Ecotoxicology and Environmental Safety 100 (2014) 218–225

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Comparative toxicity of carbaryl, carbofuran, cypermethrin and fenvalerate in Metaphire posthuma and Eisenia fetida —A possible mechanism P.N. Saxena, S.K. Gupta, R.C. Murthy n Cell Biology Division CSIR-Indian Institute of Toxicology Research, Lucknow 226001, India

art ic l e i nf o

a b s t r a c t

Article history: Received 1 July 2013 Received in revised form 8 November 2013 Accepted 11 November 2013 Available online 7 December 2013

To establish the use of Metaphire posthuma as a sensitive test model for ecotoxicological studies, acute toxicity testing of carbaryl, carbofuran, cypermethrin and fenvalerate on Eisenia fetida and Metaphire posthuma were carried out. Two different types of bioassays, contact filter paper toxicity and soil toxicity bioassays were used to determine LC50 values for these insecticides. Among the tested chemicals, carbofuran was the most toxic to both the earthworm species. In paper contact method, 72 h-LC50 values of carbofuran in M. posthuma and E. fetida were found to be 0.08 μg/cm2 and 1.55 μg/cm2 respectively while in soil test, 14-d LC50 values were 0.49 mg/kg and 21.15 mg/kg respectively. On comparing the toxicity data of these chemicals for both the earthworm species, M. posthuma was found to be more sensitive than E. fetida. Based on the acute toxicity data, the order of toxicity of insecticides in both the test procedures was carbofuran4cypermethrin4carbaryl4fenvalerate for M. posthuma whereas for E. fetida it was carbofuran4carbaryl4fenvalerate4cypermethrin. Morphological changes also appeared in the organisms exposed to these chemicals which were more pronounced in M. posthuma at lower concentrations than E. fetida in both the test procedures. The results of the present study advocates the use of M. posthuma for ecotoxicity studies, being a more sensitive and reliable model than E. fetida. Based on the data on partial atomic charges, structural features and spectroscopic studies on carbaryl and carbofuran, a possible mechanism of toxicity of carbamate insecticides in earthworm was proposed. & 2013 Elsevier Inc. All rights reserved.

Keywords: Earthworm acute toxicity Carbofuran and carbaryl Cypermethrin and fenvalerate Molecular orbital calculations FTIR spectrum Mechanism of toxicity

1. Introduction Earthworms are economically important soil invertebrates representing 60–80% of the total biomass in soil, potentially maintain structure, texture and fertility of soils by increasing aeration and drainage through their burrowing, feeding and casting activities (Edwards and Lofty, 1977).These functional characteristics confer earthworms ideal soil organisms for use in terrestrial ecotoxicology. In comparison to other terrestrial invertebrates like springtails, termites etc. (Da Luz et al., 2004; Hentati et al., 2013), earthworms are more sensitive to soil contaminating components like insecticide, industrial leachates and metals etc. thus they are considered reliable bioindicators for soil quality. Eisenia fetida is the only earthworm species so far, approved by international standards for acute toxicity testing of environmental chemicals (OECD, 1984; E.E.C., 1985). It is a compost dwelling species used for captive breeding, which is not

n Corresponding author. Present address: Scientist & Head, Chemicals and Pollutants Analysis Unit, CSIR-Indian Institute of Toxicology Research, Lucknow 226001, India. Fax: þ91 522 2611547. E-mail addresses: [email protected], [email protected] (R.C. Murthy).

0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.11.006

common in natural soils. However, its ecological representative for soil dwelling species has been controversial (Dean-Ross, 1982; Edwards and Coulson, 1992; Reinecke and Reinecke, 2004; Pelosi et al., 2013). From an ecotoxicological viewpoint, there is a great need to validate Indian species of earthworms for acute toxicity assessment of environmental contaminants for the actual reflection of ecotoxicity in Indian conditions and to compare its susceptibility to various agrochemicals. Furthermore, validation of new models for toxicity testing will certainly increase the battery of tests for ecotoxicity. Among the various earthworm species used for ecotoxicity studies, we have tested on Indian earthworm Metaphire posthuma as a possible candidate for these studies. Apart from the validation, the results from the toxicity of various insecticides may be useful to understand the ultimate impact on soil ecosystem and agriculture as these worms burrow in the soil up to a depth of 30–45 cm. Metaphire posthuma is widely distributed in various states of India and other Asian countries (Bantaowong et al., 2011; Chaudhuri et al., 2012). It has a long narrow bilaterally symmetrical and cylindrical body, well adapted for burrowing. A mature worm measures about 150 mm in length and 3–5 mm in thickness. They are hermaphrodites, but their reproduction occurs by cross fertilization through the exchange of their spermatozoa. The ova are fertilized by the sperm

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cells in the cocoon which develops into mature worm. The normal hatching period of a cocoon is 45–60 days. Insecticides are widely used in agriculture for the massive increase in food production. Two different classes of insectidescarbamate and synthetic pyrethroid were used in the present study. Carbamate insecticides are known to be strong inhibitors of acetylcholinesterase of nervous tissues and are used to control foliar feeding and soil dwelling insects.(Gupta and Sunderaraman, 1988). Synthetic pyrethroid have a higher efficiency, relatively shorter half life in soil and lower mammalian toxicity. They alter the normal function of insect nerves by modifying the kinetics of voltagesensitive sodium channels (Soderlund et al., 2002). However, high concentrations of insecticides in soil can affect earthworm population. Numerous studies on the effects of insecticides on various species of earthworms have been documented (Luo et al., 1999; Gupta et al., 2010; De Silva et al., 2010) but, the comparative studies of different insecticides on two species of earthworm are meager. Hence, a comparative study on the toxicity of different insecticides on different species becomes important in order to identify more sensitive species to be used as a model for toxicity assessments of environmental contaminants. Biological activity of tested insecticides in earthworm depends mainly on their interaction with proteins/lipids (Gupta and Sundararaman, 1988; Mosley et al., 2003a; Tripathi and Bhardwaj, 2005). Molecular parameters such as shape, size, geometry, orientation and polarizability play an important role in such interactions. Valence charge densities obtained from molecular orbital calculations and spectroscopic data can be used to understand the mechanism of action of these insecticides in earthworms at molecular level. Similar studies on bioactivity of cyclodiene insecticides and diuron have been earlier reported (Saxena et al., 2004; Saxena and Gupta, 2005). Structure dependent biological activity of carbamate insecticides have been also documented in the literature (Eya and Talcottt, 1980; Feaster et al., 1996). In the present study, we report the acute toxicity of carbaryl, carbofuran, cypermethrin and fenvalerate in two earthworm species, E. fetida and M. posthuma using contact filter paper toxicity and soil toxicity bioassays to identify the most sensitive species to be used as a model for toxicity testing of environmental chemicals. The median lethal concentration (LC50) was measured to determine the toxic levels of different insecticides. Morphological abnormalities and histopathological observations induced by insecticides in earthworms were also examined. Based on structural features, partial atomic charges and spectroscopic data, a possible mechanism of action of carbamate insecticides in earthworms has been proposed.

2. Materials and methods 2.1. Test chemicals Cypermethrin, fenvalerate, carbaryl and carbofuran with 99.9% purity were purchased from Sigma (St.Louis, MO). Stock solutions of the test compounds were prepared in acetone and diluted with deionized water to yield the final test concentrations. 2.2. Test species Two earthworm species, E. fetida and M. posthuma were chosen as the test organisms. The culture of E. fetida was obtained from an earthworm breeding farm (Rehmankhera, Lucknow) and maintained on a cow dung diet. The culture was maintained in the laboratory condition at 20 7 1 1C. Normal adult earthworms with developed clitellum and weighing 350–450 mg were randomly selected for the toxicity tests. They were separated from the cultures 3 h prior to use, rinsed with distilled water to remove cow dung and placed on a moistened filter paper at 207 1 1C in a biology oxygen demand incubator to remove their gut contents. Earthworm, M. posthuma were collected locally from the moist soil near the bank of water reservoir in the garden and kept in plastic buckets with artificial soil at laboratory conditions. They were acclimatized for one week in the untreated soil

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prior to start of the test at 207 1 1C. Healthy and clitellated earthworms with average weight 3.12 g were sorted out, rinsed with distilled water to remove soil and placed on a moistened filter paper for 24 h to evacuate their gut contents prior to experimentation.

2.3. Filter paper contact method Filter paper contact test was conducted as per OECD guidelines with slight modifications. Instead of using glass vial, 9-cm and 16 cm- petri dishes lined with filter paper were used for the study. 2 ml–4 ml of each test concentration were used to properly moisten the filter papers placed in small and bigger Petri dishes, respectively. All the Petri dishes with moist filter paper were kept open for 30 min to evaporate acetone. For each treatment including solvent control, ten replicates, each consisting of one mature worm per Petri dish was used in the experiment. More than one worm in the Petri dish was not used as the death of one worm might cause adverse effect on the other in the same dish. All the Petri dishes containing worms were kept in a biology oxygen demand incubator at 20 71 1C. After incubation period of 24, 48 and 72 h, mortality and morphological abnormality were recorded. Earthworms were considered dead when no response to mechanical stimulus by needle was observed. A range finding test on concentrations of the test substances of 0, 0.1, 1.0, 10, 100 and 1000 μg/cm2 was performed to determine concentrations causing 0 and 100% mortality. A narrow range of concentrations 0.05 μg/cm2, 0.10 μg/cm2, 0.20 μg/cm2, 0.40 μg/cm2, 0.80 μg/cm2, 1.60 μg/cm2 and 3.20 μg/cm2 and 6.40 μg/cm2 was also tested. To determine LC50, five test concentrations and a control were used.

2.4. Soil toxicity test Artificial soil test was conducted as per OECD guidelines with some modifications. Instead of ten percent sphagnum peat 30 percent cow dung manure were used for the study. This change in organic matter in the soil was made to ensure healthy growth of M. posthuma. Thus, in the soil bioassay, artificial soil medium was prepared with 30 percent cow dung manure, 20 percent clay and 50 percent fine sand. The dry constituents were blended in the correct proportion and deionised water was added to give an overall moisture content of about 35 percent of the dry weight. Calcium carbonate was used to adjust the pH 6.0 7 0.5. For each test concentration, desired insecticide was dissolved in 15 ml of acetone mixed with the moist soil. After mixing properly, the treated soil samples were kept open in plastic trays for 3 h to evaporate acetone. The moisture loss from the test soil was checked by weighing the test containers. Control soil was also treated with 15 ml of acetone in a similar manner. After providing 35 percent moisture by weight in soil, ten clitellated earthworms were placed in each beaker (high density polypropylene; 2 L capacity) containing one kg of treated soil and kept in a biology oxygen demand incubator at 207 1 1C. Mortality and abnormality were assessed after 7 and 14 days. A range of concentrations 0, 0.1, 1.0, 10, 100 and 1000 mg/kg dry soil, were used to determine a concentration range that resulted in 0–100% mortality. To determine LC50, five test concentrations and a control were used.

2.5. Histopathology The control and treated worms were sacrificed by freezing (Sharma and Satyanarayan, 2011), cut into 1 cm pieces and were fixed immediately in 10 percent neutral buffered formaldehyde solution for three days (Gupta and Sundararaman, 1990). Fixation was followed by washing in running water for 6 h. Fixed tissues were dehydrated by graded series of alcohol, cleared in xylene, embedded in paraffin wax and sections of 5 μm were cut and stained with hematoxylin and eosin. The slides were observed under light microscope (Nikon, Japan) to examine the histopathological changes.

2.6. Statistical analysis Mortality data were obtained after insecticides exposures in paper contact and soil tests, and analyzed to determine LC50 values. Trimmed Spearman–Karber method (Hamilton et al., 1977) was used to calculate the LC50 with 95% lower and upper confidence intervals. Molecular orbital calculations and FTIR spectral measurements Molecular orbital calculations were carried out using RHF theory with STO-3G basis sets available in Gamess program (Schmidt et al., 1993) to estimate partial atomic charges in carbaryl and carbofuran (carbamate insecticides). Coordinates from crystal structure data (Sandoz et al., 2000; Xu et al., 2005) have been used in the calculations. These atomic charges are very useful to determine the active site in an insecticide. Fourier transform infrared (FTIR) spectrum of carbaryl was recorded at 2 cm  1 resolution in the range 450–4000 cm  1 at Perkin Elmer 1800 spectrophotometer (Bucks,UK).

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3. Results 3.1. Contact toxicity The results for the acute toxicity of various insecticides with contact filter paper test to E. fetida and M. posthuma are presented in Table 1. The results demonstrate that the effect of all the insecticides in both the test organisms was concentration dependent and that the percentage of mortality increased with increasing concentration of Table 1 Summary of the results for the acute toxicity with contact filter paper test to Eisenia fetida and Metaphire posthuma. Insecticides

M. posthuma LC50 (95% CI) μg/cm2

E. fetida LC50 (95% CI) μg/cm2

Cypermethrin Fenvalerate Carbofuran Carbaryl

0.30 0.55 0.08 0.48

3.84 3.72 1.55 1.74

(0.22–0.39) (0.50–0.59) (0.07–0.09) (0.39–0.56)

(3.51–4.22) (2.83–4.87) (1.15–2.08) (1.43–1.89)

insecticides. Both the organisms treated with these chemicals exhibited progressive signs and symptoms like coiling, curling, extrusion of coelomic fluid with sluggish movement, segmental swellings, constrictions and degeneration (Fig. 1). In some worms, the swollen portions slowly burst causing bloody lesions and ultimately death. Symptoms of toxicity such as coiling, tremors and excessive secretion of mucous in contact method, were visible within 2 h exposure of these insecticides at 0.24 μg/cm2 concentration in M. posthuma and at 1.20 μg/cm2 in E. fetida, whereas morphological changes such as segmental swelling and bleeding sores occurred in both the test species after 48 h treatment of these toxicants through moist filter paper at similar concentrations. In some cases, the toxicity symptoms were specific to a particular insecticide e.g. the severe constriction (likely to detached) at post clitellar region in the test organisms were found at 0.09 μg/cm2 carbofuran and at 0.54 μg/cm2 carbaryl treatment for 48 h through paper contact method (Fig. 1). The percentage of mortality and abnormality induced by various insecticides was higher in M. posthuma than E. fetida. Among the insecticides tested, carbofuran was highly toxic to both the earthworm species. The order of toxicity based on LC50 values was

Fig. 1. Morphological abnormalities in M. posthuma and E.fetida. (a) Bleeding sores at clitellar region (E. fetida, 1.20 μg/cm2 carbofuran at 48 h), (b) segmental swelling (M. posthuma, 0.24 μg/cm2 carbaryl at 48 h), (c) severe constriction at post clitellar region (E. fetida, 1.20 μg/cm2 carbaryl at 48 h), (d) thinning (E. fetida, 0.18 μg/cm2 carbofuran at 48 h), (e) bleeding sores at post clitellar region (M. posthuma, 0.24 μg/cm2 fenvalerate at 48 h), (f) severe constriction at post clitellar region (E. fetida, 1.200 μg/cm2 carbaryl at 48 h) and (g) post clitellar degeneration (M. posthuma, 0.24 μg/cm2 cypermethrin at 48 h).

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as follows: carbofuran4cypermethrin4carbaryl4fenvalerate in M. posthuma and carbofuran4carbaryl4fenvalerate4cypermethrin in E. fetida.

shown in Fig. 4. The 500–1800 cm  1 range is a spectral rich region where carbaryl exhibits several sharp and intense infrared bands.

3.2. Soil toxicity

4. Discussion

The results for the acute toxicity of various insecticides with artificial soil test to E. fetida and M. posthuma are summarized in Table 2. The data exhibited a concentration and exposure time dependent relationship with all the insecticides tested. Each of the insecticides tested showed different degree of toxicity to M. posthuma and E. fetida. Morphological abnormalities such as segmental swellings, constrictions, bleeding sores, post clitellar degenerations were generally visible after 6–24 h treatment of these chemicals at much higher concentrations than contact method. At 7-day, carbofuran showed the highest toxicity to M. posthuma followed by cypermethrin, carbaryl and fenvalerate. The order of toxicity in descending order was as follows: carbofuran4cypermethrin4carbaryl4 fenvalerate in M. posthuma and carbofuran4carbaryl4fenvalerate4cypermethrin in E. fetida. At-14 day, carbofuran still exhibited highest toxicity to M. posthuma and E. fetida and the order of toxicity was similar to that at 7-day. In pyrethroid class of insecticides, at 14-day cypermethrin was 1.9 fold more toxic than fenvalerate in M. posthuma whereas in E. fetida it was 13.5 fold less toxic than fenvalerate. But in carbamates, at 14 day carbofuran was 17.5 fold more toxic than carbaryl in M. posthuma whereas in E. fetida it was 1.3 fold more toxic than carbaryl. The LC50 values obtained for different insecticides clearly reflect a better sensitivity of M. posthuma over E. fetida. The acute toxicity (LC50) values of these insecticides in soil test were higher than the values obtained by paper contact method.

The present study was carried out to compare the sensitivity of two earthworm species exposed to four different insecticides by two different methods. Similar studies on comparative sensitivity of E. fetida and Perionyx excavatus to different chemicals were also reported (De Silva and Van Gestel, 2009). E. fetida has been recommended by several guidelines as a test species for screening of agrochemicals (OECD, 1984; E.E.C., 1985; Environment Canada, 2004; ISO, 2007). However, its ecological relevance has been limited due to its rare presence in natural ecosystem and resistant behavior (Edwards and Coulson, 1992; Reinecke and Reinecke, 2004; Pelosi et al., 2013). Several earthworm species with higher ecological relevance such as Lumbricus terrestris (Mosley et al., 2003b), Apporrectodea calignosa (Hodge et al., 2000) and Lumbricus rubellus (Lukkari and Haimi, 2005) have been shown to be suitable for use in soil toxicity test. Among the tested chemicals, carbofuran was highly toxic to both the test organisms. The relative toxicity of the insecticides may be put in the order as: carbofuran4cypermethrin4carbaryl4fenvalerate in M. posthuma and carbofuran4carbaryl4fenvalerate4cypermethrin in E. fetida by both the test methods. However, the toxicity of these chemicals was more pronounced in M. posthuma than E. fetida in both the test procedures. Similar study on toxicity ranking of other insecticides based on LC50 values in P. excavatus has also been reported in the literature (De Silva et al. 2010). Comparison of the results of paper contact test with those obtained in soils clearly demonstrates that the contact test has no predictive values for the toxicity of an insecticide in soils, though it is important for the initial screening of the environmental chemicals. The differences between lowest and highest LC50 values of insecticides for M. posthuma and E. fetida in paper contact method were only 6.9 and 2.5-fold respectively while in soil they were over 38 and 26-fold. These data demonstrated that worms could tolerate higher concentrations in soil than on moist filter paper. This difference in the behavior of the insecticide may probably due to the rate of diffusion/uptake of insecticide from the medium into the body of the earthworm. It is well reported in the literature that insecticides are adsorbed on soil medium through strong binding by organic matter contents in soils (Davis, 1971; Van Gestel and Van Dis, 1988). Hence, the availability of insecticide for diffusion will be less from the soil than the impregnated filter paper. Contact filter paper test can be used as an initial screening technique to assess the relative toxicity of chemicals; however it fails to represent the situation in the soil ecosystem. Artificial soil test is more representative of natural environment of earthworms and acute toxicity data on several insecticides can be used in the ecological risk assessment on soil ecosystem. Carbamate insecticides are used to control foliar feeding and soil dwelling insects. They are strong inhibitors of acetylcholinesterase of nervous tissues in insects (Gupta and Sunderaraman, 1988). Tremors observed in the treated worms reflect the neurotoxic effect of the

3.3. Histopathological studies The histopathological examination of transverse sections of the control worm showed normal architecture of body wall showing continuous cuticular membrane, intact nature of circular and longitudinal muscles (Fig. 2). Earthworms, E. fetida exposed to 1.20 μg/cm2 of carbofuran treated with the insecticides revealed loss of normal architecture and disintegration of cuticular membrane, epidermal cells, circular and longitudinal muscles at 48 h of exposure in contact method, which may lead to bleeding and the fragmentation of the body. Similar damage of cuticular membrane and disintegration of circular and longitudinal muscles were also observed in earthworm, M. posthuma exposed to 0.5 mg/kg of carbofuran in soil medium. Similar pattern of histopathological changes were also observed with other insecticides. 3.4. Molecular orbital calculations and FTIR spectral measurements The partial atomic charges on the atoms of carbaryl and carbofuran (Fig. 3) are presented in Table 3. These electronic charges play a significant role in the biological activity of carbamate insecticides. Total electronic charge on the active wedges of these insecticides has been calculated by adding the individual charges on their atoms. The FTIR frequencies of carbaryl in the range of 450–4000 cm  1 are

Table 2 Summary of the results for the acute toxicity with artificial soil test to Eisenia fetida and Metaphire posthuma. Insecticides

M. posthuma 7-days LC50(95%CI) mg kg  1

E. fetida 7-days LC50( 95%CI) mg kg  1

M. posthuma 14-days LC50 (95%CI) mg kg  1

E. fetida 14-days LC50(95%CI) mg kg  1

Cypermethrin Fenvalerate Carbofuran Carbaryl

10.56 22.13 0.63 11.02

573.94 43.49 24.31 30.34

9.83 18.66 0.49 8.57

551.14 40.77 21.15 26.86

(8.23–13.55) (19.56–25.03) (0.42–0.96) (8.64–14.06)

(472.92–696.55) (33.72–56.08) (17.03–34.71) (24.42–37.69)

(7.77–12.96) (14.14–24.62) (0.33–0.74) (6.92–10.63)

(450.37–674.45) (31.81–52.26) (18.49–24.19) (20.45–35.28)

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Fig. 2. Histological changes in M. posthuma and E. fetida. (a) Body wall of control E.fetida showing intact nature of muscles and epidermal layer, (b) E. fetida exposed to carbofran (1.20 μg/cm2, 48 h) by contact method, (c) M. posthuma exposed to carbofuran (0.24 μg/cm2, 48 h) by contact method, (d) E. fetida exposed to fenvalerate (1.20 μg/cm2, 48 h) by contact method, (e) E. fetida exposed to cypermethrin (1.20 μg/cm2, 48 h) by contact method, (f) E. fetida exposed to carbofuran (10.5 mg/kg, 14 d) by soil toxicity bioassay, (g) M. posthuma exposed to cypermethrin (8 mg/kg, 14 d) by soil toxicity bioassay and (h) E.fetida exposed to fenvalerate (25 mg/kg, 14 ) by soil toxicity bioassay.

insecticides. The results from this study show that carbofuran and carbaryl are highly toxic to both the earthworm species even after 72 h (filter paper contact mehod) and 14 days (soil method). Our earlier studies also showed that 30 days exposure to carbaryl significantly affected reproduction in M. posthuma (Gupta and

Saxena, 2003). Lethal concentrations observed can be correlated with the field concentrations by assuming the applied dosage is homogeneously distributed over the top 2.5 cm soil layer, e.g., 0.49 mg/kg (LC50 value of carbofuran in M. posthuma) is equivalent to 0.17 kg/ha (Van Gestel, 1992). This is much lower than the actual

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223

C11H3

H3C9 C10 7

C3

9

15

10 11

14

N1 H3C2

13

H

Wedge 1

O4

C12H2

7

8

6

O5

O4

O8

13

6

O5 C3

14

16

15

N1

12

H3C2

Wedge 2

H Wedge 1

Wedge 2

Carbaryl

Carbofuran N

N

Cl Cl

C

C

CH

O

C

O C

C

O

CH

Cl

CH

O

CH

O

O

CH CH3

CH3

CH3

Acid Moiety

CH3

Acid Moiety

Cypermethrin

Fenvalerate

Fig. 3. Molecular structure of insecticides used in the study.

Table 3 Partial atomic charges on various atoms of carbaryl and carbofuran. Carbaryl

Carbofuran

Atom

Charge

Atom

Charge

N1 H1 C2 H2A H2B H2C C3 O4 O5 C6 C7 H7 C8 H8 C9 H9 C10 C11 H11 C12 H12 C13 H13 C14 H14 C15

 0.3559 0.1947  0.0780 0.0651 0.0762 0.0862 0.4105  0.2919  0.2576 0.1214  0.0733 0.0739  0.0579 0.0666  0.0617 0.0646 0.0046  0.0577 0.0620  0.0634 0.0663  0.0606 0.0642  0.0596 0.0650  0.0039

N1 H1 C2 H2A H2B H2C C3 O4 O5 C6 C7 O8 C10 C11 H11A H12B H12C C12 H12A H12B C13 C14 H14 C15

 0.4495 0.2687  0.1875 0.1223 0.1095 0.1148 0.4567  0.3203  0.2725 0.1184 0.1178  0.2478 0.1952  0.3148 0.1069 0.1020 0.1092  0.1011 0.0979 0.0979 0.0019  0.1011 0.0885  0.0963

applied concentration in the field (0.5 to 4 kg/ha). So, an extreme care should be taken for the use of these compounds in agriculture to avoid serious damage to earthworms. Synthetic pyrethroid insecticides are extensively used worldwide for agriculture pest control program due to their higher

Fig. 4. FTIR spectrum of carbaryl (4000–450 cm  1).

efficiency, relatively shorter half life in soil and lower mammalian toxicity. Pyrethroids alter the normal function of insect nerves by modifying the kinetics of voltage-sensitive sodium channels (Soderlund et al., 2002). It has been reported in the literature that most of the pyrethroid insecticides are not toxic to earthworms in artificial soil test. Our findings show that cypermethrin and fenvalerate are more toxic to M. posthuma than E. fetida. Cypermethrin was least toxic to E. fetida. This also further indicates the rational use of these compounds in agriculture to avoid damage to the soil ecosystem. Histopathological evaluation of transverse section of worm exposed to these insecticides showed that the normal architecture of body wall was largely affected. Cuticular membrane, epidermal layers, circular and longitudinal muscles were completely disintegrated leading to bleeding, constriction and fragmentation of the body. Similar histological changes were earlier reported in carbaryl and metal treated earthworms (Gupta and Sundararaman, 1988,

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1990; Lourenco et al., 2011). Such histopathological observations play a significant role in understanding the mode of action of these compounds at cellular and molecular levels. Studies on structure-activity relationships of pyrethroids demonstrate that both insecticidal and mammalian toxicity are largely dependent on molecular shape, size and sterochemical structures of acid moiety of the molecule (Soderlund et al., 2002). Structure-dependent activity in carbamates was also demonstrated by Feaster et al., 1996. In the present study, we have proposed a possible mechanism of biological activity of carbamate insecticides based on excess electronic charge on active site, orientation and spectroscopic data. The molecular mechanism responsible for toxicity of these insecticides in earthworms is not yet fully understood. However, histopathological and biochemical studies on carbamate and pyrethroid insecticides indicate that these insecticides primarily interact with body wall proteins/lipids of earthworm (Gupta and Sundararaman, 1988; Mosley et al., 2003a; Tripathi and Bhardwaj, 2005). As carbaryl and carbofuran belong to carbamate group of insecticides, carbamate moiety has been considered to be a probable active site. To demonstrate the biological activity of carbamates, the molecule of carbaryl has been divided in two parts, wedge 1 and wedge 2 (Fig. 3). Wedge 1 carrying excess charge of 0.1507 is the prospective active wedge. Wedge 1 contains highly electronegative N,O atoms and active methyl group. Wedge 2 offers a relatively planar part and plays a role of anchor support for such interactions. The molecular vibrations of active wedge atoms would polarize the earthworm body wall protein/ lipid molecules and bind there. In principle, this polarization occurs due to change in electrical polarizability during the vibration of interacting groups, thus causing disturbances. Such disturbances/ toxicity can be monitored through N–H, CH3, CQO, and C–O modes of vibrations which appear at 3315, 2942, 1713, 1418, 1388, 1363, 1011 669, 552 cm  1 in FTIR spectrum of carbaryl (Fig. 4). The assignments of the observed frequencies were made on the basis of band shape, relative peak intensity and a comparison with similar molecules (Bellamy, 1975; Saxena and Gupta, 2005; Alverdi et al., 2004). The binding of carbaryl with protein/lipids may cause destabilization of structures of these biomolecules, causing toxicity to earthworm. The same is true with carbofuran with excess electronic charge of 0.1578 on active wedge 1. However, the charge on the active wedge in carbofuran is greater than the carbaryl, which is probably responsible for greater toxicity of carbofuran in both the earthworm species. The above proposed mechanism was also supported by the studies made by Lewis et al., 1998, who analyzed the biological activity of several compounds from their molecular and electronic structure using similar molecular orbital procedure. The similar mechanism can be proposed to fenvalerate and cypermethrin insecticides (Fig. 3). In these insecticides, acid moiety may be considered to be an active site due to large polarization associated with Cl/Br, O and N atoms. These atoms are capable of polarizing earthworm body wall protein/lipid molecules and may bind there, causing toxicity.

5. Conclusion The present study suggests that the sensitivity of M. posthuma for toxicity assessment of environmental chemicals is compatible to other earthworm species. The test concentrations being less effective in E. fetida produced more pronounced toxicological effects in M. posthuma, which supports greater sensitivity of M. posthuma over E. fetida. A large difference in LC50 values in E.fetida and M. posthuma again supports the better sensitivity of M. posthuma over E. fetida. Of all the insecticides tested, carbofuran was the most hazardous to both the test organisms and would affect the earthworm populations. Molecular vibrations involving

active wedge atoms with excess electronic charge are responsible for recognition (via polarization) and binding of carbamates to earthworm body wall protein/lipid molecules, causing toxicity. The mechanism proposed can be applied to predict the toxicity potential of other carbamates.

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