Chapter III MATERIALS AND METHODS

The present investigation entitled involved a pot experiment conducted in net house followed by laboratory analysis of the plant, grain and soil samples in the Department of Soil Science and Agricultural hemistry, !nstitute of Agricultural Sciences, "anaras #indu $niversity, %aranasi& 3.1 Description of the site

3.1.1 Physiographic situation %aranasi is situated at an altitude of '(&)* meters above mean sea level and located between +,(*'- .orth latitude and '(( /0- 1ast longitude& 3.1.2 Climate %aranasi falls in a semi2arid to sub humid climate with moisture deficit index between +( 2 3(& The normal period for onset of monsoon in this region is the /rd wee4 of 5une which lasts up to end of September or sometimes extends up to the first wee4 of 6ctober& Showers of rain are often experienced during winter season& The annual rainfall of this region is about **(( mm& 7enerally, the maximum and minimum temperature ranged between +( ( 2 3+( and 8( 2 +'( , respectively& 9ay and 5une are the hottest months with maximum temperature ranging from /8( to 3+( & The cold period lies between .ovember and 5anuary with minimum temperature varying between 8( 2 *(( & The mean relative humidity is about 0': which rise to '+: during wet season and goes down to /(: during dry season& Table 3.1
&ee' o.

Meteorological data during crop growth period ( o!ember" 2##$ to March 2#1#%.
(ainfall (mm% Temperature (.). (*% +unshine ()rs.% ,!aporation (mm%

Month and Dates

Materials and Methods

Ma.. /0 /1 /2 3' 38 ,( ,* 02 1 2 3 / 0 1 2 3 $ 1# 11 12 13 9ar& ;eb& 5an& Dec& .ov& (,2** *+2*' *82+, +02(+ (/2(8 *(2*0 *)2+/ +32/* (*2() ('2*3 *,2+* ++2+' +82(3 (,2** *+2*' *82+, +02(3 (,2** *+2*' *82+, +02(* (&( +8 (&( (&( (&( (&( (&( (&( (&( (&( (&( (&( (&( /,&0 /(&0 *&3 /+&+ (&( /'&+ (&( (&( /*&/ +)&0 +3&' +,&0 +'&0 +,&+ +,&/ +/&/ ++&/ ++&, +/&0 +3&) +)&8 ++&+ +/&* +0&* +0&+ +' +0&/ /+&( /,&)

Min. *'&3 +(&, **&0 8&3 **&3 '&' *(&* )&/ ,&' )&* )&( )&+ *+&* */&* *+&* 8&8 */&( **&3 *,&+ *0&' *0&)

Ma.. )8 ', '+ )/ '( '( 8+ ')&3 )8 ') )/ )0 '( '8 '* '( '( 0, )) 0) 0(

Min. 3( 0, /0 +) /3&/ 3* 30 33&* /3 /, /( 3( /) )( ,, 3/ ,( /+ ,' /0 +' )&' +&) '&* '&+ '&* )&* 0&/ /&3 )&* 8&) 3&) ,&0 *&( ,&* /&, )&* '&3 8&( /&8 3&, '&' +&+ *&' +&* +&, +&0 +&+ *&3 +&3 '&, 8&( ,&* ,&, +&8 3&' /&8 /&8 3&) 3&8 3&3 /&) 3&8

3.2

Pot e.periment A pot experiment was conducted on wheat crop in +((82*( in the net hemistry, !nstitute of

house of Department of Soil Science and Agricultural Agricultural Sciences, "&#&$& %aranasi $&<& 3.2.1 Collection of sewage sludge

Materials and Methods

Sewage sludge was collected from 7anga water sewage treatment plant "hagwanpur, %aranasi in the month of 9ay +((8& The sewage sludge was dry at the time of collection& 3.2.2 ,.perimental soil To conduct the pot experiment, the soil sample was collected from the Agricultural =esearch ;arm, !nstitute of Agricultural Sciences, "anaras #indu $niversity, %aranasi& ;resh sewage sludge was applied in pot on basis of calculation of tonnes ha2* soil with different levels of recommended dose of .<> and rice crop was grown during 4harif +((8& After harvesting of rice, wheat crop was grown in rabi, +((8 in same pots with same levels of recommended dose of .<> and sewage sludge was not applied in wheat with a view to study the residual effect of sewage sludge on wheat&

3.1

Processing of +oil +ample ollected soil samples of the pot experiment were brought into

laboratory in a separate one 4g polythene bag and dried at room temperature& After drying, bro4en clods were ground on wooden plan4 with wooden roller and passed through a + mm sieve& The soil samples were then stored in polythene bags& The homogeni?ed soil samples were analysed for selected physico2chemical properties& 3.2.1 +oil p) A soil2water suspension was prepared in the ratio of *@+&, A*( g soil with +, mB of distilled waterC and p# was measured with the help of p# meter A hopra and >anwar, *8'+C& 3.2.2 ,lectrical conducti!ity The soil water suspension prepared for determination of p# was used to
3

Materials and Methods

estimate the electrical conductivity of the soil& Soil suspension was allowed to settle till supernatant become clear& 1lectrical conductivity was measured with the help of 1 meter and expressed as dS m2* ASpar4s, *880C& 3.2.3 4rganic Carbon (&al'ley and 5lac'" 1$3/% Procedure@ 6ne g of soil was ta4en in a ,(( mB of conical flas4& Ten mB of * . >+ r+6) solution was added and mixed& Then +( mB of onc& # +S63 was added, the flas4 was swirled +2/ times and allowed to stand for /( minutes on an asbestos sheet for the reaction& The suspension was diluted with +(( mB of distilled water& Ten mB of ',: # /<63 and * mB of diphenyl indicator were added and titrated against the solution of (&, . ;errous Ammonium Sulphate till colour changed from violet to bright green& A blan4 titration was also carried out& Calculation : 6rganic arbon in soil Ehere, D A"2TC x (&((/ x *(( + x wt& of soil

" D %olume of (&, . ;AS solution used for blan4 titration T D %olume of (&, . ;AS solution used for sample titration

3.2./ 6!ailable nitrogen Available .itrogen content in soil was determined using >Feltec Semi2 Auto .itrogen Analy?er by al4aline <otassium permanganate method as proposed by Subbiah and AsiFa A*8,0C& The method has been widely adopted to get a reliable index of .itrogen availability in soil due to its rapidity and reproducibility& Procedure@ ;ive g of soil sample was weighed and transferred in a distillation tube& The sample was moistened with , mB distilled water, washing down the soil adhering to the nec4 of flas4& Twenty five mB (&/+: >9n6 3 was added to
4

Materials and Methods

it and the distillation tube was set to the instrument& !n a +,( mB conical flas4, +( mB of + : boric acid mixed indicator was ta4en and placed under the receiver tube& Tap water was run continuously in the condenser& Twenty five mB of +&,: .a6# was suc4ed and added to the distillation tube& Then it was put on distillation for 8 min& During this process the . released in the form of ammonia is trapped in the boric acid, which develops green colour& The flas4 containing the distillate was removed& The distillate was then titrated against (&(+ . #+S63 until pin4 colour developed& Calculation 9inerali?able . A4gGhaC D AS2%C H (&(+H *3H *(0 H+&+3 *(((H,

S D Sample titration reading % D "lan4 titration reading 3.2.0 6!ailable Phosphorus Available phosphorus content of soil was determined by 6lsen-s method A6lsen, *8,3C& ;irstly reagent A was prepared by using ammonium molybdate, antimony potassium tartarate and #+S63& Then reagent " was prepared with the help of reagent A& Two gram of soil was ta4en in a *,( mB conical flas4, a pinch of Darco 720( and 3( mB of 6lsen-s reagent A(&, 9 .a# 6 /C was added to it& !t was then sha4en for /( minute on mechanical sha4er and the suspension was filtered through Ehatman .o& * filter paper& ;ive mB of filtrate was transferred in a +, mB volumetric flas4 and was acidified with +&, 9 #+S63 to p# ,&( and +( mB distilled water was added followed by 3 mB of reagent "& After waiting for *( min the intensity of blue colour was measured on spectrophotometer at ''+ nm& Simultaneously a blan4 was also run& ;irst standard reading was ta4en followed by sample reading& Calculation Available <hosphorus D = H %olume of extract H +&+3H *(0

Materials and Methods

A4gGhaC 3.2.1 6!ailable Potassium

%olume of aliIuot H Et& AgC of soil H*(0

Ehere = D g < in the aliIuot Aobtained from standard curveC Available potassium content of soil was determined by ;lame <hotometer A* . ammonium acetate extractC method A#anway and #eidal, *8,+C& ;ive g soil was transferred in a *(( mB conical flas4 and +, mB of * . ammonium acetate solution was added and it was sha4en for , minutes& The suspension was then filtered through Ehatman .o& * filter paper and potassium concentration in the filtrate was measured using flame photometer& ;irst standard reading was ta4en followed by sample reading& Calculation Dilution factor D +, G , D , times =eading of the flame photometer for the test sample D = oncentration of > in the sample from standard curve against the reading = D

Available > A4g ha2*C

D D

x , x +&( x *( x *( x *&+*

Available >+6 A4g ha2*C 3.2.2 6!ailable +ulphur

Available sulphur content in soil was determined by Turbidity method A hesin and Jein, *8,+C& ;ive g of soil was ta4en in a *(( mB conical flas4 and +, mB of (&*,: a l+ solution was added to it& Then it was sha4en for /( minutes on a sha4er and the suspension was filtered through Ehatman .o& 3+ filter paper& Then *( mB of the aliIuot was transferred to a +, mB volumetric flas4 and * g of the sieved "a l+ crystals were added and it was sha4en for * minute& 6ne ml of (&+,: gum acacia solution was added and the volume was made to the mar4& !t was sha4en for one minute and the turbidity was measured
6

Materials and Methods

after +, to /( minute on spectrophotometer, using a blue filter at a wavelength of 3+( nm& Simultaneously a blan4 was also carried following same procedure& ;irst standard reading was ta4en followed by sample reading& Calculation Available Sulphur A4gGhaC Ehere, A D S Amg B2*C in aliIuot " D ;inal volume of turbid solution AmBC D %olume of soil extractant used for extraction AmBC 1 D Air dry soil weight AgC 3.2.3 DTP6 e.tractable micronutrients and hea!y metals Available d, .i, <b, r, Kn, 9n, u and ;e in soil samples were AH "H H +&+3

DH 1

determined by the method of Bindsay and .orvell A*8)'C& !n this method, *( g of soil was extracted with +( mB DT<A extracting solution by sha4ing for + hrs on a sha4er& The suspension was then filtered and trace elements were determined by atomic absorption spectrophotometer A$.! A92808C using respective cathode lamps& 3.3 Plant analysis The plants were harvested at maturity, washed seIuentially with (&+: detergent solution, (&* . # l and finally with double distilled water& The plant material was dried at 0( for 3' h in a hot air oven& Dry plant tissue was finely grounded& 3.3.1 ,stimation of nitrogen in plant and grain sample .itrogen content in plant and grain sample was determined by 9odified >Feldahl 9ethod as per procedure outlined by 7upta A+(()C& !n a digestion tube (&, g of powdered plant straw was ta4en and *( ml of diacid solution A8@*, #+S63@# l63C was added and 4ept for overnight then *(g of sulphate mixture
7

Materials and Methods

LA+( parts >+S63C M * part catalyst mixture A+( parts uS6 3 M * part selenium powderCN was added and heating was done in a digestion chamber till a clear colourless solution appears, then cooled and filtered through Ehatman .o& 3+ filter paper in a ,( mB volumetric flas4 and made up to the volume with distilled water& Ten mB of 3: boric acid solution containing bromocresol green and methyl red indicator was ta4en in a conical flas4, outlet of distillation apparatus was dipped into boric acid solution& ;ive mB of the aliIuot was ta4en and transferred to the distillation tube of >Feltec Semi2Auto .itrogen Analy?er and *( mB of 3( : .a6# was suc4ed and added to the distillation tube& Then the instrument was put on distillation for 8 min& After completion of distillation, the boric acid was titrated against (&(+ . #+S63& "lan4 was also run and . content was calculated by formula& Calculation <ercent . in <lant material (&(+ H T H (&(*3 H ,( H,( , H (&,

T D Sample reading 2 "lan4 reading 3.3.2 Digestion for P" 7" +" 8e" Mn" 9n" Cu" Cd" Cr" and i in wheat straw and grain 6ne gram dried and powdered plant sample A+( meshC was ta4en in a ,( mB digestion tube and *( mB di2acid mixture A3@* vGv #.6 /@ # l63C was added to it and was 4ept overnight& !t was then digested on a bloc4 digester till a colourless solution was obtained& The volume of acid was reduced till the flas4 contained only moist residue& The flas4 was cooled and +, mB of distilled water was added& The solution was filtered into a ,( mB volumetric flas4 and diluted up to mar4& 3.3.2.1 Colour de!elopment of Phosphorus

Materials and Methods

Two mB of digest was ta4en in a +, mB volumetric flas4 and + drops of +, 3 di2nitrophenol indicator was added& Ammonium solution was added till yellow colour appeared and then 0 . # l was added AdropwiseC till it become colourless& Then , mB of %anadate molybdate solution was added and diluted to +, mB with distilled water, mixed well and the intensity of yellow colour was read on spectrophotometer by using blue filter at 33( nm wave length& A blan4 was also run without < solution simultaneously& ;irst standard reading and then sample reading was ta4en& <hosphorus content in straw and grain was calculated using standard curve and expressed as total < A:C& Same procedure was followed to determination of < content in grain except weight of sample in case of grain only (&+ g powdered was ta4en A5ac4son *80)C& Calculation Total < : D Abs& H dilution factor Slope of std curve H *((((

3.3.2.2 8or Potassium content in straw and grain <otassium content of plant and grain was determined by ;lame <hotometer method A5ac4son, *8)/C& !n case of potassium, digested extract was used directly for flame photometer determination of potassium& <otassium content of digested straw and grain was determined by ;lame <hotometer& > content was calculated using the standard curve and expressed as total >& Calculation = H dilution factor *((((

Total > :

= D ;lame photometer reading 3.3.2.3 8or +ulphur content in straw and grain Sulphur content in straw and grain was determined per procedure outlined by Tandon A+((*C& Ten ml of the aliIuot was ta4en in a +, mB volumetric flas4 and *&( g of the sieved "a l + crystals were added and sha4en
9

Materials and Methods

for * minute& Then * mB of (&+,: gum acacia solution was added and the volume was made up to the mar4 and it was sha4en for one minute& The turbidity was measured +, to /( minute after the precipitation, on spectrophotometer, using a blue filter at a wavelength of 3+( nm& Sulphur content of sample Astraw and grainC was calculated using standard curve and expressed as total S A:C& Calculation AH "H DH 1H*((((

Available Sulphur A:C Ehere,

A D S Amg B2*C in aliIuot " D ;inal volume of turbid solution AmBC D %olume of plant extractant used for extraction AmBC 1 D Air dry plant weight AgC 3.3.2./ ,stimation of 8e" 9n" Mn" Cu" Cd" Cr and i The content of ;e, Kn, 9n, u, d, r and .i in the straw and grain digest was determined by using atomic absorption spectrophotometer A$.! A9 O 808C as per procedure outlined by Tandon A+((*C& 3.$ +tatistical 6nalysis and :nterpretation of Data The raw data observed during the whole experiment, was put for statistical analysis by following the ;actorial software& omplete =andomi?ed Design A =DC to draw the valid differences among the treatments using S<SS

10