mixture (concentration) was divided into four equal quantities of 1 kg 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 (9.5270.25 cm) and weight (0.7270.03 g) 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 exposure tenures. 2.2. Acetylcholinesterase activity
The acute toxicity experiments were conducted in two ways, i.e., direct contact test through a filter paper method (48 h) and artificial soil test for 14-day exposure (OECD, 1984). For the filter paper contact test, the sides of flat-bottomed glass vials (8 cm in length and 3 cm in diameter) were lined with Whatman filter paper No. 1 without overlapping (the circumference of the paper is 63 cm2). The test chemical, azodrin, was dissolved in water and predetermined amounts, 0.1, 0.3, 0.6, 1.2, and 2.5 mg cm2, were loaded onto the filter paper (1 mL of 6.25, 18.75, 37.5, 75, and 125 mg L1 solution, respectively). 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 3 h to adjust to the test environment under starvation. They were then randomly divided into groups of 20 earthworms per treatment and were exposed (1 adult earthworm per vial; 9.5270.25 cm in length and 0.7270.03 g in weight) to different concentrations of azodrin described above. The artificial soil test (using an evenly blended dryweight 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 250 mg kg1) were homogeneously mixed with artificial soil. Briefly, 400, 600, 800, or 1000 mg of azodrin was dissolved in 1400 mL of distilled water and thoroughly mixed with 2.6 kg of artificial soil (dry weight) to obtain 35% moisture; pH was maintained at 6.070.5 by addition of
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 0.1 M, pH 7.5, phosphate buffer using a Potter–Elvehjam homogenizer fitted with a Teflon pestle. The homogenates were centrifuged at 10,000 g for 10 min and the resultant supernatant was recentrifuged at 10,000 g for 10 min 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 2.8 mL of 0.1 M phosphate buffer, pH 7.2, 50 mL of 0.16 mM DTNB, 50 mL of protein, and 100 mL of 0.2 mM ACT iodide as substrate. The reactions were performed at 37 C and were initiated by adding the substrate (ACT iodide). The measurement of the rate of production of thiocholine was accomplished by measurement of the continuous 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 6 min at 412 nm in a spectrophotometer (Spectra MAX Plus; Molecular Devices; supported by SOFTmax PRO-3.0). The activity was calculated as mmol/mg protein/min.
ARTICLE IN PRESS J.V. Rao, P. Kavitha / Environmental Research 96 (2004) 323–327
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) transformations, using various substrate concentrations (0.02, 0.023, 0.025, 0.030, 0.035, 0.040, 0.050, 0.065, 0.1, and 0.2 mM), 0.16 mM DTNB, and known amount of protein in 3-mL assay volume. The effect of azodrin and its mode of inhibition were assessed by adding 3.74 105, 7.47 105, 1.49 104, and 2.24 104 M 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 concentrations (Dixon and Webb, 1965). Data are expressed as mean7SE of three separate experiments, each assayed in triplicate. The experiments were repeated three times in triplicate 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 48 h for the paper contact test and at days 7 and 14 of exposure for the artificial soil test. The median lethal concentrations (LC50) were 0.4670.2 mg cm2 (2376 mg L1) for the paper contact test and 171721 and 132720 mg kg1 respectively, 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 symptoms of toxicity such as coiling, curling, and excessive mucus secretion with sluggish movements and swelling of clitellum at lower concentrations (0.1–0.6 mg cm2). Extrusion of coelomic fluid resulting in bloody lesions occurred at the higher concentrations (1.2 and 2.5 mg cm2). Morphological changes such as constriction and swelling started appearing in the anterior regions of exposed worms (0.46 mg cm2) within 12 h of exposure and degenerative changes appeared at the posterior end of the exposed earthworms after 48 h 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 autolysis from the posterior region was observed in earthworms, Polypheretima elongata, exposed to textile dyes
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Table 1 Median lethal concentration of azodrin to the earthworm E. foetida in the paper contact and artificial soil tests Median lethal concentration Paper contact method 48 h
Artificial soil method 2
0.4670.1 mg cm 2376.23 (mg L1)
7 day 14 day
171.36721.4 (mg kg1) 132.43720.8 (mg kg1)
Fig. 1. (A–C) Morphological abnormalities in earthworms after 48 h of exposure (LC50 concentration) to azodrin using the paper contact test.
(Ramaswami and Subbram, 1992). Fifty percent of the worms detached one or two of their posterior parts during 48 h of exposure (Fig. 1C). Similar symptoms were also observed when earthworms were exposed to azodrin in the artificial soil test. Numerous protrusions were observed on the anterior parts of the earthworms exposed to X150 mg kg1 azodrin. The worms exposed to 150–250 mg kg1 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 250 mg kg1) 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 behavior. The in vivo AChE activity was estimated in earthworms treated with azodrin in artificial soil after 24 h, 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 manner. The inhibition after 24 h of treatment was 11% in 100 mg kg1, 17% in 150 mg kg1, 25% in 200 mg kg1, and 31% in 250 mg kg1. 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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(Michaelis–Menten constant), was graphically determined 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 100 mg kg-1 150 mg kg-1 200 mg kg-1 250 mg kg-1
90 Mean Percent AChE Inhibition ± SE
80 70 60 50 40 30 20 10
Day-1
Day-7
Day - 14
Exposure period Fig. 2. Percentage inhibition of AchE after exposure to azodrinat 1, 7, and 14 days.
45 3.4
35
3.0 2.6
25
Km/Vmax (Slope)
1/ AChE Activity (µ moles/min/mg protein]
55
values of AChE enzyme in control and each concentration of the toxicant were derived from their regression equations, in which the 1/intercept value is the Vmax and the 1=intercept slope value is the Michaelis–Menten constant. The estimated Vmax and apparent Km values of AChE of the control earthworms were 0.415 and 0.162 mM, respectively. The kinetic constants (Vmax and 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 concentration. 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 in nature. 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 4.74 105 in 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.
15
5
-10
2.2 1.8 1.4 1.0
0
10 20 1/Substrate (mM)
0.6
30
0.2
Fig. 3. Lineweaver–Burk plots of AChE activity of earthworms in the absence and presence of azodrin as a function of substrate concentration. —&—, control; —J—, 3.74 105; —n—, 7.47 105; —,—, 1.49 104; —B—, 2.24 104. Each point indicates the activity7SE expressed in different concentrations of substrate used in the assay media.
-15
-10
-5
0
5
10
15
20
25
30
35
40
Azodrin concentration x10-5
-Ki Fig. 4. Slopes of Lineweaver–Burk plots at different concentrations of azodrin (to determine inhibitory constant (Ki )).
Table 2 Kinetic constants of earthworm AChE activity in the presence of azodrin Toxicant concentration (mol)
Intercept
Slope
Apparent Km (1/intercept) (slope)
Vmax (1/intercept)
Control 3.74 105 7.47 105 1.49 104 2.24 104
2.41 2.42 2.43 2.46 2.48
0.39 0.65 0.95 1.34 2.11
0.162 0.269 0.391 0.545 0.851
0.415 0.413 0.412 0.407 0.403
Values in parentheses indicate % increase.
(66.05) (141.36) (236.42) (425.31)
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4. Conclusions In this study E. foetida demonstrated that toxicity increased with the length of exposure to azodrin. This suggests that the toxicity is associated with accumulation 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 reproducible than was the artificial soil test. The minute and specific observations such as cuticular breakage, extrusion 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 pesticides, 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.
Acknowledgments 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.
References 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 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. Pharmacol. 7, 88–95. Finney, D.J., 1953. Probit Analysis 2nd Edition. Cambridge University Press, Cambridge, UK.
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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: Chysomelidae). 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, 265–275. 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 earthworm Polypheretima elongata. Bull. Environ. Contam. Toxicol. 48, 253–258. Rao, J.V., Swamy, A.N., Yamin, S., 1991. In vitro brain acetylcholinesterase 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 insecticides. 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 acetylcholinesterase 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.
Environmental Research 96 (2004) 323–327
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 Received 20 August 2003; received in revised form 17 February 2004; accepted 27 February 2004
Abstract 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 0.4670.1 mg cm2 (2376 mg L1) and those in the 7- and 14-day artificial soil tests were 171721 and 132720 mg kg1, respectively. The neurotoxic potentiality of azodrin was assessed by using a marker enzyme, acetylcholinesterase (AChE; EC 3.1.1.7) 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 concentrationdependent changes in the morphology and AChE activity of the earthworm E. foetida. r 2004 Elsevier Inc. All rights reserved. Keywords: AChE; Eisenia foetida; Azodrin; Morphology; Earthworm
1. Introduction Extensive usage of organophosphorus (OP) compounds 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). Vermicastings 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 Senapathi, 1986). 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 contamination in agricultural soils (Stenersen, 1979a, b). OP *Corresponding author. Fax: +91-40-2717-3387. E-mail address: [email protected] (J.V. Rao). 0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.02.014
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 3.1.1.7) 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; Vishwanathan, 1997). Further study on the influence of OPs on the kinetic properties of earthworm AChE is still warranted. 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 earthworms.
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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). Technicalgrade azodrin (purity o95%) was a gift from NOCIL, Mumbai, India. 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 1 h. Before testing, these worms were acclimatized 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 feed boxes. 2.1. Determination of median lethal concentration (LC50)
calcium carbonate (total weight of mixture=4 kg). Each test mixture (concentration) was divided into four equal quantities of 1 kg 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 (9.5270.25 cm) and weight (0.7270.03 g) 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 exposure tenures. 2.2. Acetylcholinesterase activity
The acute toxicity experiments were conducted in two ways, i.e., direct contact test through a filter paper method (48 h) and artificial soil test for 14-day exposure (OECD, 1984). For the filter paper contact test, the sides of flat-bottomed glass vials (8 cm in length and 3 cm in diameter) were lined with Whatman filter paper No. 1 without overlapping (the circumference of the paper is 63 cm2). The test chemical, azodrin, was dissolved in water and predetermined amounts, 0.1, 0.3, 0.6, 1.2, and 2.5 mg cm2, were loaded onto the filter paper (1 mL of 6.25, 18.75, 37.5, 75, and 125 mg L1 solution, respectively). 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 3 h to adjust to the test environment under starvation. They were then randomly divided into groups of 20 earthworms per treatment and were exposed (1 adult earthworm per vial; 9.5270.25 cm in length and 0.7270.03 g in weight) to different concentrations of azodrin described above. The artificial soil test (using an evenly blended dryweight 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 250 mg kg1) were homogeneously mixed with artificial soil. Briefly, 400, 600, 800, or 1000 mg of azodrin was dissolved in 1400 mL of distilled water and thoroughly mixed with 2.6 kg of artificial soil (dry weight) to obtain 35% moisture; pH was maintained at 6.070.5 by addition of
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 0.1 M, pH 7.5, phosphate buffer using a Potter–Elvehjam homogenizer fitted with a Teflon pestle. The homogenates were centrifuged at 10,000 g for 10 min and the resultant supernatant was recentrifuged at 10,000 g for 10 min 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 2.8 mL of 0.1 M phosphate buffer, pH 7.2, 50 mL of 0.16 mM DTNB, 50 mL of protein, and 100 mL of 0.2 mM ACT iodide as substrate. The reactions were performed at 37 C and were initiated by adding the substrate (ACT iodide). The measurement of the rate of production of thiocholine was accomplished by measurement of the continuous 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 6 min at 412 nm in a spectrophotometer (Spectra MAX Plus; Molecular Devices; supported by SOFTmax PRO-3.0). The activity was calculated as mmol/mg protein/min.
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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) transformations, using various substrate concentrations (0.02, 0.023, 0.025, 0.030, 0.035, 0.040, 0.050, 0.065, 0.1, and 0.2 mM), 0.16 mM DTNB, and known amount of protein in 3-mL assay volume. The effect of azodrin and its mode of inhibition were assessed by adding 3.74 105, 7.47 105, 1.49 104, and 2.24 104 M 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 concentrations (Dixon and Webb, 1965). Data are expressed as mean7SE of three separate experiments, each assayed in triplicate. The experiments were repeated three times in triplicate 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 48 h for the paper contact test and at days 7 and 14 of exposure for the artificial soil test. The median lethal concentrations (LC50) were 0.4670.2 mg cm2 (2376 mg L1) for the paper contact test and 171721 and 132720 mg kg1 respectively, 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 symptoms of toxicity such as coiling, curling, and excessive mucus secretion with sluggish movements and swelling of clitellum at lower concentrations (0.1–0.6 mg cm2). Extrusion of coelomic fluid resulting in bloody lesions occurred at the higher concentrations (1.2 and 2.5 mg cm2). Morphological changes such as constriction and swelling started appearing in the anterior regions of exposed worms (0.46 mg cm2) within 12 h of exposure and degenerative changes appeared at the posterior end of the exposed earthworms after 48 h 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 autolysis from the posterior region was observed in earthworms, Polypheretima elongata, exposed to textile dyes
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Table 1 Median lethal concentration of azodrin to the earthworm E. foetida in the paper contact and artificial soil tests Median lethal concentration Paper contact method 48 h
Artificial soil method 2
0.4670.1 mg cm 2376.23 (mg L1)
7 day 14 day
171.36721.4 (mg kg1) 132.43720.8 (mg kg1)
Fig. 1. (A–C) Morphological abnormalities in earthworms after 48 h of exposure (LC50 concentration) to azodrin using the paper contact test.
(Ramaswami and Subbram, 1992). Fifty percent of the worms detached one or two of their posterior parts during 48 h of exposure (Fig. 1C). Similar symptoms were also observed when earthworms were exposed to azodrin in the artificial soil test. Numerous protrusions were observed on the anterior parts of the earthworms exposed to X150 mg kg1 azodrin. The worms exposed to 150–250 mg kg1 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 250 mg kg1) 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 behavior. The in vivo AChE activity was estimated in earthworms treated with azodrin in artificial soil after 24 h, 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 manner. The inhibition after 24 h of treatment was 11% in 100 mg kg1, 17% in 150 mg kg1, 25% in 200 mg kg1, and 31% in 250 mg kg1. 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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(Michaelis–Menten constant), was graphically determined 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 100 mg kg-1 150 mg kg-1 200 mg kg-1 250 mg kg-1
90 Mean Percent AChE Inhibition ± SE
80 70 60 50 40 30 20 10
Day-1
Day-7
Day - 14
Exposure period Fig. 2. Percentage inhibition of AchE after exposure to azodrinat 1, 7, and 14 days.
45 3.4
35
3.0 2.6
25
Km/Vmax (Slope)
1/ AChE Activity (µ moles/min/mg protein]
55
values of AChE enzyme in control and each concentration of the toxicant were derived from their regression equations, in which the 1/intercept value is the Vmax and the 1=intercept slope value is the Michaelis–Menten constant. The estimated Vmax and apparent Km values of AChE of the control earthworms were 0.415 and 0.162 mM, respectively. The kinetic constants (Vmax and 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 concentration. 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 in nature. 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 4.74 105 in 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.
15
5
-10
2.2 1.8 1.4 1.0
0
10 20 1/Substrate (mM)
0.6
30
0.2
Fig. 3. Lineweaver–Burk plots of AChE activity of earthworms in the absence and presence of azodrin as a function of substrate concentration. —&—, control; —J—, 3.74 105; —n—, 7.47 105; —,—, 1.49 104; —B—, 2.24 104. Each point indicates the activity7SE expressed in different concentrations of substrate used in the assay media.
-15
-10
-5
0
5
10
15
20
25
30
35
40
Azodrin concentration x10-5
-Ki Fig. 4. Slopes of Lineweaver–Burk plots at different concentrations of azodrin (to determine inhibitory constant (Ki )).
Table 2 Kinetic constants of earthworm AChE activity in the presence of azodrin Toxicant concentration (mol)
Intercept
Slope
Apparent Km (1/intercept) (slope)
Vmax (1/intercept)
Control 3.74 105 7.47 105 1.49 104 2.24 104
2.41 2.42 2.43 2.46 2.48
0.39 0.65 0.95 1.34 2.11
0.162 0.269 0.391 0.545 0.851
0.415 0.413 0.412 0.407 0.403
Values in parentheses indicate % increase.
(66.05) (141.36) (236.42) (425.31)
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4. Conclusions In this study E. foetida demonstrated that toxicity increased with the length of exposure to azodrin. This suggests that the toxicity is associated with accumulation 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 reproducible than was the artificial soil test. The minute and specific observations such as cuticular breakage, extrusion 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 pesticides, 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.
Acknowledgments 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.
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