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21

Sminthurusviridis>Folsomia

3. Material and methods Experiments were conducted in both field and laboratory conditions.

3.1. Field experiments Selection of study site and its Physicography Field experiments were conducted in the grassland of Raja N. L. Khan Women’s College, Paschim Medinipur, West Bengal, India (22◦ 25’N 87◦ 19’ E). The study site is located in the laterite belt. Climatic conditions under the influence of South-West and North-East monsoon.

22

Location of Study Site (Midnapore Town, Paschim Medinipur, West Bengal, India)

Experimental site Location Field experiment had been done at the selected field site of college campus and laboratory experiment in the laboratory of Zoology department of college.

Climate The climate of the locality follows a hot tropical monsoon weather pattern. Summer last from April to mid of June with diurnal temperature ranging about 30◦C to 40◦C. Monsoon contributing the lion share of the annual rain fall of around 1500mm during mid June to late August- September. Mild winter lasts for 2-3 months during December –January having temperature ranging 10-14◦C.

Soil Soil of the site and its surroundings is basically laterite type with low organic matter. Physiochemical characteristics of soil are in chapter 4.

3.2. Method of field studies Well protected no crop fallow high land was selected for field experiment within the campus of R.N.L. Khan Women’s’ College. The area was divided into several small plots and soil was free from any previous contamination of anti biological chemicals.

3.3. Time and duration Field experiment, started in March’2011 and ended in Nov’2012.Pre-monsoon study for two consecutive year duration of each was March to May. Similarly post-monsoon period study last from September to November. Detailed time period of field experiment is in table 3.1. 23

Table-3.1 Time period of field experiment Year

Pre-monsoon

Post-monsoon

2011

March to May

September to November

2012

March to May

September to November

3.4.Experimental lay out Experimental area divided into several small plots measuring 3mx 2m having at least 1 m gap between the plots and of every side. For each herbicide 3 treated and 3 control plots were used for each period. Plots were never been used for second time which were used for earlier experiment. Plots were selected randomly for different herbicide and control. Plan of experimental lay out is in Fig. - 3.2

Fig. 3.2 Layout of Plots used for field study in each session 24

3.5. Herbicide used In the present investigation six herbicides belonging to five chemical groups viz, bipyrinidiums, dinitroanalines, acetamides,trizinones and phenoxyacetics represented by paraquat, pendimethalin,pretilachlor, metribuzin and 2,4-D(amine and sodium salt) respectively were used for preliminary screening study. All these herbicides are frequently used in agricultural field in the locality (personal interaction with the farmers). In the present study all six herbicides were used for direct toxicity bio assay. However on the basis of results of the direct toxicity bio assays and ecotoxicological relevance, other toxicity bio assays were made (both in field and laboratory) on only pendimethalin and pretilachlor representing dinitroanalines and acetamides respectively. Source of procurement of the herbicides used in the present study are given in table 3.2

Table 3.2 Details of Herbicides used in the study Chemical group of herbicide

Bipyrinidiums

Name of herbicide

Formulation

Source of procurement

Paraquat

24% SL

Krishirasayan Balasore, India

Trizinones

Metribuzin

70%WP

Krishirasayan Balasore, India

Dinitroalanines

Pendimethalin

30% EC

Krishi Rasayan Private Ltd. Jammu, India

Acetamides

Pretilachlor

50% EC

Krishirasayan Balasore, India

Phenoxyacetics

2,4-D sodium salt

80% WP

Godrej Agrovet Ltd. Mumbai, India

2,4-D amine

80% WP

KrishiRasayan Balasore, India

3.5.1. Technical information of the herbicides used Detailed technical information of the herbicides used is given in table 3.3 to 3.7 below. Chemical nature, usage and mode of action of each herbicide are given separately.

25

3.5.1.1.Paraquat: Paraquat is a non selective, fast acting weed killer, becomes biologically inactive upon contact with soil (Revkin, 1983). It is used to destroy wide range of unwanted vegetation in no crop land (Hood et al., 1963; Hood,1965; Huggins and Reganold,2008). Chemical structure of paraquat is given below. Detailed technical information is in table no-3.3

Paraquat Table: 3.3 Properties of Paraquat Properties Chemical Name: Chemical Formula: Melting Point: Solubility: Stability: days

Paraquat 1,1|- Dimethyl- 4, 4|- bipyridinium- dicholoride C12H14Cl2N2 -180ºC Soluble in water and alcohols Highly persistent in soil. Soil half-life >1000

26









3.5.1.2. Metribuzin Metribuzin is a selective herbicide used for control various types of weeds in field and crops by inhibiting photosynthesis.Metribuzin adsorb through foliage or uptake by root system and then trans- located to the other parts of the body (Chemical fact sheet, 1985). Photo-degradation and volatilization are not significant (WSSA,1989), so persists in environment. Persistence in soil depends on various climatic factors and soil characteristics (McEwen and Stephenson,1979; Hartley and Kidd, 1983; USEPA, 1988). It is reported that in animal it affect central nervous system and damaging kidney in test animal (ACGIH, 1986, USEPA, 1988). The detailed about the chemical is in table no- 3.4 The chemical structure of metribuzin is given below.

Metribuzin Table- 3.4 Properties of Metribuzin Properties Chemical Name: Chemical Formula:

Metribuzin 4–amino–6–tert–butyl–4,5-dihidro-3methylthio–1,2,4–triazin–5(4H)– one C8H14N4OS

Melting Point:

125o C – 126.5o C

Solubility:

Highly Soluble in Water

Stability:

Persists in environment, Soil half life is 1 – 6months

27









3.5.1.3. Pendimethalin Pendimethalin is a non-ionic dinitroalanine herbicide used for the selective control of grassy and broad leaf weeds in a variety of crops (Sinha, et al., 1996; Tsiropoulos and Miliadis, 1998; Bhowmik and Ghosh, 2002). It is moderately persistant in soil (Lee, et al., 2000)

Detailed technical information of pendimethalin is in table 3.5 Chemical structure of the herbicide is given below.

Pendimethalin Table 3.5 Properties of Pendimethalin



PropertiesPendimethalin Chemical Name: Chemical Formula: Melting Point: Solubility: Stability:

3,4- Dimethyl-2,6- dinitro-N_ Pentan-3-yl-alanine
C13H19N3O4
47-58oC
Soluble in Water Strongly adsorbs to soil organic matter and clay and not subject to microbial degradation. Soil half life is 90 days.





28









3.5.1.4. Pretilachlor Pretilachor is a selective herbicide of acetamidegroup , used to control various types of annual grass and board leaf weeds (Dharumarajan, et al., 2008). It is persists in soil and water for some times and also accumulate in plant parts (Asokaraja and Ali, 1995; Deepa, 2002; Tomoyoshi et al., 2004). Residual properties depend on climatic condition and soil content. Pretilachlordissipiated by photo decomposition, volatilisation and accumulate in environment (Raiet al., 1999).




Detailed technical information about pretilachlor is in table no – 3.6








Chemical structure of the herbicide in given below.

Pretilachlor

Table 3.6Properties of Pretilachlor

Properties Chemical Name: Chemical Formula:

Pretilachlor 2 – Chloro-N-(2,6diethylphenyl)N-(2-Propoxyethylacetamide)
C17 H26 Cl NO2

Melting Point:

135°C

Solubility:

Soluble in water, Benzene, Hexane, Methanol etc. Moderately stable in soil and water.Soil half life is about 10 days.

Stability:

29













3.5.1.5. 2, 4 – D 2,4 – D belongs to the phenoxy carboxylic acid group.Various from of 2, 4 – D are present, of which 3 major forms 2, 4 – D amine, 2, 4 – D sodium and 2, 4 – D ester are common in market. Sodium salt is generally more selective than amines which are more selective than ester. Ester form is not so popular to the farmers due to need of costlier solvent like petroleum oils. In the present investigation both sodium and amine of 2, 4 -D is used. In the soil 2, 4-D degraded by soil organisms though rate of decomposition depends on different characteristics of soil (Klingman, 1961; Muzik, 1970, Joy, 1980). The detailed technical information of 2, 4 – D Amine and Sodium salt is in table no. 3.7. The chemical structure of two types of 2, 4 – D are given below.














3














2, 4 – D Sodium










3




























33

33

33



2, 4 – D Amine Table 3.7Properties of 2,4-D Sodium and 2, 4-D Amine +%
,(
#



 







Sodium

C8 H5 Cl2 NaO3

200° C

 #























#




Water, Benzene 7 – 9 Days

 !


C10H13 Cl2 No3

138 – 141° C

Water, Benzene

30

7 – 9 Days


3.5.2. Application of herbicides Quality sprayer of 9 litre capacity was used for spraying herbicides in to the plots. Actual amount of herbicide required for particular plot were calculated on the basis of RAD and water requirement for uniform coverage was also calculated during pilot study. Single dose application of the herbicides was made in the morning hours. No watering was done for the next 24 hrs to avoid possible leaching and drifting of the chemical. Time of application was fixed in between 7.00A.M to 8.00 A.M.

3.6. Soil sampling Soil samples collected at random, simultaneously from the treated and control plots, at 0,3,7,15 and 30 days after application of chemicals to study selected species of collembola population. Control plots were also maintained for comparison. The time of sampling was fixed between 8.00 A.M. - 9.00 A.M. Both soil and ambient temperature was recorded at the time of sampling and soil moisture content was also measured.

Size and number of samples The size and number of samples were 5 x 5 cms of soil up to the depth of 10 cm and 8 no of samples were taken from each plot (Joy,1980; Chakravorty,1990). Samples were packed in labelled polythene packet and transfer to the laboratory for extraction.

Extraction, counting and identification ‘High gradient extraction chamber’ (Chakravorty, 1990) was used for extraction of moving micro arthropods. Animals were collected and preserved in 70% ethylalcohol in labelled specimen tubes. Sorting and counting of selected species of collembola were done by using 40X binocular microscope.

31

3.7. Laboratory Experiment 3.7.1.Test Organisms For the present investigation collembola have been selected as the test organisms. As mentioned in the previous chapter these organisms play vital role in maintaining and enhancing soil fertility and are also very susceptible non target soil organism to the herbicide pollution in agro ecosystem. Throughout the globe soil toxicologists considered these animals, one of the most important model test organisms for assessing toxicity of agrochemicals to soil ecosystem. There is a good number of works on this aspect by using collembola species in temperate countries, but much lesser in tropical ones. There are about 8000 described species of collembolaworld wide (Janssens,2010.). Indian collembola fauna represented by 299 species (Mandal and Hazra,2009).However, only a few species of collembola is used for the toxicological studies. The present study was carried out in Midnapore West district of West Bengal in India by using two native species of collembola, Xenyllawelchi and Cyphoderusjavanus. Both the species are very common in the locality. The systematic position of both the species are as follows:

3.7.2. Systematic position of the selected specimens Test Specimen-1

Test Specimen-2

Xenyllawelchi

Cyphoderusjavanus

Phylum-Arthropoda

Phylum- Arthropoda

Class- Insecta

Class- Insecta

Sub class- Apterygota

Sub class- Apterygota

Order- Hexapoda

Order- Hexapoda

Family- Arthropleona

Family- Arthropleona

Genus-Xenylla

Genus- Cyphoderus

Species-welchi

Species- javanus

3.8. Distribution and biology of selected specimens. i) Xenyllawelchi XenyllawelchiFolsom,1916 is a member of Indian collembola fauna. The species is distributed all over India. It is a common collembola species of West Bengal and also 32

commonly found in neighbouring Indian country like Bangladesh, Mayanmar, Thailand etc. The species generally located in soil with good amount of organic compost and also in organically rich system like composite pits, domestic and industrial organic waste discharge and deposit sites and also in agricultural fields receiving sewage and organic manure.

Biology:Xenyllawelchi generally remained aggregated. The adult attained the size near about 1.0 mm on average. The animal becomes sexually mature and female lays egg after 8th instars. Each stadium is determined by the presence of exuvia. The animal laid eggs in a batch. The number of eggs in a batch varied from a few to 50. The number of eggs laid by a female throughout its egg laying period is nearly 150. After egg laying period female becomes obese. Hatching success is about 85%-90%. Hatchingtakes place after 5-7 days of laying. First moult took place after 4th to 5th days of hatching. Subsequent moulting occur after every 5th to 6th days of previous moulting. Eggs are whitish in colour and more or less round in shape and gradually become enlarged. Eggs in batches are sticking together. Animal of 1st instars have little pigment in body. From 2nd instars pigmentation increased and animal becomes coloured. Over all life span varies from 110 to 120 days.

  

  
 
!

 



33

  
 




  
 


ii) Cyphoderus javanus CyphoderusjavanusBörner, 1906is also a member of Indian collembola. The species is very common and distributed all over India. The animal is edaphic and generally found in organic compost rich soil including agro-soil which receives organic manure regularly. Biology:Cyphoderusjavanus is a climbing species with long leg and larger antennae. Adult attained the size of nearly about 1.3-1.5mm in average. Animal becomes mature and female lay egg after 30-35 days of hatching. Hatching takes place after 4-5 days of egg laying and animal moults (marked by ecdycis) after 4-5 days of hatching. Subsequent moulting happens after 4-5 days of previous moulting. Mature female lays eggs one by one scatterdly on different adjoining places. Eggs are white in colour and oval in shape. Each egg attached with strata by sticky substances. Eggs gradually enlarged by increasing in size. After egg laying period female becomes obese and increased in size. Female animal lays about 20-25 no of eggs throughout its egg laying period. The average life span of the species is about 50-60 days. Both the species can be easily bred in the laboratory using variety of culture medium and has short generation time.Therefore, both the species are very appropriate for toxicity study and previously used by different researcher for soil toxicity study (Joy, 1980; Chakravorty,1990,1995).

34

  

 
 
!

 



 
 


3.8.1. Collection,extraction and identification Test specimens likeXenyllasp. and Cyphoderussp. were collected from the nearby soil having good amount of organic matter around Midnapore town (West Bengal, India) that has never been used for agricultural purpose and free from any chemical treatment. Extraction was done by using “High gradient Extractor Chamber”. After extraction animals shorted out by using hand brush and transferred to polythene jars having thin layer of sterilized (sundry technique) soil. After proper identification animals were used for culture in the laboratory.

35

3.8.2.Culture of the test specimens Inert polythene rearing vials measuring 6.5 cm in diameter and 7.5 cm in height were used to maintain stock culture on sterilized soil medium. Baker’s Yeast was given to the animals as food. Culture vials were kept inside B.O.D. incubator at 28 ± 0.5o C. Moisture of the soil was maintained by adding distled water from time to time (Bostrom and Lofs-Holmin. 1982).

  
!






 
 




!

 



3.9. Test medium Soil collected from field experimental site used as test medium for the bio assay studies. Following physiochemical properties of the test medium were determined and defaunated properly before use as medium for bio assay (Wiles and Frampton,1996). i) Soil texture following international pipette method (Piper,1942). ii) Soil pH with pH meter (Systronics model 512 SE). iii) Soil organic carbon following rapid titration method (Walkey and Black, 1934). iv) Soil water holding capacity (Lal, 1977, Viji and Rajesh, 2012)

3.10. Methods of bioassay Bioassays were done with age synchronized specimens in small inert polythene container measuring 2.0 cm diameter and 3.0 cm height treatment vials each containing 2 gm of dry test medium. Before final experiments trial experiments were conducted in the same 36

experimental vials to determine soil water dilution ratio for uniform spreading of the herbicides in the medium and moisture content of the medium. Soil moisture of the test medium was determined and kept constant throughout the experiment. Trial experiment showed that 0.7 ml of solution is required to moisten 2 gms of dry soil used as test medium. Exactly 0.7 ml of herbicide solution in different dilution was carefully administered into the vial with the help of pipette (Dripping method)(Ghabbour and Iman, 1967; Wilche, 1968; Wiles and Frampton,1996). The vials were then left undisturbed for about 30 minutes for uniform spreading of solution to the medium. The experiment was set up with three replicates for each dose of herbicide and control. Ten numbers of age synchronized test specimens were then transfer into the vials. Observations were made after every 24 hours. Those individuals who showed no apparent sign of life, even when poked with a needle were considered dead and removed. (Chakravorty, 1990) Mortality of the test specimens were recorded.

Acute toxicity studies The main objectives of the acute toxicity bioassays were to determine safe or highly toxic herbicides for both the species of test organisms (Joy, 1980). 24 hours exposures at RAD using all six selected herbicides were made for this purpose. After screening of toxic herbicides in respect of both the test species further toxicity studies were made to determine the median lethal concentration (LC50) and residual toxicity using selected toxic herbicides. Four types of bioassays were made in the laboratory and methods are described below:

i) Direct toxicity studies Direct knockdown effect of the herbicides was studied by exposing adult specimen of Xenyllawelchi and Cyphoderusjavanus at RAD doses of different herbicides (Table- 3.8). Table 3.8 RAD doses of herbicides used Herbicides (a. i. mg/kg soil) 1. Paraquat 2. Pendimethalin 3. Pretilachlor 6. 2,4-D Salt 5. 2,4-D amine 4. Metribuzin

Dose (RAD) 600.00 750.00 300.00 0.21 1200.00 1200.00

Ten numbers of specimens introduced into the treated and control vials containing 2 gms of test soil moisten with 0.7 ml of diluted herbicide solution. Finally the experimental vials were kept in a B.O.D. incubator at a constant temperature of 28േ0.5oC . 37





Mortality of the specimen was recorded after every 24 hours and dead specimens, if any, were removed from the experimental vials. Experiment was repeated three times and 24 hours mortality percentage worked out. Herbicides which result fifty percent and above mortality during 24 hours duration were considered toxic(Joy,1980) and selected for next level of toxicity study.

ii) Determination ofLC50 values Age synchronized specimens were used for the bioassay in small test vials as described earlier. Different levels of doses of the herbicides based on their recommended agricultural doses (RAD) (Table 3.9)were administered into the test vials for both the species. After application of herbicides, the vials were left undisturbed for 30 minutes for uniform spreading of chemical in the medium. Each experiment accompanied with three replicate and control set. 10 numbers of age synchronized test specimens were then transfer into the experimental vials. Finally vials were kept in a B.O.D. incubator at a constant temperature of 28േ0.5oC . Mortality of the specimens was recorded after 24 hours. The total mortality of the test specimens obtained after 24 hours of exposure was subject to probit analysis by EPA probit analysis programme, version 1.5 (USEPA, 2006) to determine LC50 value and 95% confidence limit of each selected herbicide. Table 3.9 Doses (a.i. mg/Kg soil) of the herbicides equivalents to the different levels of recommended agricultural doses (RAD) used in the acute toxicity bioassays. Herbicides RAD Pendimethalin 750.00 300.00 Pretilachlor

½ RAD 375.00 150.00

¼ RAD 187.50 75.00

1/6

RAD 125.00 37.50

1/8

RAD 93.75 50.00

1/10

RAD 75.00 30.00

iii) Determination LT50 values Bio assays to determine LT50 of the herbicide for both the test species made in the same manner with test vials as described about for LC50 value. RAD dose of herbicides applied for the bioassay. 10 numbers of age synchronized specimens transferred into the vials. Observation was made after every 30 minutes and dead individuals were removed. The time taken to achieve 50% cumulative mortality was noted and expressed as LT50 of the herbicide.

38

iv)Residual toxicity study Long term residual toxicity bio assays were carried out with the application of RAD for both selected herbicides. A total of 30 treatment vials were arranged for each herbicide. Five vials each were sampled at random at intervals of 3, 7, 15, 30, 45 and 60 days after treatment. The percentage mortality of fresh specimens introduced into these vials was noted after 24 hours. Control sets were maintained for each selected herbicide.

Chronic toxicity bioassay Objectives The main purpose of the chronic toxicity bioassays was to assess the physiological and biochemical stress caused to collembola species by long term exposure of the herbicides. Life cycle parameters like hatching success, moulting, juvenile survival, maturity percentage of juvenile, time require for maturity, life span were assessed using sub-lethal doses of herbicides. Biochemical stress was assessed from the acetylecholinesterase enzyme activity. Enzyme activity was assessed at RAD both under laboratory as well as under field conditions, whereas enzyme activity was estimated using different sub-lethal doses of herbicides only under laboratory conditions. For Xenyllawelchi experiment using sub-lethal doses was carried out by using both pendimethalin andpretilachlor but only pendimethalin was used for Cyphoderusjavanus as LC50 of pretilachlor was not achieved within the range of RAD during 24 hrs of exposure. The whole methodology of chronic toxicity was done following the guidelines of OECD, 1984.

3.11. Bioassay with sub lethal doses. Experimental vials were used as described earlier for the purpose after administering various sublethal doses of herbicides. Age synchronized test species introduced into the vials. Soil moisture of the test soil was maintained by adding herbicide solution of respective dose and by distilled water for control set. Baker’s Yeast was used as food time to time to the animal. Three replicates for each treatment were maintained and finally experimental vials were kept in B.O.D. incubator at a constant temperature of 28±0.5ºC. Different sub-lethal doses of pendimethalin and pretilachlor for Xenylla welchi and Cyphoderus javanus are in table 3.10 and 3.11.

39

Table 3.10 Different sub-lethal doses (a.i. mg/kg soil)of pendimethalin and pretilachlor for Xenylla welchi Treatment

Dose

Pendimethalin

Pretilachlor

T1 T2 T3 T4 T5 T6

0

0 0.014 0.017 0.023 0.035 0.070

0 0.005 0.006 0.008 0.013 0.025

1/ 10xLC50 1/ 8xLC50 1/ 6xLC50 1/ 4xLC50 1/ 2xLC50

Table 3.11 Different sub-lethal doses (a.i. mg/kg soil)of pendimethalin forCyphoderus javanus
Treatment

Dose

Pendimethalin

T1 T2 T3 T4 T5 T6

0

0 0.04 0.05 0.06 0.10 0.19

1/ 10xLC50 1/ 8xLC50 1/ 6xLC50 1/ 4xLC50 1/ 2xLC50





3.11.1.Parameters studied i) Hatching success Hatching success of the test specimens was determined after the egg laying of adult into the vials. Vials were carefully examined under binocular microscope and number of eggs was counted and number of eggs that hatched was also counted.

ii) Exuvia production After egg laying adult specimens were removed from the vials.After hatching juveniles appeared. 10 numbers of newly emerged juvenile was then transferred to the treatment vials and the treatment and control sets as described earlier was maintained. After every 8 hrs the vials were examined under binocular microscope and the exuvia were counted and removed, if any, produced by animals.

40

iii) Juvenile survival, percentage of juvenile become matured and time require for maturity Juvenile examined and number at different insters was recorded regularly. Juvenile becomes mature at the stage of egg laying (Bandyopadhyaya&Chowdhury, 2002).

iv) Life span The time period from the hatching to till the death of adult individual was also recorded to determine the life span of the test specimens.

v) Acetylecholinesterase activity About 500 adult test specimens of the same age and size were sorted out from the rearing vials for each treatment. The experimental animal then transferred to treatment vials and kept in BOD as described earlier and after 24 hours only live specimens are selected for AchE study (Ellman et al.,1961), standardized for collembola (Chakravorty, et al., 1995).

3.11.2.Bioassay studies with RAD in field and laboratory condition Acetylecholinesterase enzyme activity of test speciesexposed to RAD of pendimethalin and pretilachlor were determined in the laboratory as well as field condition, along with respective control.

a) In laboratory Herbicide treated experimental vials of sufficient number were kept in BOD as described earlier for determining residual toxicity. Test vials were sampled at random after 3,7,15 and 30 days. Age synchronized specimens were introduced and kept for 24 hours and after 24 hrs only live specimen were used for AchE activity estimation.

b) In field For field study test vials containing 2 gm of soil put into the field in pits dug into the ground. RAD of the respective herbicides was applied into the test vials. Treatment vials were taken to the laboratory after 3,7,15 and 30 days. Age synchronized known number of specimen was introduced into the experimental vials and kept for 24 hrs. and after 24 hrs. only live specimens were used for AchE estimation.

41

3.11.3.Bioassay studies by using more sensitive species A few bioassay experiments were done by using test medium pre treated with 1% solution of pendimethalin and pretilachlor. Soil samples were collected simultaneously both from field and laboratory condition at 3,7,15 and 30 days after treatment and mortality rate of Xenyllawelchiexposed to these samples in experimental vial were noted after 24 hours.

3.12. Analytical methods 3.12.1.Physiochemical parameters of the test soil (a) Determination of soil texture: Soil samples were dried and passed through a 2 mm sieve to remove larger particles. The texture of the soil was determined by following the international pipette method (Piper,1942). (b) Determination of soil pH: pH of the soil sample was determined with the help of glass electrode pH meter (Systronics model 512SE). Soil sample was mixed with distilled water (1:5) and shaken for half an hour by mechanical shaker. Then the solution was filtered and pH of the solution was measured directly from the instrument. (c) Determination of soil moisture: Soil moisture was measured using torsion balance moisture meter (Adair Dutt,Kolkata). Principle: The moisture content of the wet soil was directly read from the apparatus and then converted into dry weight percentage (md) using the procedure mentioned below. Requirements: i. Torsion balance moisture meter. ii.Forceps iii. Poly bag iv. Spatula v. Digging instrument vi. Weight pan

42

Procedure: The weight pan is placed in the moisture meter and the right hand knob is rotated and 0 of the movable scale is brought into the same line. Then little amount of soil sample is placed on the weight pan until the pointer comes in the same line with 0 marking at the movable scale. The infrared bulb is turned on and by rotating the knob the temperature was fixed at 105oC. After sometime the pointer shifts and the movable scale is rotated anticlockwise until it comes back to the straight line and reading from the scale is recorded. This is repeated until a constant reading is observed. Calculation: ௠

Md =ሼଵ଴଴ି௠ሽx100 m = moisture content reading; 100-m= weight of dry soil; md=dry weight percentage. (d) Determination of soil organic carbon: The organic carbon of the soil sample was determined following Walkley and Black’s rapid titration method (Walkley and Black,1934). Principle: The organic matter in the soil gets oxidized by potassium dichromate and concentrated sulphuric acid utilizing the heat of dilution of H2so4. The excess potassium dichromate, not reduced by the organic matter of the soil is determined by titration with standard ferrous ammonium sulphate [FeSo4 (NH4)2 SO4.6H2O]. Reagents: i. Standard 1N Potassium Dichromate: 49.04 g of K2Cr2O7 (oven dried at 90oC) was dissolved in distilled water and the volume was made up to one litre. ii. 0.5 N ferrous ammonium sulphate: 196 g of the hydrated crystalline salt dissolved in one liter of distilled water containing 20 ml of conc. H2SO4. iii. Diphenylamine indicator: 0.5g diphenylamine dissolved in a mixture of 20ml of water and 100 ml of conc.H2SO4. iv. Concentrated sulphuric acid (sp.gr.1.84) v. Ortho-phosphoric acid (85%). 43

Procedure: 1. 1 g soil was taken in a dry 500ml conical flask. 2. 10 ml of 1NK2Cr2O7 was pipette in and swirled a little. 3. The flask was kept on asbestos sheet. Then 20 ml of H2SO4 was added and swirled again two or three times. 4. The flask was allowed to stand for 30 minutes and then 200 ml of distilled water was added to it. 5. 10 ml of phosphoric acid was added to the solution. Then 1 ml diphenylamine indicator was added to it. 6. The contents were titrated with 0.5N ferrous ammonium sulphate solution till the colour changed from blue-violet to green. 7. Simultaneously a blank was run without soil. If more than 7 ml of the dichromate solution was consumed the determination was repeated with a smaller quantity (0-.250.50g) of soil. Calculation: Organic carbon content was calculated as follows 1ml of NK2Cr2O7=0.003g of carbon Amount of carbon present in 1 g soil =

଴Ǥ଴଴ଷൈଵ଴ሺ஻ି஼ሻ ஻ൈௌ

Where B = Volume (ml) of 0.5N ferrous ammonium sulphate required to neutralize 10 ml of 1 N K2Cr2O7, i.e. blank titration. C= Volume (ml) of 0.5 N ferrous ammonium sulphate needed for titration of soil sample (reading with soil). S= Weight (g) of the soil. e) Determination of water holding capacity of soil by Kneer-Rackzowski box (Lal, 1977; Viji and Rajesh,2012)

44

Requirements: i) Soil ii) Filter paper iii) Keen box iv) Weight pan v) Petri dish vi) Oven vii) Water viii) Watch glass ix) Spatula Procedure: The Keen box was cleaned and dried and it was weighed. Filter paper is cut according to the radius of keen box. Weight of one dry and one wet filter paper (after removing excess water) are noted separately. Dry filter paper is placed into the Keen box just above sieve. The air dried soil passed through 2 mm sieve. Then the box is filled with soil. The Keen box with air dry soil is weighed. The box is placed in petridish and gradually filled with water from the side till the water level was about 1 cm above the base of the box. The petridish is covered to prevent evaporation from soil surface and it is kept for 12 hrs or more. A continuous and shining film of water at the soil surface appears. The box is carefully removed from water and wiped dry from outside and weighed quickly. The soil of Keen box placed in watch glass carefully with the help of spatula and dried in the oven at 105oC for 10-12 hours and constant weight is recorded. Calculation: Water holding capacity of soil =

௪௘௜௚௛௧௢௙௪௔௧௘௥௜௡௧௛௘௪௘௧௦௢௜௟ ௪௘௜௚௛௧௢௙௢௩௘௡ௗ௥௬௦௢௜௟

45

x 100 %

3.12.2 Acetylcholinesterase (Ellman et al.,1961) Surviving animals were separated to estimate the acetylcholinesterase activity. 5 mg of the collembola were homogenized in 1 ml distilled

water to have a 0.5 % homogenate

o

(Chakravorty et al., 1995) at 0 C. The homogenate was centrifuged at 10,000g for 10 minutes at 40C (Remi cold centrifuge). The resultant supernatants were stored in ice and used for Acetylcholine esterase assay. Kinetic measurements were performed with

acetylthiocholine iodide as the substrate.

Reaction were performed in 300 μl of 0.1 M phosphate buffer, pH-8 containinga. 20 μl of 0.01MDTNB(5,5’ ditihio-(2- nitrobenzoic acid)) b. 20 μl of 0.075M substrate c. 10 μl tissue extracts Contents were thoroughly mixed and absorbance was measured at 405 nm in systronics UV-Vis spectrophotometer. Substrates were continued to be added before adding of the substrate till a stable reading was recorded. After addition of the substrate, the change in absorbance was recorded for a period of 10 min at an interval of 2 min. Change in absorbance per minute was thus determined. Calculation: R= 5.74 X 10-4 X A/co R= rate in moles of substrate hydrolyze/min/g tissue A= Change in absorbance/min CO= original concentration of the tissue (in mg/ml) The enzymatic activity was finally expressed in nmoles/min/mg of protein after estimation of protein content of the samples.

46

Reagents: i. 0.1 M phosphate buffer: Solution A: 5.22 g of K2HPO4and 4.68 g of NaH2PO4 were dissolved in 150 ml of distilled water. Solution B: 6.2 g NaOH was dissolved in 150 ml of distilled water. Solution B was added to solution A to get the desired pH (8 or 7) and then finally the volume was made up to 300 ml with distilled water. ii. DTNB: 39.6 mg of DTNB with 15 mg NaHCO2 is dissolved in 10 ml of 0.1 M phosphate buffer (pH 7). iii. Acetylthiocholine (ATC): 21.67 mg of ATC is dissolved in 1 ml distilled water.

3.12.3 Determination of protein (Lowry et al.,1951) This method is sensitive enough to give a moderate constant value. Protein content of the enzyme extracts were usually determined by this method. Principle: The blue colour developed by the reduction of the phosphomolybdicphosphotungstic components in the Folin-Ciocalteau reagent by the amino acids tyrosine and tryptophan present in the protein plus the colour developed by the biuret reaction of the protein with the alkaline cupric tartrate are measured in the Lowry’s method. Reagents: i. 2%Sodium carbonate in 0.1 N sodium hydroxide (Reagent A). ii. 0.5% copper sulphate solution (CuSO4, 5H2O) in 1% potassium sodium tartrate (Reagent B). iii. Alkaline copper solution: 50 ml of Regent B was mixed prior to use (Reagent C). iv. Folin-Ciocalteaureagent: Phenol reagent manufactured by Qualigens five chemicals, Glaxo India Ltd was diluted to 1:2 ratios with distilled water before use.

47

v. Protein solution (Stock standard): 50 mg bovine serum albumin (BSA) was dissolved in 50 ml distilled water. vi. Working standard: 10 ml of stock solution was diluted to 50 ml with distilled water. 1 ml of this solution contained 200 μg of protein. Procedure: 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the working standard were pipetted out into a series of test tubes. 0.5 ml of the sample extract was taken in another test tube. The volume was made up to 1 ml with distilled water. A tube with 1 ml of distilled water served as the blank. 5 ml of Reagent C was added to each of the test tube, mixed well and allowed to stand for 10 minutes. Then 0.5 ml of diluted (1:2) phenol reagent was added to each of the test tube, vortexed and incubated at room temperature preferably in dark for 30 mins. The intensity of blue colour developed was measured spectrophotometrically at 660 nm. Calculation: A standard curve was drawn using BSA and the amount of protein was calculated directly from the standard curve.

Statistical analysis Data on life cycle parameter i.e, hatching success, moulting, juvenile survival, maturity percentage, time require for maturity, life span along with AchE activity and field population density wereanalyzed for single factor ANOVA, followed by least significance difference(LSD) test to established significant variation between treatments at 5% of probability. Population of collembola species density was determined by ‫ ݕݐ݅ݏ݊݁ܦ‬ൌ

ܶ‫ݏ݈݁݌݉ܽݏ݈݈݈ܽ݊݅ܽݑ݀݅ݒ݅݀݊݅݋݈݊ܽݐ݋‬ ܰ‫݋‬Ǥ ‫݀݁ݐ݈݈ܿ݁݋ܿݏ݈݁݌݉ܽݏ݂݋‬

The density was converted into the number of individual per square meter.

48

CHAPTER- II

49

RESULTS, DISCUSSION & CONCLUSION

50

4. Results For convenience results of the field experiments considered first followed by findings of the experiments related to under laboratory conditions.

4.1. Physiochemical parameters of test medium Physiochemical parameters of field soil used as test medium are shown in Table 4.1. On the basis of the texture of soil the test medium was categorized as sandy loan type with slightly acidic in nature having moderate organic carbon content.

4.2. Environmental parameters. During the entire period of investigation monthly variation of important climatic and major edaphic parameters were recorded and shown in figure 4.1 to 4.5. Climatic parameters (Fig. 4.1 to 4.3) showed more or less uniform pattern of fluctuation during the entire study period and had a direct relationship among rate of rain fall, relative humidity and ambient temperature. Data of climatic parameters were collected from local Nature Observatory Centre situated at Midnapore College, Midanpore, paschim Medinipur, west Bengal, India. Rainy seasons were characterized by hot and humid condition and winter seasons showed dry and cold atmospheric characters with little rain fall for both the years of investigation. The edaphic parameters (Fig. 4.4 and 4.5) fluctuated without major variation between the untreated and treated plots selected for investigation for both the years. It was noticed that major environmental parameters were almost alike throughout the study period.

4.3. Findings of field experiments Results regarding the possible ill effect of chemicals pendimethalinandpretilachlor on two test species of collembola namely Xenyllawelchi and Cyphoderusjavanusare summarized below.

51

4.3.1. Effect on Xenyllawelchi Experimental findings regarding the effect of Pendimethalin and Pretilachlor on Xenyllawelchi are summarized in Table 4.2-4.5 (Fig. 4.6-4.7). The changes in the density (no/m2) of the Xenyllawelchi in the untreated and pendimethalin treated soils showed remarkable ill effect of herbicide on Xenyllawelchi. There is no significant variation of population density in different sampling day of untreated plots.

A. Effect of Pendimethalin A detailed consideration of density of Xenyllawelchi for two successive years for pre and post monsoon periods reveals a remarkable ill effect of pendimethalinon Xenyllawelchi in treated plots.

Pre monsoon Reduction of density continued upto 7th day of sampling. About 85% reduction comparing to untreated plots in 7th days sampling noticed in pre monsoon season for both the year. Recovery of population noticed from 15th day sampling. It was also noticed that the test species Xenyllawelchi is unable to complete recovery of population during the study period for both the year. For each sampling day after the application of chemicals there was significant difference between the population sampled from treated and untreated plots (Appendix-I, Table AI- I.I to I.IV).

Post monsoon For post monsoon season ill effect of the chemical to the test species was slightly different from pre monsoon. Maximum number that is about 80% population density reduction in treated plots noticed in 7th day of sampling after application of chemical comparing to the untreated plots. There is a significant difference between population of treated and control plots sampled up to 15th day after application of chemical. Recovery of population density noticed in 30th day sampling after application. This trend of population loss and recovery is more or less alike for two successive year of investigation (Appendix-I, Table AI- I.V to I.VIII).

52

B. Effect of Pretilachlor Comparison of population density (no/m2) with detailed consideration of Xenyllawelchi for two successive years for both pre and post monsoon periods for pretilachlor are as follows. The changes in the untreated and pretilachlor treated plots show significant ill effect on Xenyllawelchi. Season wise impact of chemicals is given below.

Premonsoon In premonsoon season for both the year of investigation showed maximum reduction of 80% population density in treated plots comparing to that of untreated plots in 3rd day of sampling after the application of chemicals. It was noticed that population density of 7 th day of sampling after application is still slightly lower in treated plots comparing with the sampling of 3rd day treated plots and have significant difference with the untreated plots of 7th day of sampling. In 15th day of sampling population density is still significantly lower than untreated plots though recovery started. Complete recovery of population noticed in 30th day of sampling reflecting insignificant difference between treated and untreated plots (Appendix-I, Table AI- I.IX to I.XII).

Post monsoon In post monsoon season for both the year of investigation it was noticed that ill effect of chemicals on Xenyllawelchi is slight lower than pre monsoon in respect of recovery of population density. Maximum reduction of population density is about 70% in respect of control noticed in 3rd day of sampling, in 7thday of sampling 50% less population density is counted in treated plots comparing with the untreated plots. In 15th day of sampling there is no significant difference between the population density of treated and untreated plots (Appendix-I, Table AI- I.XIII to I.XVI).

4.3.2 Effect on Cyphoderusjavanus Findings regarding the ill impact of herbicidespendimethalin and pretilachlor are summarized in table no 4.6 to 4.9.(Fig. 4.8-4.9). No significant variation of population density recorded among population in different sampling day in untreated plots.

53

A. Effect of Pendimethalin A detailed consideration about ill impact following comparision of population density (no/m2) between untreated and pendimethalin treated soil showed pendimethalin impart significant toxic impact on Cyphoderusjavanus population in chemically treated soil. Changes in population number of test species Cyphoderusjavanus between treated and untreated plots with season wise consideration are given below.

Pre monsoon In two successive year of investigation in pre monsoon season maximum reduction of population noticed in 3rd day of sampling after application and was about 70% in chemical treated plots in comparison with the untreated plots. Both 3rd and 7th day of sampling revealed that population density of treated plots significantly differ with the corresponding control plots. Although recovery of population found in progress from 15th day of sampling with a significant difference between untreated and chemically treated plots. Complete recovery was found in 30th days sampling with insignificant difference of the population density with the corresponding untreated plots (Appendix-I, Table AI- I.XVII to I.XX).

Post monsoon For both the year post monsoon period of investigation showed similar pattern of ill impact on Cyphoderusjavanus population in chemically treated soil. Maximum reduction of population is about 60% in 3rd day sampling after applicaton of chemicals in treated plots comparing with untreated plots. 7th day of sampling showed sign of recovery of population in treated plot but still have significant difference between corresponding untreated plots. Recovery of population in chemically treated plots having insignificant difference found in 15th day’s sampling (Appendix-I, Table AI- I.XXI to I.XXIV).

B. Effect of Pretilachlor Population

density

(no/m2)

change

due

to

toxic

effect

of

pretilachlor

in

Cyphoderusjavanus in soil for both the year of investigation with a season wise detailed consideration of change in population number are given below.

54

Pre monsoon For both the year pre monsoon season of study period showed ill impact of pretilachlor on Cyphoderusjavanus population density in chemically treated soil. Maximum reduction of 70% of population density noticed in 3rd days sampling in treated plots comparing with the untreated plots. A significant 60% reduction in population density found in 7th days sampling in treated plots against untreated plots. In 15th days samplingthere were found insignificant difference between population of untreated and treated plots (Appendix-I, Table AI- I.XXV to I.XXVIII).

Post monsoon In post monsoon season a maximum reduction of 50% of population noticed in 3rd day of sampling in treated pilots comparing with the untreated plots. 7th days sampling of population was shown lower number of population in treated plots but the difference is insignificant corresponding with untreated plots (Appendix-I, Table AI- I.XXIX to I.XXXII).

Hence the important findings of the experiments under field conditions are i) Herbicide chemicals pendimethalin and pretilachlor could produce very significant ill effect upon soil collembolan species namelyXenyllawelchi and Cyphoderusjavanus. ii) Of these,pendimethalin imparts more toxic effect than pretilachlor upon both the test species. iii) Among two species tested with chemicals, it was found that Xenyllawelchi population affected much than Cyphoderusjavanus population. iv) These two collembola species is sensitive against these chemicals.

4.3.3 Biochemical parameter Under biochemical parameter acetylcholinesterase enzyme activity was investigated during pre monsoon period when much toxic impact was found on population density on both the test species exposed to both the chemicals.

55

Acetylcholinesterase enzyme activity A. Xenylla welchi Acetylcholinesterase enzyme activity of Xenyllawelchi exposed to Pendimethalin and Pretilachlor in field condition (Table-4.10) was observed at RAD in different sampling day along with control. Chemical wise detailed consideration is given below (Fig.4.10).

Pendimethalin A significant inhibition of enzyme activity was found in 3rd, 7th and 15th day after application comparing with the control. 19.00%, 9.38% and 5.40% inhibition of enzyme activity was found with control in 3rd, 7th and 15th day after application. In control uniform pattern of enzyme activity was found (Appendix-II, Table AII- II.I and II.II).

Pretilachlor In case of herbicide chemical Pretilachlor significant inhibition was recorded in 3 rd and 7th day after application with 10.25% and 5.75% inhibition of enzyme activity comparing with the control. Control plots showed uniform pattern of enzyme activity in different experimental day (Appendix-II, Table AII- II.III and II.IV). B. Cyphoderus javanus Activity of enzyme acetylcholinesteraseof Cyphoderusjavanus exposed to the chemical pendimethalin and pretilachlor under field conditions was summarised in Table 4.11.

Pendimethalin Pendimethalin showed a significant inhibition of acetylcholinesterase enzyme activity of the test species on 3rd (10.00%) and 7th (6.05%) day (Appendix-II, Table AII- II.V and II.VI) comparing to the control (Fig. 4.11).

Pretilachlor There was no significant variation found regarding inhibition of enzyme activity of the test species exposed to Pretilachlor in different sample day with respective control (AppendixII, Table AII- II.VII and II.VIII).

56

4.4 Laboratory Experiments 4.4.1 Acute and residual toxicity of the herbicides to collembola A. Toxicity to Xenylla welchi Direct toxicity studies with different selected herbicides Figure 4.12 represents direct knock down effect of the agricultural doses of six herbicides chemical namely paraquat, metribuzin, pendimethalin, pretilachlor and amine & Sodium salt of 2, 4-D on Xenylla welchi. The test species commonly found in natural soil suffered complete mortality (Cent percent) within 24 hours in vials treated with pendimethalin and Pretilachlor. In Metribuzin treated vials only 20% mortality occurred in 24hrs. Among other chemicals namely Paraquat, 2,4-D amine and 2,4-D sodium salt the test species show no mortality after the exposure hour. Pendimethalin and Pretilachlor which caused more than 50% mortality selected for further investigation of acute and residual toxicity for the test species Xenyllawelchi.

Median lethal concentration (LC50) 24 hour LC50 values of the two herbicides chemical and their confidence limits are in Table 4.12 as summary of these two chemicals Pendimethalin with 190.0 gma.i./ha was found less toxic than Pretilachlor 72.734 gmai/ha. However, both the values were much less than their respective RAD (1.25 Kg ai/ha, 0.5 Kg ai/ha)

Median lethal time (LT50) Time required for 50% cumulative mortality of the test species Xenyllawelchi at RAD dose of chemicals taken for study are in Fig 4.13. The herbicide Pretilachlor attained the fastest LT50 (110 minutes) followed by Pendimethalin with LT50 (140 minutes).

Residual toxicity Residual toxicity on the basis of bio activity of test species Xenyllawelchireveald that herbicide chemical Pendimethalin is more persistent in soil than Pretilachlor (Fig 4.14). Mean mortality value for Pretilachlor declined from 58% to 25% in 15 days and further reduced to 15% in 30 days. But for Pendimethalin 55% mean mortality recorded in 15 days and declined to 45% in 30 days further 25% mean mortality recorded in 45 days. 57

B. Toxicity to Cyphoderus javanus Direct toxicity studies of different selected herbicides 24 hrs direct knockdown effect of six selected herbicides namely paraquat, metribuzin, pendimethalin, pretilachlor, 2,4-D amine and sodium salt at their respective RAD dose on Cyphoderusjavanus summarised below (Fig 4.15). Test species suffered 75% mortality within 24 hrs in vial treated with Pendimethalin. Among other chemicals pretilachlor show 30%, paraquat 5%, metribuzin 4%, 2,4-D amine 6% and 2,4-D sodium 4% mortality at their respective RAD dose treated vials during 24 hrs exposure. On the basis of the result of direct toxicity study pendimethalin and pretilachlor selected for further investigation of acute and residual toxicities.

Median lethal concentration (LC50) 24 hrs. LC50 values of the two herbicides chemical and their confidence limit are summarised in table4.13 .Pretilachlor do not achieve LC50value in 24 hrs. Pendimethalin appeared as toxic with LC50 value 581 gma.i./ha. LC50 value is lower than its corresponding RAD.

Median lethal time (LT50) Time required for 50% commulative mortality at RAD of selected chemicals are summarized in table 4.14 LT50 value of Pendimethalin is 7 hrs where as in Pretilachlor treated vials 50% cumulative mortality do not achieved during the exposure time period of 24 hrs duration ( Fig. 4.16).

Residual toxicity Residual toxicity on the basis of bio activity of Cyphoderusjavanus showed maximum mean mortality 27% for pendimethalin in 3rd day declining to 13% in 7 days and further reduced to 10% in 15 days and declining upto 7% in 30 days. Mean mortality value recorded maximum as 7% in 3rd day for Pretilachlor and declining at 3% in 7th day (Fig 4.17)

58

4.4.2 Chronic toxicity of herbicides to test species. 4.4.2.1 Toxicity to Xenyllawelchi A. Life cycle parameter Hatching success Mean value of eggs laid per female specimen and hatching success were 156 ± 10.98 and 90.60± 1.30 respectively. Hatching success in different sub lethal doses of chemicals pendimethalin and pretilachlor and control set are summarized in Table 4.15. Pendimethalin In T2hatching success attained almost the same value as control. The values showed a significant 63.30% (T6), 50.00% (T5), 35.55% (T4) and 26.66% (T3) reduction in hatching success for Pendimethalin comparing with respective control (Appendix-III, Table AIIIIII.I and III.II) Pretilachlor In respect of control a significant reduction were found in T6 (44.10%), T5 (24.44%), T4 (8.00%) and T3 (5.00%) (Appendix-III, Table AIII- III.III and III.IV). Exuvia production Production of exuvia of the test species Xenylla welchi in untreated and different sub lethal doses of Pendimethalin and Pretilachlor treated vials are given in Table 4.16. Pendimethalin It was observed that juvenile survive only T1, T2 and T3 vials. In T1 exuvia produced every after 5.5 ± 0.5 days. In T3 and T2 exuvia production significantly increased 32% and 48% per week respectively (Appendix-III, Table AIII- III.V and III.VI). Pretilachlor It was found that juvenile did not survive in T5and T6dose. A significant increase of exuvia production 28% in T2, 25% in T3 and 15% inT4 dose in respect of T1were observed (Appendix-III, Table AIII- III.V and III.VI).

59

Survival of juvenile (upto 1st moult)

Survival of juvenile in untreated and treatment of chemicals Pendimethalin and Pretilachlor was observed for upto 1st moult and shown in Table 4.17. Detailed considerations of survived juvenile specimen of Xenylla welchi in respect of two herbicides are given below. Pendimethalin Juvenile died before 1st moult in T4, T5 and T6. In T2 and T3a significant number of 41% and 67% juvenile died without moulting comparing with T1 where 84% hatched specimen remain alive after 1st moult (Appendix-III, Table AIII- III.VII and III.VIII). Pretilachlor In Pretilachlor treated vials, test animals (Juvenile) do not survived upto the 1st moult in T5 and T6. In T2, T3 and T4a significant number of 28%, 42% and 64% respectively juvenile died without moulting comparing with T1 (Appendix-III, Table AIII- III.VII and III.VIII). No. of specimen attain maturity stage Test specimen in different treatment of sub lethal doses of two chemicals pendimethalin and pretilachlor and in untreated vials are observed from Juvenile to maturity (egg laying) stage and number of animal attain maturity stage was recorded and summarised in Table 4.18.Detailed consideration of survived matured animal of each chemical are given below. Pendimethalin Test specimen only survived in T1, T2 and T3. In T2 and T3 about 38% and 75% less animal attained the age of maturity respectively comparing with the T1 having significant variation (Appendix-III, Table AIII- III.IX and III.X). Pretilachlor Test species do not survived in T5 and T6. Comparing with the T1a significant number i.e., about 17% less animal attain maturity stage in T2. In T3 and T4 it was observed that 24% and 34% less animal survived upto maturity stage against their respective control (Appendix-III, Table AIII- III.IX and III.X).

60

Time (in days) required for attaining maturity Time took to become mature for the test specimen Xenylla welchi reared in untreated and different sub lethal doses of two chemicals are given in Table 4.19. Chemical wise considerations in respect of different sub lethal doses are given below. Pendimethalin In T1 about 42.20 ± 0.7 days required for reaching egg laying stage. In T2 and T3 about 16% and 24% less time required for maturity having significant variation in respect of T1(Appendix-III, Table AIII- III.XI and III.XII). Pretilachlor T4, T3 and T2 required 19%, 16% and 12% significant less time for test species becoming maturity in respect of T1 (Appendix-III, Table AIII- III.IX and III.X). Life span (days) Life span of Xenylla welchi in untreated and treated vials of different sub lethal doses of two chemical is summarized in Table 4.20. Pendimethalin Life span of test animal become shorter in treated vials. 28% and 36% shorter life span is observed in T2 and T3 comparing with theT1 having significant variation(Appendix-III, Table AIII- III.XIII and III.XIV). Pretilachlor In case of Pretilachlor 29%, 24% and 16% significant shorter life span is observed in T4, T3 and T2 respectively comparing with the T1(Appendix-III, Table AIII- III.XIII and III.XIV).

B. Biochemical parameter Acetylcholinesterase enzyme activity Under bio chemical parameter activities of acetylcholinesterase enzyme of the test species Xenyllawelchiexposed to Pendimethalin and Pretilachlorin RAD and in different sublethal doses are summarized in Table 4.21 and Table 4.22.A detailed chemical wise 61

consideration of enzyme activity in untreated and treated sets is given below. Uniform level of enzyme activity found in control for both the chemical tested (Appendix-IV, Table AIV- IV.I to VI.III). Pendimethalin Recommended agricultural dose In control mean value of acetylcholinesterase enzyme activity was found to be 960.25± 1.50 n molethiocholine/min/mg proteins. The enzyme activity was found inhibited throughout the investigation period by Pendimethalin significantly in 3rd (32%), 7th(21%) , 15th(17%) and 30th (7%) day after application. Sub lethal doses Significant inhibition wasnot found in any treatmentin respect of T1. Pretilachlor Recommended agricultural dose In case of Pretilachlor significant inhibition of enzyme activity between control and treatment of 3rd(26%), 7th(15%) and 15th(7%) day after application was observed. Sub lethal doses Significant inhibition was not found in any treatment in respect of T1.

4.4.2.2 Toxicity to Cyphoderusjavanus Life cycle and bio chemical parameters wereobserved only in chemical pendimethalin in different sub lethal doses of using Cyphoderusjavanusas because LC50 was not achieved within the range of RAD of pretilachlor during 24 hrs. exposure.

A. Life cycle parameters Hatching success Hatching success of eggs of the test species Cyphoderusjavanus exposed to untreated and different sub lethal doses of herbicide chemical Pendimethalin is summarized in Table 4.23.Hatching success in untreated vials was found to be 80.00±1.00 percent. T6, T5, 62

T4and T3 recorded a significant 80.9%, 74.3%, 46.2% and 21.9% reduction in hatching success respectively comparing with the T1(Appendix-III, Table AIII- III.XXV and XXVI). Exuvia production Exuvia production (individual/week) of matured test specimen in untreated and sub lethal doses of chemical treated vials was summarized in Table 4.24. Untreated vials specimen produced 1.40 ± 0.32 exuvia per individual specimens per week. Where as in treated vials significant increase of exuvia production observed. Animal do not survived in T5 and T6. About 30%, 25% and 16% increase of exuvia production was observed in T4, T3 and T2 respectively per week (Appendix-III, Table AIII- III.XXVII and III.XXIII). Survival of juvenile upto 1st moult Findings regarding survival of juvenile upto 1st moult is in Table 4.25.Juvenile do not survive up to 1st moult and died without moulting in T5 and T6. In T1 about 74.36 ± 3.82 juvenile survived at least up to 1st moult. A significant reduction in survival of juvenile up to 1st moult in T4 (59%), T3 (39%) and T2 (20%) in respect of control (Appendix-III, Table AIII- III.XXIX and III.XXX). .No of individual attain maturity Percentage of test specimen Cyphoderusjavanus attain maturity stage (egg laying) in untreated and different treatment of sublethal doses of chemical Pendimethalin are summarized in Table 4.26.In T1 78.25 ± 3.65 percent animal attain maturity stage. A significant reduction in number of animal maturity was to be found in T4 (36%), T3(17%) and T2 (8%) in respect of T1 (Appendix-III, Table AIII- III.XXXI and III.XXXII). Time (day) required for attaining maturity (egg laying of female) Time required for egg laying of female individual of the test species Cyphoderus javanus in untreated and different sub lethal doses of treated vials are summarized in Table 4.27. Maturity time in control found to be 38.62 ± 0.25 days. In T4, T3 and T2 animal took about 16%, 11% and 5% less time respectively for maturity having significant difference with T1 (Appendix-III, Table AIII- III.XXXIII and III.XXXIV). 63

Life span in days Table no 4.28 shown life spans of test species in untreated and different treatment of sub lethal doses as in summarized form. It was found that in control set average life span of Cyphoderus javanus is 52.85േͳǤ͵͸ǤA significant reduction of 7% in T2, 13% in T3 and 17% in T4 were noticed (Appendix-III, Table AIII- III.XXXV and III.XXXVI).

B. Bio chemical parameter Acetylcholinesterase enzyme activity Acetylcholinesterase enzyme activity in untreated and treatment with pendimethalin (at RAD and different sub lethal doses) and pretilachlor (at RAD) using Cyphoderusjavanus were summarized in table 4.29 and Table 4.30.Findings of enzyme activity are given below. Pendimethalin Recommended agriculture dose A significant inhibition of enzyme activity was found up to 15th day experiment. 6.05%, 9.00% and 15.00% inhibition of enzyme activity were found in 15th, 7th and 3rd day respectively after application of chemical in respect of control (Appendix-IV, Table AIVIV.V and IV.VI). Sub lethal doses Significant inhibition of enzyme activity was not found in any treatment (T2 to T6) comparing with T1 (Appendix-IV, Table AIV- IV.VII). Pretilachlor Significant inhibition was not found at recommended agricultural dose in any treatment comparing with control (Appendix-IV, Table AIV- IV.V).

4.4.3. Bio assay studies using more sensitive species Xenylla welchi Findings of bio assay experiments employing more sensitive species Xenylla welchi both in field and laboratory condition are summarized in Table 4.31.

64

A clear decrease in the persistence of Pendimethalin and Pretilachlor were found in both the samples collected from field and laboratory condition but Pendimethalin persisted with high toxicity than Pretilachlor. Results were used to predict the respective herbicide chemical residue in unknown soil sample. For example Table- 4.31 showed Pendimethalin caused 25% mortality in field and 45% mortality in laboratory condition for 30th day sample. This could be extrapolated to equivalent concentration or bioactivity of herbicide with the help of standardised mortality rate of the species given in table – 4.32. Here, 25% mortality corresponds to 0.24 gm/litre and 45% mortality corresponds to 0.36 gm/litre. So, it may be proposed that sample collected from the field and laboratory condition have toxicant equivalent to 0.24 gm/litre and 0.36 gm/litre respectively for of herbicide Pendimethalin. The difference in the results is obviously due to the variation of decomposition rate of chemical under laboratory and field condition.

4.4.4. Comparison of effect regarding acetylcholinesterase enzyme activity between field and laboratory condition A. Xenylla welchi Acetylcholinesterase enzyme activity of the test species Xenyllawelchi exposed to the chemicals Pendimethalin and Pretilachlor in the laboratory and field condition have been summarized in Fig 4.18-4.20. Recommended agricultural dose of Pendimethalin produced similar inhibition effect both in field and laboratory condition. In laboratory condition significant inhibition of enzyme activity was found entire the study period i.e., upto 30th day while in field condition significant inhibition of enzyme activitywas found upto 15th day. In both laboratory and field condition enzyme activity reduced on 3rd day in respect of control and gradually increased there after towards the level of control (Appendix-V, Table AV- V.1 and V.II). In case of Pretilachlor exposure, enzyme activity showed similar significant inhibition both in laboratory (up to 15th day) and field (up to 7th day) condition (AppendixV, Table AV- V.III and V.IV).

65

B. Cyphoderusjavanus Test species Cyphoderusjavanus when exposed to RAD of Pendimethalin both in field and laboratory condition acetylcholinesterase enzyme activity found to be inhibited (Fig. 4.21) both in field and laboratory conditions. Significant inhibition of enzyme activity was found upto 15th day in laboratory and upto 7th day in field conditions (Appendix-V, Table AV- V.V and V.VI). In

Pretilachlor

exposure

of

test

species,

significant

inhibition

of

acetylcholinesterase enzyme activity was not found both in field and laboratory condition in respect of corresponding control (Appendix-V, Table AV- V.VII). Both the test species show similar trend of enzyme activity exposed to the chemical. In each cases enzyme activity reduced maximum in 3rd day after application of chemical and gradually increased there after towards the level of control. In laboratory condition inhibition percentage and duration for enzyme activity to reach upto the level of control is higher than field condition. Significant inhibition of acetylcholinesterase enzyme activity was not found in different sub lethal doses (T1to T6) tested for both the chemical on two test species. So, extrapolation for detecting persistent toxicant chemical in unknown field soil sample may not be possible below the range of RAD by using inhibition of acetylcholinesterase enzyme activity.

66

TABLES Table no.-4.1Physiochemical parameters of test medium

Properties


Value

Texture(g%) Clay Silt Sand pH Organic carbon Water holding capacity

17.5 11.9 70.6 5.78±0.25 1.45±0.32 34.00%




Table- 4.2A comperison of density of Xenyllawelchi (no/m2) during 1st year (Pre monsoon) in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachor treated (T-Pret) plots at RAD. Sampling interval In days 0 ՜ 3 7 15 30 45

UT 1860.00 േ 2.02a

T-Pend 1860.15 േ 0.25a

T-Pret 1860.20 േ 1.50a

372.00േ1.50b369.15േ0.00b 1860.00 േ 1.50a a 1859.25േ3.04 278.15േ1.00c 275.50േ1.75c 1860.15േ5.20a1116.05േ2.00d 952.10േʹǤͷͲd 1860.20േ3.35a 1205.00േͳǤ͹ͷe 1865.25േʹǤͷͲa a f 1550.00േ2.50 1862.35േ1.50a 1861.35േͳǤͷ0

1. Values are expressed as mean േS.D. 2. 8 no of samples taken from each plot, mean value of 3 plots were considered. 3. Different superscripts in the same column denote significant difference at P൏0.05 4. ‘՜’ indicates the stage at which herbicide was applied.




67

Table- 4.3A comparison of Xenyllawelchi (no/m2) during 2nd year pre monsoon in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachlor treated (T-Pret) plots at RAD. Sampling intervals UT T-Pend T-Pret In days 0 1860.00േ1.50a1860.50േͳǤͲͲa1860.25േ2.50a ՜ 3 1859.05േ1.00a 370.05േ2.00b371.05േ2.50b 7 1860.15േ1.05a 275.15േ1.50c 277.50േ1.00c 15 1860.25േ2.50a1120.50േ2.00d948.00േ2.50d 30 1860.00േ2.00a 1210.00േ2.50e1860.05േ1.00a 45 1861.15േ2.50a 1575.25േ3.00f2895.66േ10.40a 1. Values are expressed as mean േS.D. 2. 8 no of samples taken from each plot, mean value of 3 plots were considerd. 3. Different superscripts in the same column denote significant difference at P൏0.05 4. ‘՜’ indicates the stage at which herbicide was applied.


Table-4.4A comparison of density of Xenyllawelchi (no/m2) during 1st year post monsoon in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachlor treated (T-Pret) plots at RAD. Sampling intervals UT T-Pend T-Pret In days 0 2810.50േ2.50a2806.00േ1.50a2808.50േ1.00a ՜ 3 2805.00േ1.00a702.50േ1.00b845.50േ1.50b 72808.00േ2.50a 562.00േ1.75c1405.00േ1.00c 15 2806.05േ1.50a 1580.50േ2.00d2805.00േ2.00a 30 2808.00േ2.00a 2825.75േ2.50a 2822.00േ2.50a 45 2809.00േ2.75a 2820.50േ1.00a2840.00േ2.00a 1. Values are expressed as meanേS.D. 2. 8 no of sample taken from each plot, mean value of 3 plots were considered 3. Different superscripts in the same column denote significant difference at P൏0.05 4. ՜Indicates the stage at which herbicide was applied.

68

Table-4.5A comparison of density of Xenyllawelchi (no/m2) during 2nd year post monsoon in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachlor treated (T-Pret) plots at RAD. Sampling interval UT T-Pend T-Pret In days 0 2808.50േ2.50a 2806.00േ2.50a2808.00 േ1.50a ՜ 3 2810.00േ1.50a700.50േ1.00b 848.05േ1.00b 7 2806.55േ2.00a 560.00േ2.50c1408.25േ2.05c 15 2808.00േ2.50a 1775.25േ2.00d2805.00േ1.00a 30 2810.00േ2.55a 2810.20േ2.55a 2825.15േ2.75a 45 2810.00േ1.00a 2825.00േ1.56a2830.05േ1.00a

1. Values are expressed as meanേS.D. 2. 8 no of sample taken from each plot, mean value of 3 plots were considered 3. Different superscripts in the same column denote significant difference at P൏0.05 4. ՜Indicates the stage at which herbicide was applied.

Table- 4.6A comparison of Cyphoderus javanus (no/m2) during 1st year pre monsoon in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachlor treated (T-Pret) plots at RAD Sampling interval UT T-Pend T-Pret In days 0 990.25േ1.00a 989.25േ1.00a 990.25േ1.25a ՜ 3 991.00േͳǤͷ0a 297.25േ1.25b298.25േ1.50b a 7 990.50േ1.25 301.00േ1.00c396.00േ1.00c 15 989.25േ2.00a 445.20േ1.45d988.25േ1.52a 30 990.00േ1.00a 992.00േ1.00a 995.00േ1.25a 45 991.00േ1.50a 995.25േ1.00a998.25േ1.00a 1. Values are expressed as meanേS.D. 2. 8 no of sample taken from each plot, mean value of 3 plots were considered 3. Different superscripts in the same column denote significant difference at P൏0.05 4. ՜indicates the stage at which herbicide was applied.

69

Table- 4.7A comparison of Cyphoderusjavanus (no/m2) during 2nd year pre monsoon in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachlor treated (T-Pret) plots at RAD Sampling interval UT T-Pend T-Pret In days 0 990.00േ1.00a 990.25േ1.50a 991.25േ0.75a ՜ 3 990.00േ1.50a 295.00േ1.25b 299.00േ1.00b a c 299.25േ2.05 398.25േ1.50c 7 989.25േ2.00 982.50േ1.25d 15 988.75േ1.50a 455.00േ2.00d 30 991.00േ1.00a 989.25േ0.75a 992.35േ2.55a a a 45 990.25േ0.75 995.50േ1.25 998.00േ2.00a 1. Values are expressed as meanേS.D. 2. 8 no of sample taken from each plot, mean value of 3 plots were considered 3. Different superscripts in the same column denote significant difference at P൏0.05 4. ՜indicates the stage at which herbicide was applied.



Table-4.8 A comparison of density of Cyphoderusjavanus (no/m2) during 1st year post monsoon in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachlortreated (T-Pret) plots at RAD. Sampling interval UT T-Pend In days 0 1500.25േ1.00a 1501.25േ1.20a1500.25േ1.05a ՜ 3 1500.00േ1.50a 601.25േ1.00b 752.00േ1.50b 7 1502.25േ1.75a 885.50േ1.50c1490.25േ1.00a 15 1498.25േ2.50a 1498.25േ2.00a1502.00േ1.25a 30 1500.00േ1.05a1501.00േ1.00a 1509.25േ1.00a 45 1499.25േ2.50a1525.25േ1.50a 1508.00േ1.00a

T-Pret

1. Values are expressed as meanേS.D. 2. 8 no of sample taken from each plot, mean value of 3 plots were considered 3. Different superscripts in the same column denote significant difference at P൏0.05 4. ՜indicates the stage at which herbicide was applied.


70

Table-4.9A comparison of density of Cyphoderus javanus (no/m2) during 2ndyear post monsoon in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachlortreated (T-Pret) plots at RAD. Sampling interval UT T-Pend T-Pret In days 0 1502.00േ1.00a1500.00േ1.50a1501.25േ1.25a 3 1500.05േͳǤʹͷa599.25േ1.00b 750.00േ1.00b 7 1499.75േ1.00a888.00േ1.25c 1485.00േ1.50c 15 1501.00േ0.75a 1492.25േ2.50a 1502.25േ1.25a a a a 30 1500.25േ1.20 1499.25േͳǤͷ͸ 1525.00േ1.05 45 1498.75േ2.50a1520.75േ2.00a1520.25േ1.00a 1. Values are expressed as meanേS.D. 2. 8 no of sample taken from each plot, mean value of 3 plots were considered 3. Different superscripts in the same column denote significant difference at P൏0.05 4. ՜indicates the stage at which herbicide was applied.






Table- 4.10Acetylecholinesterase enzyme activity of Xenyllawelchiunder field condition at RAD of herbicides(n mole thiocholine/min/mg protein). Treatment Days after application 0 3 7 15 30

Pendimethalin 960.25േ1.50a 777.50±1.25b 870.25±1.00c 908.50±1.00d 959.50±1.25a

% inhibition 0.00 19.10 9.38 5.40 0.00

Pretilachlor 960.25±1.50a 861.50±1.25b 905.25±1.00c 958.50±1.25a 960.00±1.00a

% inhibition 0.00 10.25 5.75 0.00 0.00

1. Values expressed as mean േS.D. 2. Different super scripts in the same column denote significant difference at P൏0.05.




71

Table-4.11Acetylcholinesterase enzyme activity of Cyphoderusjavanus in field condition at RAD of pendimethalin and Pretilachlor(n mole thiocholine/min/mg protein). Treatment Pendimethalin Days after application 0 3 7 15 30

% inhibition Pretilachlor

958.00±1.00a 0.00 864.25േ1.50b10.00 901.50±1.25c 6.05 956.25±1.00a0.00 956.75±1.25a 0.00

% inhibition

958.00േ1.00a 0.00 956.25േ1.25a 957.50േ1.50a 957.25േ1.00a 958.00േ1.25a

0.00 0.00 0.00 0.00

1. Values expressed as mean േS.D. 2. Different super scripts in the same column denote significant difference at P൏0.05.

Table – 4.12 24 hours LC50 values (gma.i./ha) of two selected herbicides for Xenylla welchi Herbicides

24 hrs. LC50

95% confidence limit

Pendimethalin

190.000

159-223

Pretilachlor

72.734

57.041-87.950

Table: -4.13 24 hrs LC50 value of two selected herbicide of Cyphoderus javanus. Herbicide Pendimethalin Pretilachlor

24 hrs LC50 (aigm/ha) 581

95% confidence limit 465-782

Not achieved in 24 Hrs.

72

Table-4.14 Mean lethal time of Cyphoderusjavanus at RAD doses of two selected herbicides. LT50 at RAD

Herbicide

7 hrs.

Pendimethalin Pretilachlor

Not achieved in 24 Hrs.

Table – 4.15Hatching success (%) of Xenyllawelchi at control (T1) and different sub lethal doses (T2-T6) of two selected herbicides. Treatment

Pendimethalin

Pretilachlor

T1

89.0േ1.30a

90.6േ1.30a

T2

85.0േ2.50a

88.33േ2.88a

T3

66.66േ2.80b

T4

58.33േ1.50c

83.33േ2.50c

T5

45.0േ2.00d

68.33േ2.25d

T6

32.66േ2.51e

50.66േ1.15e

84.66±2.00b

1. Values are expressed as mean േS.D. 2. Different superscript letters in the same column denote significant difference at P൏0.05 3. n=10 individual per vial, mean value of 3 vials were considered.


73

Table –4.16 Exuvia production per individual per week of Xenyllawelchi exposes to control and different sub lethal doses (T2-T6) of two selected herbicides. Treatment

Pendimethalin

Pretilachlor

T1

1.27േ1.00a

1.27േ1.00a

T2

1.88േ1.25b

1.62േ1.00b

T3

1.68േ1.00c

1.58േ1.00c

T4

0.00

1.47േ1.00d

T5

0.00

0.00

T6

0.00

0.00

1. Values are expressed as mean േS.D. 2. Different superscript letters in the same column denote significant difference at P൏0.05 3. n=10 individual per vial, mean value of 3 vials were considered.


Table –4.17 Survival of juvenile percentage (upto 1st moulting) of Xenylla welchi at control (T1) and different sublethal doses (T2-T6) of pendimethalin and pretilachlor.

Treatment

Pendimethalin

Pretilachlor

T1

84.00േ1.0a


84.00േ1.0a

T2

49.00േ1.0b

60.00േ1.0b

T3

27.3Ͳ േ1.5c

49.00േ1.0c

T4

0.00

30.00േ1.0d

T5

0.00

0.00

T6

0.00

0.00

1. Values are expressed as mean േ S.D. 2. Superscripts in the same column denote significant difference at 3. n=10 individual per vial, mean value of 3 vials were considered. 74

P൏0.05

Table -4.18 No of animal (%) attain maturity of Xenyllawelchi at control (T1) and different sub lethal doses (T2-T6) of Pendimethalin and Pretilachlor.

Treatment

Pendimethalin

Pretilachlor

T1

79.00േ1.0a


79.00േ1.0a

T2

49.00േ1.0b

65.00േ1.0b

T3

20.00േ1.0c

60.7Ͳ േ1.5c

T4

0.00

50.00േ1.0d

T5

0.00

0.00

T6

0.00

0.00

1. Values are expressed as mean േ S.D. 2. Superscripts in the same column denote significant difference at P൏0.05 3. n=10 individual per vial, mean value of 3 vials were considered.

Table- 4.19 Time (days) require for attaining maturity of Xenylla welchi at control (T1) and different sub lethal doses (T2-T6) of herbicide Pendimethalin and Pretilachlor. Treatment

Pendimethalin

Pretilachlor

T1

42.20േ0.7a

42.20േ0.7a

T2

35.00േ0.5b

37.00േ0.5b

T3

32.00േ0.5c

35.80േ0.8c

T4

0.00

34.00േ0.5d

T5

0.00

0.00

T6

0.00

0.00

1. Values are expressed as mean േ S.D. 2. Superscripts in the same column denote significant difference P൏0.05 3. n=10 individual per vial, mean value of 3 vials were considered. 75

Table-4.20Life span (indays) of Xenyllawelchi at control (T1) and different sublethal doses (T2-T6) of Pendimethalin and Pretilachlor Treatment

Pendimethalin

Pretilachlor

T1

110.3Ͳ േ1.5a

110.3Ͳ േ1.5a

T2

79.30 േ1.5b

92.40േ1.0b

T3

70.00േ2.0c

83.60േ1.5c

T4

0.00

78.10േ1.0d

T5

0.00

0.00

T6

0.00

0.00

1. Values expressed as mean േ S.D. 2. Superscripts in same column denote significant difference at P൏0.05 3. n=10 individual per vial, mean value of 3 vials were considered.


Table-4.21Acetylcholinesterase enzyme activity of Xenyllawelchiunder laboratory condition

at

RAD

of

herbicides

pendimethalin

and

pretilachlor

(n

mole

thiocholine/min/mg protein). Treatment (Days after

Pendimethalin

% inhibition

Pretilachlor

% inhibition

application) 0

960.25േ1.50a

3

645.10±1.00

b

759.25±1.25

c

790.50±1.75

d

890.20±1.00

f

7 15 30

0.00
32.80 21.00 17.70 7.30

960.25±1.50a

0.00

706.25±1.25

b

26.40

816.50±0.50

c

15.00

940.50±1.25

d

7.29

960.25±1.50

a

0.00

1. Values expressed as mean േS.D. 2. Different super scripts in the same column denote significant difference at P൏0.05. 76

Table-4.22 Acetylcholinesterase enzyme activity of Xenyllawelchiunder laboratory condition at sub lethal of herbicides pendimethalin and pretilachlor (n mole thiocholine/min/mg protein). Treatment

Pendimethalin

%

Pretilachlor

% inhibition

inhibition T1

:71-11>2-11

































1-11





:71-11>2-11





































1-11

T2

959.25>2-36

































1-11

959.25>2-11





































1-11


T3

960.00>1-61

































1-11

T4

959.25>2-11

































1-11

959.25>2-36




































1-11

T5

959.25>1-61

































1-11

959.25>2-36





































1-11

T6

960.00>1-36

































1-11

960.00>1-36



































1-11

960.00>1-61





































1-11

1. Values expressed as mean േS.D. 2. Different super scripts in the same column denote significant difference at P൏0.05.

Table-4.23 Hatching success (%) of Cyphoderusjavanus at control (T1) and in different sublethal doses (T2-T6) of Pendimethalin. Treatment

Pendimethalin

T1

80.00േ1.00a

T2

78.75േ1.50a

T3

63.25േ1.25b

T4

43.00േ1.00c

T5

20.50േ1.50d

T6

15.25േ1.05e

1. Values are expressed as mean േS.D. 2. n= 30 eggs per vial, mean value of 3 vials were considered 3. Different superscript letters in the same column denote significant difference at p൏0.05

77

Table- 4.24 Exuvia production of Cyphoderusjavanus in control (T1) and different sub lethal (T2-T6) doses of Pendimethalin. Treatment

Pendimethalin

T1

1.40േ0.32a

T2

1.49േ0.22a

T3

1.75േ0.18b

T4

1.63േ0.18c

T5

0.00

T6

0.00

1. Values expressed as mean േ S.D. 2. n=10 individual per vial, mean value of 3 vials were considered. 3. Different superscripts in the same column denote significance difference at p൏0.05


Table-4.25 Survival of juvenile upto 1st moult of Cyphoderus javanus in control (T1) and different sub lethal (T2-T6) doses of pendimethalin. Treatment

Pendimethalin

T1

74.36±3.82a

T2

59.58±4.58b

T3

45.28±3.62c

T4

30.00±4.82d

T5

0.00

T6

0.00

1. Values expressed as mean േS.D. 2. n=10 juvenile per vial , mean value 3 vials were considered. 3 Different superscripts in the same column denote significant difference at p൏0.05 78

Table-4.26 Percentage of matured individual of Cyphoderusjavanus reared in control (T1) and different sublethal (T2-T6) doses of pendimethlin. Treatment

Pendimethalin

T1

78.25±3.67a

T2

72.00±1.25b

T3

65.38±2.24c

T4

50.46±1.67d

T5

0.00

T6

0.00

1. Values expressed as mean േS.D. 2. n=10 individual per vial, mean value of 3 vials were considered. 3. Different superscripts in the same column denote significant difference at P൏0.05

Table-4.27 Time required for attaining maturity (egg laying) of Cyphoderus javanus in control (T1) and different sublethal doses (T2-T6) of pendimethalin. Treatment

Pendimethalin

T1

38.62±0.25a

T2

36.00±0.06b

T3

34.85±0.18c

T4

32.68±0.58d

T5

0.00

T6

0.00

1. Values expressed as mean േS.D. 2. n=10 individual per vial, mean value of 3 vials were considered. 3. Different superscripts in the same column denote significant difference at P൏0.05.

79

Table-4.28 Life span of Cyphoderus javanus in control (T1) and different sublethal doses (T2-T6) of Pendimethalin Treatment

Pendimethalin

T1

52.85±1.36a

T2

48.68±1.28b

T3

45.00±1.00c

T4

43.38±1.12d

T5

0.00

T6

0.00

1. Value expressed as mean േS.D. 2. n=10 individual pervial, mean value of 3 vials were considered. 3. Different superscripts in the same column denote significant difference at P൏0.05

Table-4.29 Acetylecholinesterase enzyme activity of Cyphoderusjavanus at RAD of pendimethalin and Pretilachlor under laboratory conditions.

Treatment (Days after

Pendimethalin

% inhibition

Pretilachlor

% inhibition

application) 0

958.00±1.00a

0.00

958.00±1.00a

3

816.25±1.25b

15.00

955.25±1.50a

0.00

7

873.50±1.50c

9.00

953.50±1.25a

0.00

15

901.00±1.00d

6.05

955.25±1.00a

0.00

30

956.25±1.25a

0.00

958.00±1.00a

0.00

1. Values expressed as mean േS.D. 2. Different super scripts in the same column denote significant difference at P൏0.05.

80

0.00

Table – 4.30Acetylcholinesterase enzyme activity of Cyphoderusjavanus in different sublethal doses of Pendimethalin under laboratory conditions. Treatment

Pendimethalin

T1

958.00±1.25a

0.00

T2

958.00±1.50a

0.00

T3

957.25±1.25a

0.00

T4

958.00±1.50a

0.00

T5

957.75±1.25

a

0.00

957.25±1.00

a

0.00

T6

% inhibition

1. Values expressed as mean േS.D. 2. Different super scripts in the same column denote significant difference at P൏0.05.

Table – 4.31 Mortality % of Bio assay exposing Xenyllawelchi to Pendimethalin (2.5 gm/litere) and Pretilachlor (1.0 gm/litre) Experimental Condition

ChemicalAge of treated soil 3

7

15

30

45

Pendimethalin

63

55

40

25

00

Pretilachlor

70

46

15

00

00

Pendimethalin

70

65

55

45

25

Pretilachlor

80

58

25

15

00

Field

Laboratory

81

Table – 4.32Standardised mortality rate (24 hrs.) of Pendimethalin and Pretilachlor on Xenyllawelchi (From probit analysis) Pendimethalin

Pretilachlor

Dose (g/l)

Mortality %

Dose (g/l)

Mortality %

0.07

1.00

0.02

1.00

0.12

5.00

0.03

5.00

0.15

10.00

0.05

10.00

0.18

15.00

0.66

15.00

0.24

25.00

0.08

25.00

0.30

35.00

0.11

35.00

0.36

45.00

0.14

45.00

0.38

50.00

0.15

50.00

0.79

85.00

0.37

85.00

0.92

90.00

0.45

90.00

1.21

95.00

0.63

95.00

1.95

99.00

1.15

99.00

82

FIGURES Ambient Temperature 51

→

41

Temperature in oC

46

36 .3122)-& #-

31

.3122
!-
& #-

26

$.3123
)-
& #-

21

$.3123
!
& #-

6 1 1

3

5

7

9

21

23

25





&


o

Fig. - 4.1 Monthly variation of mean ambient temperature in C (Max. & Min.)during study period.

Rainfall 61 56










→

51 46 41 36

$.3122

31

$.
3123

26 21 6 1 1

3

5

7

9

21

23

25







Fig. - 4.2 Monthly variation of mean rainfall in cm. during study period.

83




Humidity :1 91 81  #
/


71 61 $.3122
' &*
=

51

$
3123
' &*=

41 31 21 1 1

3

5

7

9

21

23

25










Fig.- 4.3 Monthly variation of mean humidity % during study period.

Moisture content of soil 27

 
/




25 $
3122
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%"%
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#"&%

23 21

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3123
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"
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#"&%

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Fig. - 4.4 Monthly variation of mean moisture (%) content of soil of treated and untreated plots of field.

84

Soil temperature



 








→

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3122
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o

Fig.- 4.5 Monthly variation of mean soil temp.in C of treated and untreated plots of field during study period. 3111

 
#
)'+*

2911 2711 2511 .3122

2311


.3123

2111

.!.3122

911

.!
3123

711

.$&-
3122

511

.$&
3123

311 1 1

4

8

26

41

56

#




2

Fig.- 4.6A Comperison of density of Xenyllawelchi (no/m ) during Pre monsoon (Two successive year) in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachor treated (T-Pret) plots at RAD.


85


4611

 
#
)'+*

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2611

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3123

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.$&-
3122 .$&
3123

611 1 1

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56

#




2

Fig. – 4.7 A Comperison of density of Xenyllawelchi (no/m ) during Post monsoon (Two successive year) in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachor treated (T-Pret) plots at RAD. 2311

 
#
)'+*

2111 911

.3122 
.3123

711

.!.3122 .!
3123

511

.$&-
3122 .$&
3123

311 1 1

4

8

26

41

56

#




2

Fig.- 4.8 A Comparison of density of Cyphoderus javanus (no/m ) during Pre monsoon (Two successive year) in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachor treated (T-Pret) plots at RAD.



86

$#
!#


''


2911 2711 2511 2311

.3122

2111


.3123 .!.3122

911

.!
3123

711

.$&-
3122

511

.$&
3123

311 1 1

4

8

26

41

56

#






Fig.- 4.9 A Comparison of density of Cyphoderusjavanus (no/m2) during Post monsoon (Two successive year) in different sampling days between untreated (UT) with pendimethalin treated (T-Pend) and pretilachor treated (T-Pret) plots at RAD.

$#
!#
)

''
*

2311

2111

911 "!&$"

711

!-
$& $&-
$&

511

311

1 1

4

8

26

41

#



Fig- 4.10Acetylcholinesterase enzyme activity of Xenylla Welchiunder field conditions at RAD of herbicide pendimethalin and pretilachlor (n mole thiocholine/min/mg protein).

87

$#
!#


''


:91 :71 :51 :31 :11

"!&$"

991

!-
$&

971

$&-
$&

951 931 911 1

4

8

26

41

#






Fig.- 4.11 Acetylcholinesterase enzyme activity of Cyphoderus javanus under field conditions at RAD of herbicide pendimethalin and pretilachlor (n mole thiocholine/min/mg protein).

+,
&
#
/   !
#
)/*

231 211

91 71 51

35
$%-
"$&&*
=

31 1

Fig. - 4.12 24 hrs mortality percentage of X.welchi using RAD of different selected herbicide

88

231

  !
#
)/*

211 91 71

! &! $&"$

51 31 1 1

61

211

261

311

361



" )  *

Fig.-4.13 Mean Lethal time to cause 50% mortality (LT50) of Xenylla welchi at RAD of two selected herbicides.
:1 91

)/*
#


81 71 61 ! &!

51

$&"$

41 31 21 1 1

4

8

26

41

56

#



Fig.– 4.14 Residual toxicity of Pendimethalin and Pretilachlor at RAD on Xenylla welchi.

89

  !
#
)/*

+,
&
#
/ 231

211 91 71 51 35
$%-
"$&&*
=

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1

Fig. - 4.15 24 Hrs cumulative mortality % of Cyphoderusjavanus using RAD of selected herbicides.

91   !
#
)/*

81 71 61 51

! &!

41

$&"$

31 21 1

1 2 3 4 5 6 7 8 9 : 21 22 23 24 25 26 27 28 29 2: 31 32 33 34 35


)
 *

Fig. - 4.16 Mean lethal time of Cyphoderusjavanus at RAD doses of two selected herbicides.

90

41

#
)/*

36 31

26

#! &! $&"$

21 6 1 4
*

8
*

26
*

41
*

56
*

#



Fig.- 4.17 Residual toxicity of Pendimethalin and Pretilachlor at RAD on Cyphoderus javanus

$#
!#


''


2311 2111 911 "!&$"

711

! &! 511

$&"$

311

1 1

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#



Fig.-4.18 Comparison of acetylcholinesterase enzyme activity under laboratory conditions between Pendimethalin and Pretilachlor in respect of control on Xenyllawelchi at RAD.

91

$#
!#


''


2311 2111 911 "!&$"

711

! &!
511

! &!


311 1 1

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Fig. 4.19 Comparison of acetylcholinesterase enzyme activity under field and laboratory conditions of pendimethalin in respect of control on Xenyllawelchiat RAD.

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!#


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2311 2111 911

"!&$"

711

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511

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Fig.-4.20Comparison of acetylcholinesterase enzyme activity at RAD of Pretilachlor under field and laboratory conditions in respect of control on Xenyllawelchi.


92

$#
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Fig. - 4.21 Comparison of acetylcholinesterase enzyme activity under field and laboratory condition at RAD of Pendimethalin on Cyphoderusjavanus.

93

5. Discussion 5.1. Variation of toxicities of Herbicides used Toxic effect of herbicides on soil organisms depend on different factors such as chemical nature and formulation of the chemical, soil characteristics, uptake routes of exposed organism along with it’s morphology, different climatic and edaphic factors etc (Zimdahl et al., 1984; Zwieten, 2004; Adl and Coleman, 2005; Piola et al., 2009). The present preliminary screening study of six herbicides has been done by using sandy loam soil as test medium. Comparing the ill effect of six herbicides in the present investigation, it was found that recommended agricultural dose (RAD) of Pendimethalin and Pretilachlor produced a direct knockdown effect (>50%) on the test species Xenylla welchi but other test species Cyphoderus javanusfaced similar effect against Pendimethalin only. Slightly less than 50% mortality was observed in case of C. javanus against pretilachlor exposure. Similar knockdown effect of different herbicides on Collembola has been noticed by several workers (Johnson et al., 1955; Joy, 1980; Park and Lee, 2005; Amorim et al., 2005). Species specific variation of toxic effects on Collembola has also been found (Wiles and Frampton, 1996; Frampton, 1997; Ponge et al., 2013; Amorim et al., 2005; Pereira et al, 2011). On the other hand both the test species exposed to RAD of herbicides Paraquat, Metrabuzin, 2, 4-D amine and sodium salt exhibited 0-20% mortality. As uniform test medium used for the experiment of each and every herbicide tested the variation of mortality of test species was found to be related with species and chemical used. Variation of toxicity of herbicides on collembola and other soil insect is also evident from the study done by several workers (Freemark and Boulin, 1995; Wiles and Frampton, 1996; Ponge et al, 2013; Renaud et al., 2004).

5.1.1. Field Experiments For the assessment of toxic hazards due to herbicides on non target soil organisms, in-situ field bio-assays are very essential. There are few limitations and advantages of field bioassay (Wiles and Jepson, 1992). Effects of experimental parameters are one important aspect which influences the findings of the field bio assay. These aspects were considered in the present investigation by incorporating detailed local climatic and edaphic 94

characteristics throughout the investigation period. Sufficient control plots were maintained for each and every herbicide chemical used for the investigation. During the present investigation period more or less uniform pattern of fluctuation of climatic parameters were recorded. A direct relationship among rainfall, relative humidity and ambient temperature were found. Soil organic carbon, pH of the soil etc, remained more or less stable in the experimental field. An inverse relationship between soil temperature and moisture was recorded during the study period. Various soil edaphic and climatic factors regulate the soil collembola population which has been noticed by other investigators too. Soil moisture content (Parvez and Sharma, 2004b), organic carbon (Adl and Coleman, 2005), pH (Hutson, 1978), rainfall (Huhta and Hanninen, 2001) have significant influence on collembola population fall in line with the present investigation. In the present study, population of two soil collembola species was investigated. It was observed that an increase in the population of both the test species during post monsoon compared to the pre monsoon period. This type of seasonal fluctuation of soil collembola population had been observed by other workers (Christiansen, 1964, Sinha et al., 1988, Parvez and Sharma 2004a & 2004c). Ilyas and Parvez (2011) established a direct negative effect of high ambient temperature and low soil moisture content on collembola population. Bandhopadhay and Chowdhury, (2002) also showed negative correlation of soil collembola population with soil temperature. In low soil pH at rainy season (Jaeger and Eisenbeis, 1984) density of soil collembola population increased (Hutson, 1978; Ilyas and Parvez, 2011). Maria et al., (2004) also found the peak diversity of soil microarthropods having direct relation with soil water content and soil temperature. Effect of Herbicides A total of two herbicides were used for the present field experiments and findings of each herbicide are discussed with a few references. Pendimethalin Pendimethalin produced a temporary ill effect on both the test species population i.e., XenyllawelchiandCyphoderusjavanus. Specific and seasonal variations on toxic effect were noticed. Intensity of ill impact was more in case of X. welchithanC. javanus along with

high

impact

during

premonsoon

than

postmonsoon

season.

In

premonsoonseasonXenylla welchi unable to recover its population density during 95

investigation period while in post monsoon period complete recovery of population density occurred within 30th day after application of chemical. Cyphoderus javanus showed complete recovery within 30th and 15th day after application of chemical in pre monsoon and post monsoon season respectively. Velcheva et al., (2012) showed the toxic impact of Pendimethalin on the community structure of soil meso-fauna and observed negative correlation between the residual amount of herbicide in the soil and the abundance of the taxa recorded. Yusaf et al., (2013) showed low soil microbial activity in Pendimethalin exposure. Early recovery and less toxic effect on earthworm (Lumbricidae) population density during post monsoon period in Pendimethalin treated soil were found by Velcheva et al., (1997 & 1999) and Krogh et al., (2007). Bandopadhyay and Choudhury (2009) showed the linear negative relation between soil moisture and soil Pendimethalin in autumn than hot summer in field soil. These findings fall in the line of our investigation. Pretilachlor Temporary ill effect on population density of both the test species was observed in case Pretilachlor treatment with slight variation in respect of test species and season of treatment. Xenyllawelchi showed reduction of population density upto 7th day after treatment and population density became more or less equal with the control within 30th day after application of chemical in premonsoon season but during post monsoon Xenyllawelchi recovers its population density slight earlier than premonsoon i.e., within 15th day after application of chemical. Intensity of toxicity relating to Pretilachlor on Cyphoderusjavanus in respect of population density is slightly low in contrast with Xenylla welchi in respect of recovery time. Cyphoderusjavanus recovered its population density compared with corresponding control within 15th and 30th days after application of chemical in post and pre monsoon period respectively.Chen et al., (1999) found the ill impact of Pretilachlor on predatory insect population of Nilaparvatalugens. Residual toxicity of the chemical in soil depends on edaphic factors and climatic parameters (Rai et al., 1999; Prakash and Suseeladevi, 2000; Deepa, 2002; Quayle et al., 2007; Dharumarajan et al., 2008) which fall in our line of investigation in respect of seasonal variation of toxicity of the chemical. However,

96

negative impact of different herbicides like Simazin (Edwards, 1970), Atrazin (Jansech, et. al., 2006), Pendimethalin (Kartalska et al., 2009) on soil microorganisms were recorded.

5.1.2. Effect on Acetylcholinesterase enzyme (AchE) activity For both the test species X. welchi and C. javanus the acetylcholinesterase enzyme activity was inihibited in Pendimethalin treated field population. Inhibition found on 3rd, 7th and 15th day after application of the chemical in X. welchi and on 3rd and 7th day after application of chemical for C. javanus. For Pretilachlor only the test species X. welchi showed inhibition after treatment of chemical on 3rd and 7th day. Boily et al., (2013) observed actylcholinesterase inhibition in honey bee (Apis melifera) under field condition exposed to glyphosate herbicide. However,acetylcholinesterase enzyme

activity in micro-organism including soil

collembola was recorded in laboratory experiment using pesticides including herbicides (Chakravorty et al., 1995; Boonthai et al., 2000; Campero et al., 2007).

5.2. Laboratory Experiments 5.2.1. Acute and Residual Toxicity 5.2.1.1. Acute Toxicity Based on LC50 value of two herbicides selected in the present investigation, it was found that the test species X. welchi was most affected by Pretilachlor followed by pendimethalin; while the test species C. javanus was susceptible to Pendimethalin only with respect to their recommended agricultural doses (RAD). It was also found that X. welchi was more susceptible to the chemical Pendimethalin than C. javanus. Ecological hazards of these chemicals can only be estimated by comparing with the quantity of the herbicides applied in the agricultural fields. Comparing the LC50 value of these chemicals with their respective recommended agricultural dose, it was found that Pretilachlor possessed greater ecological risk to X. welchi having LC50 value 0.145 than Pendimethalin having LC50 value equal to 0.152 of RAD of respective chemicals. LC50 value of these herbicides to C. javanus were equal to 0.581 of RAD for Pendimethalin while LC50 value was not established during 24 hrs exposure time period within the range of RAD. From the LC50 value it was also revealed that the test species X. welchi is more vulnerable to Pendimethalin than C. javanus. When X. welchi was exposed to recommended agricultural 97

dose of two chemicals, Pretilachlor achieved fastest LT50 (110 mints.) than Pendimethalin with LT50 value of 140 mints. In case of C. javanus LT50 value achieved at 7 hrs when exposed to Pendimethalin, whereas in case of Pretilachlor exposure, the test species do not achieved LT50 value during 24 hrs exposure time. Toxicity of Pendimethalin and Pretilachlor was reported by Haque et al., (2011) on X. welchiin laboratory conditions. Park and Lee (2005) demonstrated the toxicity of Pendimethalin and Metilachlor, one of the analogues of Pretilachlor, on collembola species Proisotomaminuta using artificial sea salt solution and established seven day LD50 value 10.4 and 12.4 gm/lit for Pendimethalin and Metolachlor respectively. However, acute toxicity of Pendimethalin in soil nematode, aquatic daphnia was reported (Shah et al., 1997). Strandberg et al., (2004) showed the acute toxic effect of the chemical Pendimethalin in rat. Acute toxicity of Pretilachlor was reported in case of daphnia, honey bee, earthworms, birds and mammals (PPDB, 2014). Maryam et al., (2013) determined acute toxicity of Pretilachlor on fingerlings of a major carp Ctenopharyngodonidella and 96 hrs LC50 value was recorded as 1.43 mg/lit. Takahasi et al., (2007) and Satyabani et al., (2011) also reported the toxicity of Pretilachlor on freshwater major carp. Tilak et al., (2007), Butchiram et al., 2009) also reported the toxicity of Butachlor and Alachlor (both are analogues of Pretilachlor) on fish. All these findings fall in line with our findings related to the acute toxicity of Pendimethalin and Pretilachlor on X. welchi and C. javanus. But due to the difference of exposure period, physio-chemical properties of the test medium, exposed organism and formulation of the chemicals the data could not be compared with the experimental data of Park and Lee (2005), Shah et al., (1997) and PPDB, (2014). Pendimethalin was found highly toxic for both X. welchi and C. javanus. For both the species LC50 value of the chemical was very much lower than the Recommended Agricultural Dose. LC50value for Pendimethalin was nearly 1/6th and ½ of recommended agricultural dose for X. welchi and C. javanus respectively. X. welchi was found to be highly sensitive against the chemical Pendimethalin with faster LT50 (140 mins) than C. javanus with LT50 value 7 hrs. Species specific variation in toxic response against particular chemical was reported (Wiles and Frampton, 1996; Ponge et al., 2013) in different species of collembolan may be due to bio-availability of the chemical, body size and morphology of the exposed species (Wlies and Frampton, 1996).

98

Pretilachlor was found to be toxic for X. welchi with LC50 about 1/7th of Recommended Agricultural Dose of the chemical along with LT50 value 110 minutes. C. javanus species did not achieve LC50 and LT50 value within 24hrs exposure time period. Thus, Pretilachlor, though highly toxic to X. welchi was considered moderately toxic for C. javanus. Tomlin (1975) and Frampton (1994) also found interspecific variation of insecticides on collembola species. X. welchi faced severe knockdown effect on the exposure of both the chemical Pendimethalin and Pretilachlor with higher initial toxicity of Pretilachlor than Pendimethalin in respect to their Recommended Agricultural Dose. Thus, results of the present study indicated that application of agricultural doses of Pendimethalin and Pretilachlor might eliminate the natural collembolan fauna from soil. 5.2.1.2. Residual Toxicity It was revealed from the results of the long term residual toxicity studies of the two herbicides tested; Pendimethalin was more persistent in soil than Pretilachlor. Pendimethalin with soil half life of 90 days (PPDB, 2014), does not go rapid soil microbial degradation (Bandyopadhyay and Choudhury, 2009) with little effect of soil type on microbial degradation process (Zimdahl et al., 1984). From the present study it was found that negative toxic impact of Pendimethalin on X. welchi and C. javanus exhibited beyond 60 and 30 days respectively after application of the chemical. Comparing long term toxicities of Pendimethalin on different species of soil organisms (Kartalska et al., 2009; Velcheva et al., 2012), it was found that high negative impact observed till the 100th day after application of the chemical; though influence was species specific and was related with other physical factors (Velcheva et al., 2012). The herbicide Pretilachlor exhibited slightly low toxicity beyond 30 days for X. welchi and 7 days for C. javanusin present investigation. Soil half life of the chemical Pretilachlor is about 10 days (Dharumarajan et al., 2008) and dissipated readily by photodecomposition, microbial degradation and volatalization (Tomoyoshi et al., 2004) and persisted in soil upto 45 days after application (Dharumarajan et al., 2008).

5.2.2. Chronic Toxicity 5.2.2.1.Hatching Success Comparing with the respective control in the present investigation, hatching success of both the test species X. welchi and C. javanus exposed to Pendimethalin showed

99

significant inhibition except the T2dose. In case of X. welchi exposed to Pretilachlor maximum significant reduction of hatching success was found in T6dose followed by T5 and T4 dose in comparison with respective control. Fecundity has been reported to be a sensitive parameter to estimate the toxic impact of pesticides on soil collembola (Chakraborty and Joy, 1993). In the present study, effects of Pendimethalin on the hatching success of both the test species were more severe and ill effect of the chemical was found at doses much lower than the respective RAD. Similar effect was also found in X. welchi exposed to pretilachlor. Chakravorty and Joy (1993) reported the significant reduction of hatching success in Cyphoderus species exposed to insecticides. Haque et al., (2011) found reduction of hatching success in X. welchiexposd to herbicides. Jager et al., (2007) reported ill effect of Chlorpyrifos on egg Production of Folsomia candida. Survival of Juvenile upto 1st month In the present study it was found that hatched juvenile did not survive even upto 1st moult exposed to T4, T5 and T6 dose forX. welchi and T5 and T6 dose forC. javanus to Pendimethalin. Significant reduction in number of survived juvenile was found in all treatment doses i.e., T2, T3 and T2, T3 and T4 for X.welchi and C. javanus respectively for the chemical Pendimethalin. Hatched juvenile of X. welchi survived atleastupto 1st moult at doses T2, T3 and T4 having significant reduction with respective control exposed to Pretilachlor. Number of Juvenile survived at least up to maturity stage Juvenile survived at least up to maturity stage (egg laying stage of female having significant difference with respective control in T2, T3 and T2, T3 and T4 for X.welchi and C. javanus respectively exposed to Pendimethalin. In case of Pretilachlor exposure significant reduction of juvenile number attaining maturity stage was found in X. welchi at T2, T3 and T4 doses comparing with control. Miguel (2010) reported lower number of juvenile by Folsomia candida exposed to herbicide glyphosate. Reduction in population number of soil collembola were reported due to exposure to herbicides (Frampton et al., 2006; Lins et al., 2007; Greenslade et al., 2010), agree with our findings of ill impact of herbicides on juvenile, ultimately afeect the population number of the species exposed.

100

Moulting/Exuvia Production It was found in the present investigation that exuvia production significantly increased in T2, T3, and T2, T3 and T4doses inX.welchi and C. javanus respectively exposed to Pendimethalin comparing with respective control. Significant increase of exuvia production was also found in X. welchi exposed to T2, T3 and T4 doses of Pretilachlorcomparing with control. Moulting found in collembolan throughout life (Chakravorty and Joy, 1993). Exuvia production has been reported one of the important sensitive parameter to estimate the toxic impacts of pesticides on soil collembola (Chakravorty and Joy, 1993; Haque et al., 2011). Eijsackers (2009) reported increased moulting frequency in springtail Onychiurusquadriocellatus at sublethal doses of herbicide 2, 4, 5-T. Maturity and Life Span Significant early maturity and shorter life span in T2, T3 and T2, T3 and T4 for X.welchi and C. javanus respectively exposed to the chemical Pendimethalin comparing with respective control. Similar effect of Pretilachlor exposure to X. welchi found at doses T2, T3 and T4 with respect to control. Reproduction has been reported to be the more sensitive parameter for early detection of toxicity on collembola (Crouau and Moiä, 2006). Eijsackers (2009) and Haque et al., (2011) reported early maturity and shorter life span of soil collembola exposed to sublethal doses of herbicides. Ill effect on reproduction, early maturity and shorter life span were noticed in different investigation using Folsomia candida exposed to herbicides (Jager et al., 2007; Staempfli et al., 2007; Miguel, 2010). Muthukaruppan et al., (2005) and Dasgupta et al., (2012) found negative impact on reproduction of earthworm Perionyx sp. exposed to different pesticides. 5.2.2.2. Biochemical Parameter Acetylcholinesterase Enzyme Activity For both the test species, X. welchi and C. javanus activities of acetylcholinesterase enzyme showed significant inhibition exposed to Pendimethalin at Recommended Agricultural Doses only. No significant inhibition was recorded at sublethal doses of the chemical. Significant inhibition of acetylcholinesterase enzyme activity on Pretilachlor

101

exposure was found only at Recommended Agricultural Dose in X. welchi. Test species C. javanus not significantly affected by Pretilachlor exposure relating to acetylcholinesterase enzyme activity. Effects of herbicide on the activities of acetylcholinesterase enzyme on soil collembola is quite a recent approach. Generally, cholinesterase enzyme activity is used in vertebrate to detect exposure of chemicals like pesticides (Edward and Fisher, 1991). Chakravorty et al., (1995) noticed inhibition of acetylcholinesterase enzyme activity on soil collembolan Cyphoderus sp. exposed to insecticides methylparathion and carbaryl. Boonthai et al., (2000) found inhibition of AchE on different insect exposed to atrazine. Belden and Lydy (2000) and Jin-clark et al., (2002) reported inhibition of acetylcholinesterase activity by herbicide atrazine in combination with insecticides in midge, Chironomustentans. Campero et al., (2007) found AchE inhibition activity on damselfly Coenagrionpuella. Panda and Sahu (2004) reported little inhibition of AchE activity by herbicide Butachlor which is an analogue of Pretilachlor on Oligochaeta, Drawida willsi.

5.3. Comparative

findings

of

herbicides

effect

on

acetylcholinesterase enzyme activity between field and laboratory For both test species X. welchi and C. javanusacetylcholinesterase enzyme activity was inhibited significantly both in field and laboratory condition at Recommended Agricultural Dose exposed to Pendimethalin. However, inhibition of enzyme activity is significantly higher in magnitude and duration after the application of the chemical under laboratory conditions

than

under

field

conditions.

Comparatively

less

inhibition

of

acetylcholinesterase enzyme activity were found on both the test species exposed to the chemical Pretilachlor. Although, significant inhibition on acetylcholinesterase enzyme activity were found only in X. welchi exposed only at Recommended Agricultural Dose of Pretilachlor.Similar trend of inhibition activity of AchE enzyme were found with higher inhibition under laboratory condition in respect of control relating with magnitude and effect after application of the chemical.

102

6. Conclusions: Following conclusions were drawn from the results of the present study1.

Persistence of pretilachlor was for much shorter period than pendimethalin both under field and laboratory conditions.

2.

Both the herbicides have negative toxic impacts on the population of both the test species.

3.

Among two test species Xenylla welchi is more susceptible to both the herbicide chemical than Cyphoderusjavanuswhich results in early recovery of population.

4.

Seasonal variation of toxic effect of a chemical under field conditions due to change of environmental parameters.

5.

Findings of the present investigation clearly indicate species specific variation of toxic response against same toxic chemicals both under field and laboratory.

6.

For assessing risk of application of herbicides in agro-ecosystems, LC50 value alone is not adequate. It is necessary to compare LC50 value of the chemicals with their respective recommended agricultural doses for non-target soil organisms to estimate the ecological safety of the herbicide chemicals.

7.

In present investigation comparing the LC50 value with the respective recommended agricultural doses it was found that pretilachlor and pendimethalin were most dangerous for soil collembola population.

8.

From the preliminary screening study it was revealed that herbicides like paraquat, 2,4-D amine, 2,4-D sodium salt and metribuzin appeared quite safe for non-target soil collembola population in agro-ecosystem.

9.

Chronic effect on different life cycle parameter of both the test species at sub-lethal doses also indicated that both pendimethalin and pretilachlor herbicides were more dangerous for the soil collembola species.

10. Among two species Xenyllawelchi appeared highly sensitive against both the herbicides pendimethalin and pretilachlor than the species Cyphoderusjavanus and

103

can be used as sensitive species for early detection of pollution in agro-ecosystem by employing various life cycle parameters at sub-lethal doses. 11. Acetylcholinesterase enzyme activity of the more sensitive species Xenyllawelchi may be used as biomarker for the toxicity of herbicide chemical pendimethalin in agro-ecosystem. 12. Further experiments on this biomarker enzyme may be used for predicting effects of herbicides in field conditions. 13.

By employing sensitive Xenylla welchi species on unknown soil sample prediction of herbicide containment may be possible by using mortality chart obtained from probit (for determining LC50) and the mortality chart prepared by using known percentage of herbicide solution.

14. Comparing of data regarding acetylcholinesterase enzyme activity between field and laboratory conditions it was revealed that ill effect of herbicide was much lower both in magnitude and duration under field conditions than under laboratory conditions. 15.

Considering all the data generated in the present study it was concluded that application of pendimethalin as weed killer in agricultural field was ecologically more dangerous than pretilachlor for soil ecosystem. Collembola species Xenyllawelchi appear as potential bio-sensor species than Cyphoderusjavanus for herbicide pollution in agro-ecosystem relating to pendimethalin and pretilachlor.

104

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