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Life Science Research and Sustainable Development ISBN: 978-98-84663-33-9

litter), moisture (20g %) and temperature (25+2°C). Earthworms were removed from culture pots
and kept half immersed in glass petriplates containing 30ml of tap water in 25+2°C temperature
for 24 hours to evacuate their guts (Dash and Patra, 1977). The study was carried out in plastic
culture pots under laboratory conditions following the protocol of Panda and Sahu (2002). In
brief, three heavy metals namely mercuric chloride (HgCl2), Copper sulphate (CuSO4) and Zinc
sulphate (ZnSO4) respectively used as the test chemicals were obtained from Ranbaxy Chemicals
Ltd. The pesticides were chosen on the basis of their extensive use in this area.
Toxicity tests using of adult (10cm length and 3 gm in weight) Perionyx excavatus were
conducted for 24 to 96 hours with different concentrations of each heavy metal (mg/kg dry soil
equivalent) applied to the soil as per the method used by Dash and Patra (1977). The
concentration were prepared in dilution of acetone and sprayed on the soil surface. After
evaporation of the solvent, the treated soil was thoroughly mixed to distribute the pesticide
evenly and enough water was added to bring the moisture content up to the field capacity. The
same procedure with pure acetone was applied to prepare the controls. Ten healthy gut cleared
earthworm were added to each pot. The experiment was maintained at 20 % soil moisture at 250
+2°C soil temperatures. Earthworm deaths were recorded and probit method of Finney (1971)
was followed to calculate LD50 value for adult earthworm, Panda and Sahu (2002). Calculation
of the Regression line: Assessment of median lethal dose LD50
The experiment was carried out for finding the range of concentrations for confirmatory
evaluation. The mortality was recorded for earthworm Perionyx excavatus at 24, 48, 72 and 96 h
exposure to mercuric chloride, copper sulphate and zinc sulphate, were corrected for natural
response by Abbott's formula (Abbott, 1925).
Abbott's formula:

The corrected mortality data was analyzed following the method of Finney (1971) to
determine the LD50 values. The LD50 values were obtained by probit regression line, taking test
concentration and corresponding percent mortalities on log value and probit scales respectively.
Straight line (regression line) was drawn between the points, which represent the survival
percentage versus concentration (APHA, 1989). From the points at which this line intersects the
50 percent survival line, a perpendicular line drawn to the concentration ordinate, indicates the
LD50 dose of that particular period. By graphical interpolation LD50 values were fixed and their
fiducial limits 95% upper and lower confidence limits were also calculated.
Results:
Results obtained from the present investigation clearly shows at the earthworm Perionyx
excavatus survived well from 1 to 6 mg /100 gm soil for 24 h, 1 to 4 mg/100 gm soil for 48h, 1 to
3 mg /100mg soil for 72 h, 1 to 1.5 mg /100 gm soil for 96 h for mercuric chloride. They also
survived well from 20 to 55 mg/100 mg of soil for 24 h, 20 to 30 mg /100 gm of soil for 48 h, 10 to
15 mg/100 gm of soil for 72, 5 to 10 mg /100gm of soil for 96 h for copper sulphate and 100 to 180
mg /100 gm of soil for 24 h, 50 to 60 mg /100 gm of soil for 48 h, 20 to 30 gm of soil for 72h and
10 to 15 mg/100 gm of soil for 96 h respectively for zinc sulphate.
The survival rate of earthworm gradually decreased in all media with an increase in
concentration of heavy metals. The observed lethal dose (LD50) for 24, 48, 72 and 96 h were

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