it gives the range to be used for the definitive acute toxicity test. Six concentration
levels of 15, 23, 34, 50, 75 and 112 mg a.i./kg (dry weight of natural soil) along with
control were used for determining 0% and 100% mortality at 7th and 14th day. Three
replicates were used for each test.
20.2.6 Acute Toxicity Test
Four concentration levels of MP 2% dust a.i. along with control were selected for the
acute toxicity test. The concentration levels were 25, 40, 55 and 70 mg a.i./kg of
dried natural soil. Three replicates were used for each dose. The suspended solution
of MP 2% dust a.i. was thoroughly mixed in the natural soil. For this test, adult
earthworms (with clitellum) having a weight between 300 and 450 mg were used.
Earthworms that were to be used were acclimatized for 24 h in natural soil before the
start of the test. Ten earthworms were added in each reactor after one day. The
earthworm mortality and biomass change were monitored at 7th and 14th day after
proper cleaning and drying of earthworms on the Whatman filter paper. The reactors
examined on the 7th day were replaced by the earthworms after the counting and
weighing process was completed. The median lethal concentration (LC 50 ) was
determined in this acute test as per the procedure mentioned in the ISO 11268-1
(ISO 2012).
20.2.7 Chronic Toxicity Test
Six concentration levels of MP of 2.5, 4.0, 6.4, 10.2, 16.3 and 26.2 mg a.i./kg of
dried natural soil along with control were selected for the test. Ten earthworms were
placed in each reactor. Three replicates were used for each dose and the test was
administered for the duration of 28 and 56 days. According to the OECD 222 test
(OECD 2016), the feeding material, i.e. powdered septic tank sludge was added on
the day after the addition of the earthworms. Around 5 g of sludge as feed was spread
over the soil surface in each reactor and was moistened by adding water. The
production of cocoons from mature earthworms requires a time span of 28 days.
During the period of 28 days, 5 g of sludge as feed was added once a week. The
mature earthworms were removed on the 28th day from the reactors and the change
in biomass was noted after cleaning and drying the earthworms on Whatman filter
paper. The number of cocoons produced in each reactor was counted and left in the
reactors for the remaining 4 weeks without any further addition of feeding materials.
According to Shi et al. (2007), the weight of the earthworms was measured and used
for computing growth inhibition. The growth inhibition is given by:
20 Effect of Methyl Parathion on the Growth and Reproduction of Eisenia. . .
355
levels of 15, 23, 34, 50, 75 and 112 mg a.i./kg (dry weight of natural soil) along with
control were used for determining 0% and 100% mortality at 7th and 14th day. Three
replicates were used for each test.
20.2.6 Acute Toxicity Test
Four concentration levels of MP 2% dust a.i. along with control were selected for the
acute toxicity test. The concentration levels were 25, 40, 55 and 70 mg a.i./kg of
dried natural soil. Three replicates were used for each dose. The suspended solution
of MP 2% dust a.i. was thoroughly mixed in the natural soil. For this test, adult
earthworms (with clitellum) having a weight between 300 and 450 mg were used.
Earthworms that were to be used were acclimatized for 24 h in natural soil before the
start of the test. Ten earthworms were added in each reactor after one day. The
earthworm mortality and biomass change were monitored at 7th and 14th day after
proper cleaning and drying of earthworms on the Whatman filter paper. The reactors
examined on the 7th day were replaced by the earthworms after the counting and
weighing process was completed. The median lethal concentration (LC 50 ) was
determined in this acute test as per the procedure mentioned in the ISO 11268-1
(ISO 2012).
20.2.7 Chronic Toxicity Test
Six concentration levels of MP of 2.5, 4.0, 6.4, 10.2, 16.3 and 26.2 mg a.i./kg of
dried natural soil along with control were selected for the test. Ten earthworms were
placed in each reactor. Three replicates were used for each dose and the test was
administered for the duration of 28 and 56 days. According to the OECD 222 test
(OECD 2016), the feeding material, i.e. powdered septic tank sludge was added on
the day after the addition of the earthworms. Around 5 g of sludge as feed was spread
over the soil surface in each reactor and was moistened by adding water. The
production of cocoons from mature earthworms requires a time span of 28 days.
During the period of 28 days, 5 g of sludge as feed was added once a week. The
mature earthworms were removed on the 28th day from the reactors and the change
in biomass was noted after cleaning and drying the earthworms on Whatman filter
paper. The number of cocoons produced in each reactor was counted and left in the
reactors for the remaining 4 weeks without any further addition of feeding materials.
According to Shi et al. (2007), the weight of the earthworms was measured and used
for computing growth inhibition. The growth inhibition is given by:
20 Effect of Methyl Parathion on the Growth and Reproduction of Eisenia. . .
355