INTRODUCTION:

The earthworms represented a major proportion of total biomass of terrestrial invertebrates up to 80% which play an important role in ingestion of large quantity of decomposed litter, manure and other organic matter and convert it into rich top soil (Sandoval et al 2001). Earthworms are regarded as a reference compartment to observe soil contaminant bioavailability and are used to evaluate the lethal and sub lethal effects of chemicals contaminants and pollutants (Rida 1997).

Eguchi et al (1995) reported that earthworms are considered not only composting agents but also nature’s ploughs, aerators, moisture retainers, crushers, and biological agents. Vermicasting have led to significant increases in the yields of several crops, with significant reductions in pesticides use and almost zero chemical fertilizer imputes (Dash and Senapathi 1986).

Earthworms play an important role in soil fertility and they are also important contributors to the recycling of carbon and nitrogen in the ecosystem and most suitable bio-indicator organisms for testing chemicals in soil (Edwards and Lofty 1977; Callahan 1988; Goats and Edwards 1988; Cock et al 1980; Gobi et al 2004). Generally, herbicides manifest low toxicity on earth worms, but indirectly can produce the reduction of the populations by decreasing the organic matter input and weed coverage. Earthworms can contribute extensively to soil formation through consumption of dead plant and animal matter, mixing of the particles during digesting, depositing their casts throughout the soil column and improving aeration and drainage of the soil burrowing (Kavitha et al 2011). Earthworms are also important contributors to the recycling of carbon and nitrogen in the ecosystem. This makes them one of the most suitable bioindicator organisms for testing chemicals in the soil (Callahan 1988; Goats and Edwards 1988).

Reproduction in earthworms is peculiar because of hermaphroditism (Kale et al 1982, Julka 1988; Kaushal and Bisht 1992; Kaushal et al 1995). Gobi (2010) reported that the percentage of clitellum development decreased with increasing concentration of butachlor. Use of specific herbicides, fungicides and insecticides in the agricultural field can be highly toxic to earthworm and they will suppress or nearly eliminated earthworm population (Williamson 2000). Earthworm have been used a model animals for the study the effect of agrochemicals on soil fauna. Herbicides have been adverse effect on the survival of earthworm (Van- Gestrel and Van Dis 1988; Ribidoux et al 1999). The herbicide 2,4-D has very toxic for growth and reproduction of earthworm Eisenia foetida (Helling et al 2000; Zhou et al 2007; Corriela and Moreira 2010).

2,4-D is an herbicide and secondarily a plant growth regulator. Xiao et al (2006) reported that the herbicide acetochlor caused adverse effect on the sperm count and DNA in Eisenia foetida. Several other studies have demonstrated the lethality of herbicide and pesticide to earthworm and their histopathological effects (Gupta and Sundarman 1988; Sorour and Larink 2001; Lady and Link 2003; Gobi et al 2004; Rombke et al 2007; Moish 2009). 2,4-D has been shown to have negative impacts on a number of groups of animals. 2,4-D exposure reduced hatchling success and caused birth defects in birds, (Duffard et al 1981). The toxicity of 2,4-D to fish is variable, with the ester form of 2,4-D expressing greater toxicity than other forms. A product of the breakdown process of 2,4-D is dicholorophenol. Which is extremely toxic to earthworms, 15 times more toxic than 2,4-D itself (Roberts Dorough 1984). Singh and Kumar (2014) reported that the earthworm Eutyphoeus waltoniis found abundantly in agricultural fields of different localities of eastern Uttar Pradesh. Eutyphous waltoni is the standard test organism used in terrestrial ecotoxicology because it can easily feed on a variety of organic wastes.

The aim of present study was to investigate the sub lethal effect of LC₅₀ herbicide 2,4-D of in different combination of buffalo dung with agro wastes of as feed materials under laboratory conditions.

MATERIAL AND METHODS:

The cultured earthworm Eutyphoeus waltoni were used for the experiment. The buffalo dung, wheat straw and gram bran were collected from different part of Gorakhpur district of U.P. India. Commercially available herbicides 2,4-D (2’-4’ dicholorophynoxy acetic acid) was purchased by Earth Care Pvt. Ltd Kolkatta (India) and used in the experiment at different concentrations at laboratory conditions. The LC₅₀ of 2,4-D of different combinations of feed materials were in observed previous work.

Measurement of Reproduction and Development of Eutyphoeus Waltoni:

The experiment for study of reproduction and development of earthworm Eutyphoeus Waltoni were performed by the method of Chauhan and Singh (2012).The experiment were conducted on cemented earth surface. One kg of different combinations of animal dung, agro wastes in different ratio were kept on 30 × 30× 10 cm in bed form at room temperature in dark. Buffalo dung is used as control. The vermicomposting beds were turned over manually every 24 hours for 10 days in order to eliminated volatile substances. After treatment 20% and 60% of LC₅₀ of 2,4-D of different combination of feed materials of all exposure periods, ten adult earthworms were incubated in each vermibed for the observation of clitellum development, cocoon production, hatchlings and survival of hatchlings. The clitellum development was observed after one week of earthworms inoculation. After this, cocoons were observed each and every day until cocoon production become ceased (about 13- 14 weeks). After isolation, cocoons were freshly laid inside a Petri dish containing moist filtered paper at 30±2 °C and 70±5 % RH. Hatching of cocoons were determined after stipulated incubation and number of progeny emerged per cocoon were recorded. The percent survivability of hatchlings was recorded after hatching from cocoons in same treated feed materials.

Each experiment was replicated at least six times. Data were analyzed by student t- test and analysis of variance (ANOVA) in between different exposure periods and different combinations of feed materials for all parameters. After 13 weeks of experiment, the treated earthworms were transferred into untreated same feed materials of buffalo dung and agro-wastes for the observation of withdrawal of clitellum development, cocoon production/worm, hatchlings and survival of hatchlings.

RESULTS:

In control earthworms clitellum development (days), cocoon production/earthworm, hatchlings/cocoon and survival of hatchlings was 26.29±0.53, 32.39±0.54, 3.79 and 2.68, respectively (Table 1-6). The reduction in clitellum development (days), cocoon production/ earthworm and hatchlings/cocoon as well as survival of hatchlings were observed in treatment of sub lethal exposure of LC₅₀ of 2,4-D of different combination of feed materials of all exposure periods during the experiment. The treatment of 20% and 60% of 24h LC₅₀ of 2,4-D of BD+WS+GB caused a maximum reduction in clitellum development 28.08±0.57, 31.38±0.61, cocoon production/ earthworm 28.32±0.56, 26.25±0.44 and hatchling/cocoon 87.10% and 70.25% as well as survival of hatchling 78.69% and 71.09%, respectively (Table 1). The time and dose dependent effect were observed after treatment of sub-lethal exposure of (20% and 60%) of LC₅₀ of all exposure periods of different combinations of buffalo dung with agro wastes on the clitellum development, cocoon production and hatchlings of cocoon as well as survival of hatchlings of earthworm Eutyphoeus waltoni (Table 1-6).

Table 1. Effect of sub lethal exposure of 24h LC₅₀of herbicide 2,4-D of different combinations of feed material of buffalo dung with agro-wastes on the clitellum development, cocoon production, hatchling, and survival of hatchling of earthworms Eutyphoeus waltoni.

Combinations of wastes

Sub lethal Concentration of 24h LC₅₀of herbicide

2,4-D

Clitellum development

(in day)

Cocoon production

/worm

Hatchling

/ Cocoon

Survival of hatchling

Control

26.29±0.53

32.39±0.42

3.79 (100)

2.68

(100)

20% of LC₅₀

(110.02mg/kg)

34.53±0.55*

20.19±0.53*

2.13

(56.12)

1.24

(46.23)

60% of LC₅₀

(330.06mg/kg)

37.23±0.53*

18.23±0.53*

1.79

(47.14)

1.20

(44.85)

BD+Ws

20% of LC₅₀

(108.04mg/kg)

32.38±0.62*

21.52±0.76*

2.47

(65.10)

1.54

(57.61)

60% of LC₅₀

(324.12mg/kg)

34.29±0.58*

20.19±0.59*

1.92

(50.67)

1.40

(52.09)

BD+Gb

20% of LC₅₀

(108.08mg/kg)

33.64±0.58*

22.14±0.55*

2.09

(55.04)

1.33

(49.76)

60% of LC₅₀

(324.26mg/kg)

36.22±0.58*

20.32±0.52*

1.78

(46.84)

1.27

(47.22)

BD+Ws+Gb

20% of LC₅₀

(102.16mg/kg)

28.08±0.57

28.32±0.56

3.30

(87.10)

2.11

(78.69)

60% of LC₅₀

(306.48mg/kg)

31.38±0.61*

26.25±0.44*

2.66

(70.25)

1.91

(71.09)

BD=Buffalo dung, Ws=wheat straw, Gb=Gram bran. Each value is Mean ±SE of six replicates. Value in parentheses are percent change with control taken as 100%.* Significant (p< 0.05, t –test) difference between control and treated groups for clitellum development and cocoon production.

Table 2. Effect of sub lethal exposure of 48h LC₅₀of herbicide 2,4-D of different combinations of feed material of buffalo dung with agro-wastes on the clitellum development, cocoon production, hatchling, and survival of hatchling of earthworms Eutyphoeus waltoni.

Combinations of wastes

Sub lethal concentration of 48 h LC₅₀of herbicide

2,4-D

Clitellum development

(in day)

Cocoon production

/worm

Hatchling

/Cocoon

Survival of hatchling

Control

26.29±0.53

32.39±0.42

3.79

(100)

2.68

(100)

20% Of LC₅₀

(97.06mg/kg)

35.91±0.51*

19.05±0.34*

1.99

(52.44)

1.21

(45.21)

60% of LC₅₀

(291.18mg/kg)

38.42±0.52*

17.33±0.52*

1.67

(44.15)

1.15

(43.01)

BD+Ws

20% of LC₅₀

(91.34 mg/kg)

33.91±0.82*

20.45±0.62*

2.52

(66.48)

1.53

(56.98)

60% of LC₅₀

(274.03mg/kg)

33.39±0.56*

19.35±0.79*

1.99

(52.46)

1.41

(52.71)

BD+Gb

20% of LC₅₀

(92.14mg/kg)

35.06±0.57*

23.19±0.51*

2.34

(61.81)

1.30

(48.56)

60% of LC₅₀

(276.43mg/kg)

37.42±0.63*

21.16±0.56*

2.15

(56.82)

1.22

(45.69)

BD+Ws+Gb

20% of LC₅₀

(94.91mg/kg)

29.33±0.68

29.85±0.53

3.29

(86.76)

2.06

(76.92)

60% of LC₅₀

(284.74mg/kg)

32.33±0.57*

26.92±0.40*

2.65

(70.04)

1.88

(70.01)

BD=Buffalo dung, Ws=wheat straw, Gb=Gram bran. Each value is Mean± SE of six replicates. Value in parentheses are percent change with control taken as 100%.* Significant (p< 0.05, t –test) difference between control and treated groups for clitellum development and cocoon production.

Table 3. Effect of sub lethal exposure of 72h LC₅₀of herbicide 2,4-D of different combinations of feed material of buffalo dung with agro-wastes on the clitellum development, cocoon production, hatchling, and survival of hatchling of earthworms Eutyphoeus waltoni.

Combinations of wastes

Sub lethal Concentration of 72 h LC₅₀ of herbicide 2,4-D

Clitellum development

(in day)

Cocoon production

/worm

Hatchling

/Cocoon

Survival of hatchling

Control

26.29±0.53

32.39±0.54

3.79

(100)

2.68

(100)

20% of LC₅₀

(85.09mg/kg)

37.01±0.53*

18.62±0.44*

1.94

(51.22)

1.21

(45.02)

60% of LC₅₀

(255.27mg/kg)

38.41±0.52*

17.11±0.41*

1.60

(42.23)

1.12

(41.79)

BD+Ws

20% of LC₅₀

(79.90mg/kg)

34.34±0.56*

21.64±0.68*

2.46

(64.87)

1.39

(51.92)

60% of LC₅₀

(239.72mg/kg)

36.47±0.52*

20.85±0.67*

2.34

(61.82)

1.32

(49.31)

BD+Gb

20% of LC₅₀

(80.82mg/kg)

35.36±0.59*

24.19±0.56*

2.23

(58.88)

1.14

(42.68)

60% of LC₅₀

(242.46mg/kg)

37.23±0.53*

22.20±0.57*

2.09

(55.20)

1.08

(40.31)

BD+Ws+Gb

20% of LC₅₀

(81.92mg/kg)

29.91±0.57

30.12±0.51

3.25

(85.74)

1.96

(73.05)

60% of LC₅₀

(245.76mg/kg)

32.32±0.53*

27.55±0.57*

2.65

(70.01)

1.85

(69.08)

BD=Buffalo dung, Ws=wheat straw, Gb=Gram bran. Each value is Mean±SE of six replicates. Value in parentheses are percent change with control taken as 100%.* Significant (p< 0.05, t –test) difference between control and treated groupsfor clitellum development and cocoon production.

Table 4. Effect of sub lethal exposure of 96h LC₅₀of herbicide 2,4-D of different combinations of feed material of buffalo dung with agro-wastes on the clitellum development, cocoon production, hatchling and survival of hatchling of earthworms Eutyphoeus waltoni.

Combinations of wastes

Sub lethal Concentration of

96 h LC₅₀of herbicide 2,4-D

Clitellum development

(in day)

Cocoon production

/worm

Hatchling

/Cocoon

Survival of hatchling

Control

26.29±0.53

32.39±0.42

3.79

(100)

2.68

(100)

20% of LC₅₀

(72.12mg/kg)

37.42±0.48*

16.53±0.53*

1.86

(48.95)

1.20

(44.75)

60% of LC₅₀

(216.36mg/kg)

39.11±0.51*

14.18±0.39*

1.54

(40.52)

1.12

(41.62)

BD+Ws

20% of LC₅₀

(68.53mg/kg)

35.36±0.54*

22.32±0.50*

2.40

(63.23)

1.30

(48.67)

60% of LC₅₀

(205.59mg/kg)

38.45±0.57*

21.02±0.55*

2.30

(60.78)

1.20

(45.68)

BD+Gb

20% of LC₅₀

(70.68mg/kg)

35.58±0.48*

24.92±0.52*

2.20

(57.95)

1.16

(43.25)

60% of LC₅₀

(212.05mg/kg)

38.63±0.52*

22.91±0.53*

2.11*

(54.76)

1.09

(40.65)

BD+Ws+Gb

20% of LC₅₀

(68.79mg/kg)

30.04±0.01

30.58±0.53

3.13

(82.48)

1.90

(70.71)

60% of LC₅₀

(206.39mg/kg)

34.15±0.55*

27.96±0.28*

2.62

(69.09)

1.74

(65.01)

Table 5. Effect of sub lethal exposure of 120h LC₅₀of herbicide 2,4-D of different combinations of feed material of buffalo dung with agro-wastes on the clitellum development, cocoon production, hatchling and survival of hatchling of earthworms Eutyphoeus waltoni.

Combinations of wastes

Sub lethal Concentration of

120 h LC₅₀of herbicide 2,4-D

Clitellum development

(in day)

Cocoon production

/worm

Hatchling

/Cocoon

Survival of hatchling

Control

26.29±0.53

32.39±0.42

3.79

(100)

2.68

(100)

20% of LC₅₀

(61.81mg/kg)

37.97±0.49*

16.11±0.52*

1.75

(46.23)

1.17

(43.75)

60% of LC₅₀

(185.43mg/kg)

40.92±0.46*

13.22±0.51*

l.49

(39.22)

1.08

(40.22)

BD+Ws

20% of LC₅₀

(57.81mg/kg)

35.36±0.52*

22.65±0.52*

2.34

(61.73)

1.23

(45.89)

60% of LC₅₀

(173.43mg/kg)

37.49±0.48*

20.18±0.45*

2.17

(57.22)

1.15

(43.08)

BD+Gb

20% of LC₅₀

(60.83mg/kg)

36.40±0.17*

24.98±0.17*

2.11

(55.62)

1.07

(40.02)

60% of LC₅₀

(182.49mg/kg)

39.92±0.52*

23.04±0.53*

1.95

(51.47)

1.06

(39.58)

BD+Ws+Gb

20% of LC₅₀

(59.94mg/kg)

30.59±0.58

31.05±0.51

3.06

(80.67)

1.75

(65.32)

60% of LC₅₀

(179.84mg/kg)

34.65±0.49*

37.89±0.52*

2.56

(67.47)

1.62

(60.44)

Table 6. Effect of sub lethal exposure of 240h LC₅₀of herbicide 2,4-D of different combinations of feed material of buffalo dung with agro-wastes on the clitellum development, cocoon production, hatchling and survival of hatchling of earthworms Eutyphoeus waltoni.

Combinations of wastes

Sub lethal Concentration of

240 h LC₅₀of herbicide 2,4-D

Clitellum development

(in day)

Cocoon production

/worm

Hatchling

/Cocoon

Survival of hatchling

Control

26.29±0.53

32.39±0.42

3.79

(100)

2.68

(100)

20% of LC₅₀

(61.93mg/kg)

36.25±0.53*

15.23±0.50*

1.75

(46.06)

1.15

(43.09)

60% of LC₅₀

(185.79mg/kg)

38.33±0.26*

14.24±0.51*

1.44

(38.05)

1.08

(40.13)

BD+Ws

20% of LC₅₀

(50.52mg/kg)

37.41±0.37*

26.72±0.51*

2.28

(60.16)

1.16

(43.28)

60% of LC₅₀

(151.57mg/kg)

39.28±0.52*

27.56±0.53*

1.94

(51.25)

1.08

(40.28)

BD+Gb

20% of LC₅₀

(54.30mg/kg)

35.49±0.56*

26.41±0.53*

1.98

(52.21)

1.06

(39.53)

60% of LC₅₀

(162.92mg/kg)

37.26±0.38*

25.54±0.49*

1.83

(48.18)

0.96

(35.91)

BD+Ws+Gb

20% of LC₅₀

(53.79mg/kg)

28.39±0.19

33.95±0.46

2.87

(75.82)

2.00

(74.81)

60% of LC₅₀

(161.38mg/kg)

31.62±0.38*

37.98±0.62*

2.48

(65.39)

1.77

(65.96)

Summary of computation of analysis of variance (ANOVA) of the Clitellum development and time with combinations

20% of LC₅₀ of 2,4-D
Source of variation	d.f.	S.S.	Variance	F-ratio	P<0.01	P<0.05
Between treatment	5	478.1	95.6	74.1	4.10	2.71
Between Time	4	66.0	16.5	12.8	4.43	2.87
Error	20	25.8	1.3
Total	29	569.8
60% of LC₅₀of 2,4-D
Between treatment	5	669.8	134.0	118.8	4.10	2.71
Between Time	4	55.2	13.8	12.2	4.43	2.87
Error	20	22.6	1.1
Total	29	747.5

F-value significance at P<0.01; *Not significant; ** significant at P<0.05

Summary of computation of analysis of variance (ANOVA) of the data Cocoon production and time with combinations

20% of LC₅₀ of 2,4-D
S. variation	d.f.	S.S.	Variance	F-ratio	P<0.01	P<0.05
Between treatment	5	776.9	155.4	57.7	4.10	2.71
Between Time	4	10.7	2.7	1.0*	4.43	2.87
Error	20	53.8	2.7
Total	29	841.4
60% of LC₅₀of 2,4-D
Between treatment	5	933.4	186.7	45.3	4.10	2.71
Between Time	4	43.5	10.9	2.6*	4.43	2.87
Error	20	82.3	4.1
Total	29	1059.2

F-value significance at P<0.01; *Not significant; ** significant at P<0.05

Summary of computation of analysis of variance (ANOVA) of the hatchling and time with combinations

20% of LC₅₀ of 2,4-D
Source of variation	d.f.	S.S.	Variance	F-ratio	P<0.01	P<0.05
Between treatment	5	14.4	2.9	422.6	4.10	2.71
Between Time	4	0.2	0.1	8.5	4.43	2.87
Error	20	0.1	0.0
Total	29	14.8
60% of LC₅₀of 2,4-D
Between treatment	5	17.3	3.5	257.7	4.10	2.71
Between Time	4	0.1	0.0	2.4*	4.43	2.87
Error	20	0.3	0.0
Total	29	17.7

F-value significance at P<0.01; *Not significant; ** significant at P<0.05

Summary of computation of analysis of variance (ANOVA) of the survival of hatchling and time with combinations

20% of LC₅₀ of 2,4-D
Source of variation	d.f.	S.S.	Variance	F-ratio	P<0.01	P<0.05
Between treatment	5	9.6	1.9	171.6	4.10	2.71
Between Time	4	0.1	0.0	3.2**	4.43	2.87
Error	20	0.2	0.0
Total	29	9.9
60% of LC₅₀of 2,4-D
Between treatment	5	10.6	2.1	401.8	4.10	2.71
Between Time	4	0.2	0.0	9.5	4.43	2.87
Error	20	0.1	0.0
Total	29	10.9

F-value significance at P<0.01; *Not significant; ** significant at P<0.05

It is evident from results, among all treatment of sub-lethal exposure of LC₅₀ of different feed materials, the tertiary combination of buffalo dung with wheat straw and gram bran have more potency to increase the tolerance power of earthworm Eutyphoeus waltoni in agricultural fields. The use of vermicompost of buffalo dung with wheat straw and gram bran is beneficial for better productivity of crops as well as provided better nourishment to enhance the population and tolerance power against herbicides and other chemicals.

ACKNOWLEDGEMENT:

Authors are thankful to Prof. D. K. Singh, Department of Zoology, Deen Dayal Upadhyaya Gorakhpur University, Gorakhpur 273009, U.P. India, for design and analysis and interpretation of data of the manuscript.

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Received on 24.10.2014 Modified on 01.11.2014

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