40

CHAPTER III

MATERIAL AND METHODS

A. Location and research period

The research was conducted in the Karanganyar district from April

2015 till January 2016. Karanganyar is located in Central Java province in

the slope of Lawu Mountain. This Rdistrict is bordered by Sragen district in

the North, East java province (east side), Wonogiri and Sukoharjo districts in

the south, and Surakarta and Boyolali districts in the west part respectively.

The average altitude in the district is roughly 511 m above the sea level; the

lowest elevation is 90 meters in Jaten, whereas reach 2000 meters in

Tawangmangu sub district. The district comprises different soil types, such as

Alfisols, Andosols,Ultisols, Alluvial and Inceptisol; and the maximum annual

precipitation can reach 7,231.4 mm. The study area is characterized by

irregular topography including hills, rolling, vales, and alluvial plains

(Department of Statistic of Karanganyar, 2014)

The research involved four villages namely Kemuning, Bakalan,

Tamansari, and Sambirejo, respectively. The main economic activities in

these villages are modest business in local markets as well as agriculture. The

agricultural system comprises rice fields grown in the alluvial lands. The

yields are partially sold in the local markets, while the remnant is kept for

family’s consumption. Apart from rice fields, there are some diversified rain

fed crops grown in the highlands, including maize, cassava, and beans. The

farmers also grow fruit trees such as Avocado, papaya, and Orange tree,

which are used to enrich the local diets. In addition, the fruit’s tree also

represent a source of income to the local people, although seasonally. Across

the Sub-districts there are also some woody species used to produce furniture

and in house construction, and is believed that it contributes with

considerable percentage to the local incomes. The medicinal and spice herbs

are produced in agroforestry systems mixed with woody stypes and crops.

41

Figure 9. Research Location

42

The cultivation of medicinal herbs across the investigated villages

is one of the economic activitieswhich is currently gaining power. These

herbs have been domesticated for decades as basic ingredients in the local

cuisines. However, in the last decade their cultivation has been massively

intended to supply market’s demand. Furthermore, the herbs are grown in

agroforestry systems where they take advantage of the shade offered by trees.

Usually the medicinal plants production covers small land areas because they

do not represent the main crop.

According to the Department of Agriculture of Karanganyar, the total

production of Ginger in the regency reached 133.6 ton in the year 2014. In the

same year the total production of turmeric by sub district was reported as

follows: 446.31 ton for Kerjo, 71.7 ton for Jumantono, and 404.132 tons for

Jumapolo. However, the data of Ngargoyoso were not revealed. Regarding

the production of Javanese Ginger, the production was 104.5 tons for

Jumapolo, 67.6 ton for Jumantono, and 31, 6 ton for Kerjo. The data of

Ngargoyoso once again are not available. For the year 2015, the global

production reported until July for three herbal plants was 335,5 Ton, 50.6 ton,

and 123.3 ton corresponding to Ginger, turmeric, and Javanese ginger

respectively

B. Research design

1. Research type

This research relied on quantitative and qualitative methods

including field observations, site description and soil sampling. The

sampling process based on the soil units previously identified on the map.

Formal interviews were conducted including practices of herbs production,

and socio-economic aspects.

2. Population and samples

The population in this study is represented by every medicinal

plants farms belonging to the members of the herbal plants association

(Biofarmaka). From this population, sampling sites were selected

purposively based on soil type and slope of terrain. The samples are every

soil cores collected from the herbal plants farms. The choice for purposive

43

sampling is due to the fact that across the research place not all farmers

produce medicinal plants, and also among those who produce medicinal

plants just few of them adhered organic farming practices.

3. Variables of study

This research is based on three variables namely Dependent

variables, independent variables, and supporting variables.

a. Dependent variables

Are all those which are influenced by independent variables. In

this research dependent variables include cadmium content,Cation

exchange Capacity-CEC, herbaltypes, pH and efficiency of binding

agents.

b. Independent variables

Define all those variables that exert influence over the

dependent variables. On the other hand, the features of independent

variables are somehow expressed on the dependent variables. In this

study are taken as independent variables, Soil texture, soil type, pH,

Soil organic matter, CEC.

c. Supporting variables

Comprise of those variables that exert indirect influence over

the dependent and independent variables. Usually their help to predict

the behavior of the two types of variables. Thus, this study consider as

variables of support, precipitation, fertilizers, geology, slope, and

temperature.

4. Data collection and procedures

The data used in this research are as follows:

a. Primary data

Primary data was obtained directly from the object of study

through field observation, sampling and interviews across the four sub

districts in the Karanganyar district. Field observation included among

other parameters, the inputs applied in the production process, yield,

and preparation of organic fertilizers and bio-pesticides, annual

incomes. The sampling procedure aims to collect representative soil

cores and herbal plant samples.

44

b. Secondary data

Secondary data were obtained from third sources. Data

regarding the production of herbal plants in the mentioned sub districts

were obtained from the Agricultural office of Karanganyar; data

related to precipitation, temperature, geology, soil units, topography

were obtained from the related institutions. Literature review has been

done to complement the research.

c. Sampling methods

The sampling process was proceeded with two methods

namely, Soil map unit and elevation sampling in order to catch patterns

of variability. Sampling points were established across the identified

soils units in each sub district. However, the establishment of the plots

followed a purposive sampling method because the study is intended

exclusively to the biofarmaka clusters, and these are just found in

certain zones. On undulating terrains (elevation sampling), samples of

soil and plants were collected using stratification (Fiure 11). The

stratification was horizontal to catch differences resulting from

gradient, while the vertical stratification aims to catch variability of

Cadmium content and other parameters within the soil layers.

In each sampling point inside of the plot, 2 sub-samples were

taken using an auger. The first subsample was collected from the first

layer (0-15 cm of depth) and the second sub-sample was taken from

the second layer (15-30 cm of depth). In this case is taken into

consideration 30 cm of the topsoil recommended by many scientists as

ideal for the analysis of heavy metals concentration. This sampling

depth also is based on the principle of that many plants uptake the

nutrients within 30 cm. On the other hand, soil organic matter, which

binds considerable amount of heavy metals, is found in large

percentage in the topsoil. In addition, is also important to mention that

the herbs in study, establish their rhizomes in the first 15-20 cm of soil

depth.

45

The sub-samples collected using a Zig-zag technique (Figure

11), were mixed in two buckets according to the depth, then two (2)

composite samples with 500g each other were collected from the

buckets and packed in plastic bags for lab analysis.

The number of sub-sampling points depended on the size of the

plot or herb’s field. However, 6 to 15 cores are recommended per area,

and every point is equidistant. The plant sampling (rhizomes) also

obeyed the stratification method, but differently from the soil cores;

only one composite sample of rhizomes was taken from each field

(figure10)

Figure 10. Soil Sampling Using an Auger

On the left side: Sampling scheme for a sloped field with

distinct upper, middle, and lower areas. Circles represent sub-sample

locations which are composited for each of the three areas. On the right

side: grid-sampling in the field. Each sample is analyzed separately to

evaluate field variability.

46

Figure 11. Sampling Scheme (Harrell, 2014)

From the four sub districts a total of 28 sampling points

were established. These sampling points are distributed as follows: 6

sampling points for Ngargoyoso (Kemuning); 9 sampling points for

Kerjo (Tamansari), including one (1) control point as well; 6 points

for Jumantono (Sambirejo), and 7 sampling points for Jumapolo

(Bakalan), respectively. Control points were also established based on

those areas which are lying fallow at least for more than 10 years.

47

5. Material

Table 1. Materials used in the field and laboratory

For field

word Guiding Material Lab Experiment Reagents

Auger Administrative

map AAS Dolomite

Knife Geological map Tubles Charcoal

Buckets Land use map Destillation

chamber Organic fertilizers

Shovel soil unit map Shaker (vortrex) Three types of soil

Hoe GPS pH meter HNO3,

HClO4,CaCl2

Tape Topographical map

H3BO3, Zinc, HCl

Camera Clinometer

Nacl, NH4OAc

Plastic bags

Rhizomes of herbs

C. Laboratory methods

1. Atomic Absorption spectrometer (AAS)

Cadmium is ubiquitous in the environmental ecosystems and is not

degradable at all. It can be found in soils, sediments, water, air, and food.

However analytical methods are available for the analysis of cadmium in

all of these environmental media. Such methods have sensitivity to

measure levels and detect elevated and lower concentrations.

In this research the analysis of cadmium content in soil samples

and rhizomes of medicinal herbs was performed using Atomic-

Absorptionspectrophotometer (AAS). The cadmium concentrations

obtained were compared with the European Union, Indonesia, and WHO

standards for limits of Cadmium in food and herbal drugs. The technique

of AAS has its origin in 1666 with Isaac Newton who used a prism to

separate colors of the solar spectrum. By 1900s many experiments with

AAS were performed, such as the study of atomic absorption spectra of

metals in Iron group carried out by Angerer in 1924. However, the first

real application of AAS to chemical analysis was made by Walsh in 1955.

Atomic Absorption Spectrometry is a process involving the absorption by

free atoms of an element of light at a wavelength specific to that element,

48

or simply it means by which the concentration of the metal can be

measured.

In AAS, emission, absorption and fluorescence, energy is put into

the atom population by thermal electromagnetic, chemical and electrical

forms of energy and are converted into light energy by various atomic and

electronic processes before measurement. The AAS is useful not only for

the identification but also the quantitative determination of many elements

present in samples. The method is sensitive and is able to detect down

small amount of metals around 0.001pp-1ppm. (Thermo Fisher Scientific-

AAS Methods Manual, Issue 4 (08/04), 2008, United Kingdom,

www.thermo.com)

2. Determination of Soil pH

The soil pH was measured through a pH meter in soil solution to

estimate the dominance of charges in the soil (if the soil can hold cations

or anions). The pH determined in this research was pH (H2O). The

procedure consisted in weighing out 5 grams of soil sample using an

analytical weighing scale, and then was put into a flask bottle (three

replications for each soil sample brought from the field). Were added 10

ml of distilled water into the flask bottle containing soil and then, the

flasks were shaken for 30 minutes using a vortex shaker. After 30 min the

flasks were taken out from the shaker and let static overnight to have all

the particles precipitated. Finally, the pH values were recorded using a pH

meter. Before starting to read the pH of the samples, the pH meter was

calibrated using a neutral solution (pH 7 buffer) by introducing the

electrode into this solution. Then the electrode was rinsed with distilled

water and wiped with Kim wipe. Again, the electrode was submersed into

pH 4 buffer, and when the pH icon stopped flashing, the electrode was

rinsed with distilled water and then dried with Kim wipe. During the

measurement, the electrode was soaked into distilled water after each

measurement, and regularly Kim wipe was used to clean the electrode

(Figure 12).

49

Figure 12. pH meter

3. Determination of Soil Organic Matter.

To measure soil organic carbon, Walkey-black method was used.

The determination of soil organic matter was performed weighing out 0.1

g of soil sieved with 0.5 mm mesh, and then transferred into flask bottle.

Then 1.5 ml of potassium dichromate was added and shaken, then

followed by an addition of 2.5 ml of concentrated Sulfuric acid (1%), and

the solution was swirled and allowed to cool. The sample must be cooled

as a result of the exothermic reaction when the potassium dichromate

(K2Cr2O7) and Sulphuric acids (H2SO4) are mixed), prior for adding water

to halt the reaction. To continue, 7.5 ml of distilled water were added up to

a volume of 20 ml, and then the solution was allowed to stay overnight in

order to allow the solution to become clear. In the next day, the clear

solution was used to proceed with measurements using a

spectrophotometer method, with wavelength of 651 nm (Tiessen and Moir,

1993 cited in Brian A, 2002).

The chemistry of this extraction procedure is as follows:

2Cr2O7

2-

+3 C0

+ 16H+

= 4Cr 3+

+ 3CO2 + 8H20

Organic karbon content (%) = ppm curve x ml extract x 1000 ml-1

x

100 x mg soil-1

x fc

= ppm curve x 20 x 1000-1

x 100 x 100-1

x

fc

= ppm curve x 0.02 x fc

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Description

Ppm curve = content obtained from the relationship between content

of linear curve and the reading after blank correction.

100 = Conversion to percentage

Fc = Correction factor of water content (=100/(100 - % water

content)

Organic matter content (%) =1.724 x % Organic carbon

a. Preparation of soil samples

The collected samples were brought to the laboratory and

initial treatment was done for the preservation and further analysis

according to the following methods. The soil samples were treated by

adopting the method recommended by Saeed and Rafique cited in

Sqbal et al., 2011. Firstly, soil samples were air dried for a day and

solid particles like litter, roots were removed. Secondly, the samples

were dried in oven at 105oC till a constant weight was acquired. Then

about half of each sample was ground in a wooden mortar. After

grinding, each sample was passed through 0.5 and 2.0 mm nylon mesh

sieve. Finally, soil samples were repacked, labeled and preserved for

further analysis.

b. Preparation of plant samples

The sample preparation technique for plants involved steps

such as, washing, drying (figure 13), grinding, packing, labeling, and

storage. The plant samples were prepared according to the method

recommended by Jones and Amma cited in Sqbal et al., 2011. The

plant (rhizomes) samples were firstly washed with detergent then

washed with water and air dried, and then were dried in an oven at 70-

80oC until a constant dry weight was achieved. Secondly, dried

samples were ground through a wooden mortar to make a fine powder.

Finally, the powder was passed through 0.5 mm nylon mesh sieve and

packed in polythene bags to prevent absorption of water from the

humid environment (Garcia and Báez, 2012).

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Figure 13. Air dried Rhizomes indoor

4. Analysis of Soil Cation Exchange Capacity (CEC)

The cation exchange capacity was determined by ammonium

acetate (pH 7) method. Five grams (5 g) of soil sample sieved with 0.5 mm

mesh were weighed out using an analytical weighing scale (figure 14), and

then transferred into a flask bottle. To this 5 g soil were added 50 ml of

ammonium acetate, and the solution was shaken for 30 minutes (fig. 15).

Figure 14. Shaker

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Figure 15. Analytical weighing Scale

After having been shaken for 30 minutes, the solution was allowed to stay

overnight to allow the particles to precipitate. The solution was filtered using a

filter paper and then the soil contained in the funnel was washed with 10 ml of

alcohol. The filtrate was discarded and the flask bottle was replaced by a clean

one. Furthermore, the filter paper’s surface was washed with 50 ml NaCl/ KCl

10% (Figure 16).

Figure 16. Filtration Process and Washing with alcohol

The filtrate was taken to the distillation chamber (Figure 17). In

this process 5 ml of NaOH and 2 grains of Zinc (Zn) were added to the

distillation flask containing the solution. In addition was prepared a

reservoir solution of 10 ml H3BO31% and the mixed solution. After that,

53

the distillation flask was heated and the distillation stopped when the

volume of the solution reached 40 ml. Finally, 10 ml was taken from 40

ml, and the titration process was performed by adding chloride acid. In the

end a volume of 0.1 N HCl was recorded.

Figure 17. Distillation Chamber

The Catio exchange capacity (CEC-cmol (+)/Kg) was calculated

using the formula as follows.

CEC (cmol(+)kg-1

) = (Vt-Vb) x N HCl x 0.1 x 1000 g/soil sample (g) x Fd x

Fc

Description

Vc = Titration volume

Vb = Blanco volume

0.1= Conversion factor from mmol to cmol

Fd = Dillution factor (40 ml/ 10 ml)

Fc = Correction factor of water content (= 100 / (100 - % moisture)

5. Analysis of soil texture

Soil samples collected from two depths 0-15 cm and 15-30 cm

respectively were analyzed to determine the distribution of soil mineral

particles. The percentages of sand, silt and clay were determined using the

Pipette method. Pipette method is an exact and accurate method, however

is time consuming. To ensure good dispersion of soil mineral particles and

54

consequently their precise estimation using this method is necessary to

remove the portion of organic matter. The dispersion of soil sample allows

that each particle is settled individually instead of forming an aggregate

clump. The destruction of organic matter was done through a pre-treatment

with hydrogen peroxide and manganese oxide. Furthermore, the soil

sample was treated with hydrochloric acid to decompose carbonates. The

use of hydrogen oxide (H2O2) decomposes organic matter slowly in soils

with high manganese oxide content. As a result, soils with high amount of

manganese oxide and lower organic matter need not treatment for organic

matter removal. To resolve the inhibitor characteristic of manganese oxide,

acetic acid (CH3COOH) is used followed by hydrogen peroxide to convert

manganese oxide into manganous form, which does not interfere with the

destruction of organic matter through hydrogen peroxide.

The particle size was determined by weighing out 10 grams of soil

sieved with 2 mm mesh and transfer into a beaker also called shaker cup.

To this amount were added 50 ml of distilled water and 15 ml of hydrogen

peroxide (H2O2 10%), stirred for 5 minutes and left overnight until the

reaction got finished (figure 21 ), aiming to destroy organic matter. Then,

20 ml of H2O2 (30%)were added, then heated till boiling (figure 19) and

left static till get cool again. The heating process was stopped when the

foam disappeared. Furthermore, 20 ml of HCl (2N) were added, then

heated till boiling (Figure 18), and allowed to cool (Figure 20). The

addition of HCl aims to decompose carbonates. This step was followed by

an addition of distilled water into the beaker (Volume: 1000/500 ml). Once

the soil particles got settled, waste water (suspended particles) were

disposed. The disposal was performed 4 times till the water turned clear.

55

Figure 18. Heating the Solution of

HCl

Figure 19. Heating the Solution of

H2O2

Figure 20. Cooling of Solutions and H2O2

56

Figure 21. Reaction of H2O2 before the heating process

Moreover, 10 ml of Tetrasodium pyrophosphate (Figure 22 and 23)

were added and followed by an addition of distilled water up to the limit of

the measuring tube (Volume: 1000/500 ml). Then the solution was stirred

for a minute and left static for 1 minute as well. Having finished this step,

an empty cup with known weight was prepared, and then 25 ml of the

solution were pipettedand poured into the cup. The cup containing the

solution was taken into the oven, and then weighed out after getting dried.

Figure 22. Filtration Process

Figure23. Solution with Na4P2O7

57

The percentages of each soil particles (silt, clay and sand), were finally calculated

using the equations as follows to determine the final. The textural triangle was

also use to determine the textural composition (Figure 24). The number 20 is the

conversion factor obtained from 25 ml pipetted from 500 ml.

% clay = (20 x mass of clay in aliquot / total mass of soil) x 100 %

% silt = (20 x [mass of silt + clay - mass of clay] / total mass of soil) x

100%

% sand = 100 % - (% silt + % clay)

Figure 24. Textural Triangle

6. Analysis of Cadmium content in soil samples

Cadmium content in soil samples was determined through

spectrophotometer method. The determination was done by weighing out

0.5 grams of soil sample sieved with 0.5 mm mesh, and put into a

destruction flask. Were added 1ml and 3 ml of nitric acid (HNO3) and

HCLO4, respectively, and the solution was allowed to stay overnight. In

the next following day the mixtures were submitted to destruction chamber

till the orange smoke has disappeared, and the solution was cooled. Then,

were added 8.7 ml of distilled water up to 10 ml of the water volume, and

the solution was shaken for 30 minutes using Vortex shaker. After the

solution having been left static was filtered using a filter paper. After

58

filtration, the filtrate was diluted up to 4 times. The procedure consisted in

adding 3 ml of distilled water to 1 ml of the extract. The measurement was

performed with an Atomic Absorption Spectrophotometer (AAS) with 228

nm of wavelength.

7. Analysis of Cadmium content in plant tissues.

Cadmium content in plant tissues collected from rhizomes of three

herbaceous plants namely, turmeric, ginger and Javanese ginger, was

determined through spectrophotometer method. The determination was

done by weighing out 0.1 grams of ground plant sample and put into a

destruction flask. Were added 1ml and 0.3 ml of nitric acid (HNO3) and

HCLO4, respectively, and the solution was let static overnight. In the next

following day was carried out the destruction process. Fist was heat at

90oC for 90 minutes, and then at 110

oC for 75 minutes. The digestion

temperature was raised again to 130oC for 150 minutes, and then to 150

oC

for 60 min till the orange smoke has disappeared. When the orange smoke

disappeared, the chamber temperature was raised to 170oC for 70 min till a

white smoke was formed. The digestion finished with the formation of a

white precipitated with 1.3 ml of solution. Finally the solution was cooled.

Then, were added 8.7 ml of distilled water up to 10 ml of the water

volume, and the solution was shaken for 30 minutes using Vortex

shaker.After the solution having been left static was filtered using a filter

paper. After filtration, the filtrate was diluted up to 4 times. The procedure

consisted in adding 3 ml of distilled water to 1 ml of the extract (Figure

26). In the Cd content was measured through an Atomic Absorption

Spectrophotometer (AAS) with 228 nm of wavelength (Figure 25).

59

Figure 25. Analysis of Cadmium using AAS

Figure 26. Extraction of Cadmium from soil and Rhizomes

60

Figure 27. Destruction process of the samples

The Cd concentration in soils and plant tissues was calculated as follows:

= ppm curve x ml extract x 1000-1

x 1000g x mg sample-1

x fc

ml extract = dillitued volume (after the last digestion the remained volume

s dilluted with distilled water).

Fc = Correction factor of water content (=100/ (100 - % water content)

8. Lab experiment of immobilization of Cadmium in soils.

The immobilization of cadmium was performed with three binding

agents, namely fermented organic fertilizer (animal manure), dolomite,

and activated carbon (Charcoal). Three different soil types such as,

Inceptisols (Kemuning), reddish brown Alfisols (Bakalan) and

Ultisols(Tamansari) were used. The Cadmium concentrationsin soils were

previously determined to know the current soil condition.

a. Incubation process

To study the immobilization of cadmium, a solution made up

by deionized water (8ml), soil (5 g) and dolomite (0.2 g) was prepared

in a 50 ml test tube. The solution was shaken on a gyratory shaker at

100 rpm at room temperature for 16 hours. In order to validate the

61

results, each treatment was carried out in triplicate (modified from

Low and Lee, 1994).

A laboratory experiment on the effect of fermented organic

fertilizer and activated carbon (charcoal) amendment on Cd

immobilization was conducted. Each mixture was made up of 1g

organic fertilizer, 5 g of soil and a mixture solution of, HNO3(1ml) and

KCLO4 (3 ml). The mixture was initially digested at 80oC for 2 hours

and further at 110oC till the sample reaches a volume of 4 ml in the test

tube of 50 ml. For Cadmium extraction was used a solution of 0.02

mol L-1

CaCl2. The use of charcoal followed the same process used for

fermented organic fertilizer. Both treatments with organic fertilizer,

activated carbon and dolomite, after having been shaken for 16 hours

were incubated for 24 days at room temperature (modified from

Gomes et al., 2011) to allow the reactions to occur.

b. Extraction of Cadmium

The extraction of Cadmium was done with 0.5 N CaCl2 by

shaking the soil mixture with 20 ml CaCl2 solution for 1 hour. The soil

mixture was then centrifuged and the supernatant was analyzed for its

Cadmium concentration using an AAS (Low and Lee, 1994). The soil

pH analysis upon thetreatments, the incubation process also took 24

days with distilled water and any acid was added. Finally the pH

values were measure to assess the effect of such binding agents on soil

pH.

The amount of cadmium immobilized by the binding agents

was calculated using the following formula.

Where Cl and Cs are Cd concentrations in the supernatant

(mg/l) and soil (mg/kg); while Vl and Vs are the volume of supernatant

(L) and soil mass (Kg), respectively. (Wuana et al., 2010)

62

Cs =(Cl x Vl)/ms

Cl = Concentration of Cadmium in supernatant (mg/l)

Cs concentration of cadmium in soil (mg/kg)

Vl =Volume of supernatant (l)

ms = dry mass of the soil (kg)

c. Mechanism of precipitation of Cd with the dolomite

Zhan (1986) states that under higher soil pH (>8) and high

concentrations of cadmium in soils, the use of inorganic reagents as

Carbonates and phosphates leads to precipitation of cadmium in the

form of CdCO3 and Cd3(PO4)2. However, such precipitation takes

place as follows:

First: Take place the replacement of the counter cation Ca2+

by Cd2+

.

Second: Then is followed by chemisorptions of Cd2+

on CO32-

and

PO43-

Third: The new formed compounds are taken down soil profile.

D. Process of immobilization of Cd by organic materials and activated

carbon

Soil organic matter (humus) and clay (colloids) contain particles which

are negatively charged. These particles attract cations positively charged such

as Ca2+,

Cd2+,

Zn2+,

Mg2+,

etc. Apart from these particles, organic matter also

contains organic ligands with chelating properties. These ligands are

determinant for immobilization of cadmium in soils; however they are

strongly influenced by soil pH. Dolomite reacts with CO2 and H2O in the soil

to produce bicarbonate ion (HCO3-) that can remove Hydrogen (H

+) and

Alumium (Al3+

) ions from the soil solution, thereby increasing the soil pH.

The raise of pH prevent the availability of Cadmium through adsorption

process.

E. Statistical Analysis

Analysis of Variance (Multivariate ANOVA) was performed, using

Minitab Software version 16. Difference between mean values were

63

determined using Tukey’s advanced test at p<0.05 for the data obtained from

laboratory experiment (Immobilization of cadmium). The data analysis with

different sample size was done through 2 tailed t-Tests. The t-Test was

followed by correlation test to understand the relationship between the

variables, and whether is positive or negative as well as, to understand the

strength of the relationship. The variables which showed a statistically

significant correlation were submitted to stepwise regression. Analyses of

Stepwise regression intended to identify the most dominant variable and/or

variables that impact on Cadmium adsorption, desorption and its availability

to plants.