chapter iii material and methods a. location and research...
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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.
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Figure 9. Research Location
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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
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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.
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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.
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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.
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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.
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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,
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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).
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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,
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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
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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.
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Figure 18. Heating the Solution of
HCl
Figure 19. Heating the Solution of
H2O2
Figure 20. Cooling of Solutions and H2O2
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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
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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
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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).
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Figure 25. Analysis of Cadmium using AAS
Figure 26. Extraction of Cadmium from soil and Rhizomes
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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
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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)
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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
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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.