International Journal of Scientific & Engineering Research, Volume 4, Issue 12, December-2013 1641

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The Effect of Cow Manure Application on the Distribution Fractions of Heavy

Metal Lead in Contaminated Soils

Ghodsie Hoseinian Rostami1, Ahmad Gholamalizade Ahangar2, Amir Lakzian3


The accumulation of heavy metals in soil is a drastic environmental risk. It is well known that metals are present in soil in different chemical forms, which influence their reactivity and hence their mobility and bioavailability. A study, to determine the effect
of Cow Manure on Lead availability and their redistribution amang soil fractions, pot experiments were conducted. Chemical properties such as pH, EC, CEC and Lime of
concerned soils are also analyzed. Cow Manure (0, 1, 5%) was added to soil contaminated with different concentrations of Pb (0, 100, 200mg kg-1 soil, added as pb(NO3 ) 2 ) at 60% field moisture. The amount of Pb was determined from the soil after
4 amounths of incubation time using sequential extraction procedure. Tessier method
was exerted to decompose the metal content into exchangeable, acid extractable, reducible ,oxidizable fractions and Residual fraction was determined in aqua regia digest. Fractionation studies on soils amended by different amounts (1, 5%) of Cow Manure with single metal nitrate, showed that, after foure months, Pb were bound mostly to the acid-extractable, reducible and oxidisable fractions in amended soil (p<0.05). Also the application of Cow Manure (CM) levels significantly decline the exchangable and residual fractions (p<0.05). Whereas in control soil, pb was mainly occurred in the acid-extractable and residual fractions.
Keywords: Fractionation, Metal Bioavailability, Pollution
Sequential Extraction.

1. MSc Student, Department of Soil Sciences, Faculty of Soil and Water Engineering, University of Zabol

*Corresponding author email:

2. Assistant Professor, Department of Soil Sciences, Faculty of Soil and Water Engineering, University of


3. Professor, Department of Soil Sciences, Agriculture Faculty, Ferdowsi University of Mashhad, Iran

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Food security in the developing world population with limited land
resources, so that it has the least effect on their environmental impact, is one of
the important issues. Increased industrial activity associated with the
production of contaminants including heavy metals is a fundamental problem
in today's growing human (22).
Heavy metal polluted soils can be an
environmental tension and a potential financial liability to landowners. However, the evaluation of heavy metal polluted soil has received much attention in the last few decades. According to the United Stated Environmental Protection Agency (USEPA), Pb is the most common
soil labile fraction is taken into account because this fraction is often called, by extension, the bioavailable fraction (13).
By assessment of total heavy metal content one could not obtain suitable information about the hazards of bioavailability, the capacity for remobilisation and the behavior of the metals in the environment. Metal speciation, is taken from the fractionation of the total metal content into exchangeable (bound to exchangeable sites of clay minerals), acid extractable (bound to carbonates and hydroxides), reducible (bound to Fe/Mn oxides), oxidisable (bound to organic matter/ sulfides) and residual (bound to clay minerals) forms. The chemical forms of the metal control its


heavy metal contaminant in the
environment (16) because of the
potential harmful effects it may have on food quality and health of soil (25). It is a non-essential element in metabolic processes and maybe toxic even when absorbed in small amounts. However, Pb is strongly adsorbed in soil particles (33), organic matter, carbonate and phosphate minerals (19,44), and hydrous Fe and Mn oxides (5). Proper evaluation of the impact of heavy metals on the natural environment is possible on the basis of knowledge about their forms and bindings with soil components found. Sequential extraction is a technique of the above information, enabling survey and quantitative assessment of various forms of the same chemical element (14).
Metal in soils can be divided into two
fractions (28): (i) inert fraction, assumed as the non-toxic fraction, and (ii) the labile fraction, assumed to be potentially toxic. To assess the availability of heavy metals, only the
bioavailability or mobility (37).
The determination of specific
chemical species or binding forms is difficult and often virtually impossible. For this reason, sequential extraction procedures are commonly applied because they provide information about the fractionation of metals in the different lattices of the solid sample, which is a good compromise method that gives information on the environmental contamination risk (27,30). Method developed by Tessier et al., (35) is the one widely used for this purpose.
The bioavailable fraction can differ
from one metal to another and from one receptor to another. The availability of metals for plants and micro-organisms in soil depends on the composition of the different compartments of soil such as carbonates, (oxy) metal hydroxides, organic matter and silica (42).
Soil organic matter is considered as
one of the pillars of soil fertility. Organic soil amendments can ameliorate metal toxicity to plants by

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redistributing metals to less available fractions. The distribution of metals is significantly influenced by soil pH and organic matter content. The up-take of many heavy metals can be increased by the reduction of pH within soil. Heavy metal bioavailability increases with the decrease of pH. Decreasing pH will in turn increase the solubility of metals.


The analyzed soil sample was taken from the 0 to 30 cm layer of agricultural
soil in sistan va baluchestan, southeast
Iran. The soil samples were collected
destructively and air-dried, ground and sieved to 2 mm for the analysis of available pb and different fractions of Pb. The experiment totally contained 9 treatments. The 9 of the treatments spiked with a series of pb concentration
Heavy metals bind to the surface of soil particles, results in a reduction of availability (22). The aim of this study is the assessment of the potential effects of organic amendment cow manure on soil chemical properties, mobility index and distribution of Lead fractionation, which will be estimated by a series of sequential extractions. bottles for metal content determination. The conventional method developed by Tessier et al., (35) was followed for the sequential extraction (table 2). The residual and total metal contents were determined in aquaregia digest (11,31). The total metal content in fresh samples were extracted with aqua regia.
The extraction procedure was as follows:
(1) Exchangeable: About 1.0 g sample was extracted at room


(0, 100, 200 mg kg-1 soil, added as
pb(NO3 ) 2 ) and incubated in a greenhouse at 250C for two weeks. During the incubation, soil moisture
was maintained about 60% of water holding capacity. After incubation, the soil was air dried, crushed, passed through a 2mm sieve, and CM were added to the soil (3 replicates per treatment) at 1 and 5% w/w and the third part was kept as the control. The
samples were incubated at 250C for 4
month at 60% of water holding capacity. Some of the relevant chemical and physical properties of soil and cow manure were given in Table 1.
The samples was transported with an mass of 1.0 g oven dried sample. The selective extraction was carried out in polypropylene centrifuge tubes of 50 mL capacity. After each successive extraction, the supernatant liquid was removed after centrifugation at 4000 rpm for 15 min and diluted to volume. The residue was washed with 8ml of distilled water and again centrifugation. The extracts were stored in polythene
temperature with 8 mL of 1 M
MgCl2 (pH 7) and continually shaken for a period of 1 hour (at
(2) Fraction bound to carbonates: Residue from above step (1) was leached at room temperature whit 8 mL of 1M of NaOAC (pH=5 adjusted whit HOAc) and continually shaken for a period of 5 hours (at 22°C).
(3) Fraction bound to Fe and Mn
oxides and hydroxides: The residue left after fraction 2 was extracted with 20 mL of 0.04M NH2 OH.HCl in 25% (v/v) HOAc agitated for 6 hours at
(4) Fraction bound to sulfides and to organic matter: 3 ml of 0.02M HNO3 and 5 ml of 30% H2 O2 were added to the residue left after fraction 3 (pH= 2, adjusted with HNO3 ); heated at 85 ºC for
2 h with occasional agitation. A
second 3 ml aliquot of 30%
H2 O2 was added and heated at

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85 ºC for 3 h with occasional agitation. After cooling, 5 ml of
3.2M NH4 OAc in 20% (v/v) HNO3 were added into the cooled mixture; diluted to 20 mL; agitated for 30 min and centrifuged.
(5) Residual fraction: Residue from (4) was digested with 8 mL of aqua regia (HCl and HNO3 , 3+1 v/v) for 2 h at 120 ºC.
(6) Total fraction: Fresh sample, 1.0
g, was digested with 8 mL of aqua regia (HCl and HNO3 , 3+1 v/v) for 2 h at 120 ºC.

Table 1. Characteristics of the initial soil, Cow Manure used for the pot experiment (dry weight basis)



Cow Manure

Sand (%)



Silt (%)



Clay (%)









EC (dSm-1)



CEC (cmolc kg-1)



OMI(%) JS0.6E7 R12.5

Total Pb (ppm) 3.98 2.55

Lime (%) 20 -

Table 2. Protocol for sequential extraction procedure

Step no. Fractions Reagents pH Temperature (0C) Time shaking

1 Exchangable 1 M MgCl2 7 25 1h

2 Acid Extractable 1 M Na OAc 5 25 5h

3 Reducible 0.1 M NH2OH_HCl 2 96 6h

4 Oxidisable 30% H 2 O 2 2 85 3h

5 Residual HNO 3 –HCl - 120 2h

F1 : exchangeable

F2 : acid extractible fraction: metals bound to carbonates

F3 : reducible fraction: metals bound to hydrated oxides of iron and manganese

F4 : oxidise fraction: metals bound to organic matter

F5 : residual fraction

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2.1. Statistical analysis

Data were analyzed by one-way ANOVA

procedure using SPSS software and standard deviation (SD) was used to indicate variability. Significances were based on probability (P ≤ 0.05) level for Least Significant Difference (LSD).


3.1. Soil pH and EC

Figure 1A shows the effect of CM

amendment on the soil pH after 4 months incubation. The application of CMR1R and CMR2R levels, relative to the control, had significant effect on the soil pH. The increased values of pH at CMR1

Rand CMR2R levels, was 6.35% and

11.35%, respectively (p<0.05) (figure
1A). An increase of soil pH following
organic matter addition could be largely due to addition of basic cations and
decomposition, although adsorption of H+ ions, development of reducing conditions due to increased microbial activity and displacement of hydroxyls
from sesquioxide surfaces by organic anions could contribute also (39). Clemente et al., (10) deduce a proximate respect between soil pH and sulphate content, the sulphate formation being rebounded in an increase in EC. Fig 1B shows that, for the manure- amended soil, there was an increase in soil EC with time. It may have been caused partly by the high EC of the manure, and partly by the mineralisation of its organic matter and the resultant formation of soluble salts. Abbaspour et al., (1) represented that reason decrease in the soil pH may dissolve some precipitated minerals, as a result,
increase the soil EC.
production of NH3 during


B 1



0.4 c


A 8.4

8 b


b 7.6 c




Figure 1. Effect of Cow Manure on soil pH (a) and EC (b) after 4 months of incubation time (Mean values followed by different letters are

significantly different (p < 0.05) by LSD’s test). Control (CM0), Cow Manure 1% level (CM1) and Cow Manure 5% level (CM2).

3.2. Distribution of chemical fractions of pb in the cow manure treatment

The sequential extraction used in this
study is proper to indirectly assess the potential mobility and bioavailability of heavy metals in the soils. The five chemical fractions are operationally recounted by an extraction sequence that follows the order of decreasing solubility.

The exchangeable fraction (FR1R) contains metal elements in the ionic form, which have a high mobility and can be drained by water. The fraction related to the carbonates (FR2R) is easily extractable at pH

= 5 and can be accumulated in the plants
(bioavailability) (35). The fraction related

to the oxides of iron and manganese (FR3R)

and that related to organic matter (FR4R)

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contain the metals enclosed in the matrix. The action of these metals in the vegetable-growing environment requires relatively long time, which makes the toxicity of these metals conditional. The fraction (F5 ) contains the inert metals.
Counting that bioavailability is related
to solubility, metal bioavailability
decreases in the following order: exchangeable> carbonate> Fe-Mn oxide> organic> residual. This order is just a generalization and presents only qualitative information about metal bioavailability. Based on the above notification, we can further assure that metals in the nonresidual fractions are more bioavailable than metals associated with the residual fraction. The nonresidual fraction is the aggregate of all fractions except the residual fraction. In the ecological background, the mobile fractions rather than the total metal amount
application of CM at 1% level, led to a significant decrease of F1 and F5 fractions, relative to the control, and the proportions of these two fractions were 9% and 20%, respectively (p<0.05) and at 5% level, the proportions of these two fractions were
9.8% and 27%, respectively (p<0.05) (fig
2b). However, no significant difference
was observed between the two CMs at 1% and 5% levels. McLaren and Crawford, (21) and Williams et al., (40,41) suggested that low mobility (F1 ) was due to specific adsorption of the metals. Also, Morsy Khaled, (23) stated that by addition of the sludge to the soil, the water extractable fraction of heavy metals (Zn, Cu, Pb, and Ni) was drastically low compared to the total content. They suggested that this could be related to the alkalinity of the soil. The effects of organic amendments on heavy metal availability also pertain drastically upon the degree of humification


are important. Moreover the chemical
species play an important function in the
transfer of metals along the plant↔soil↔animal↔water↔human
chain (31). To comment the phenomena of mobility and bioavailability of heavy
metals with sequential extractions, we now the distribute of Pb in different fractions in
Table 2.
The comparison of the various fractions
concentration is demonestrated in fig 2. The application of amendments to soils that can immobilize heavy metals in situ may prepare a expense effective and tolerable solution for the remediation of contaminants in soils. In this study, the ability of CM in reducing Pb availability in a heavily contaminated soil and possibility of regeneration was studied. The
of their organic matter and their effect
upon soil pH (4,24,32,38). This may have
been a consequence of the immobilization of Pb by humified OM, in the Cow manure-treated soil. Moreover, cow manure increase Lead acid Extractable, Reducible and Oxidisable fractions (p<0.05). This significantly increases pb concentration relative to the control, at 1% and 5% levels, for F2 section was 17.6% and 25.5% respectively, and for F3 , was
33% and 35%, respectively and in F4
section was respectively 21.5% and 35.7%
(fig 2B). It is presumed that Pb availability
in soils reduces via formation of OM-Pb complexes. Abbaspour et al., (2) also deduce that alfalfa powder used for three soils significantly reduced EDTA- extractable Pb.

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Figure 2. Effect cow manure (A) and Lead (B) on the Distribution Fractions. (Mean values

followed by different letters are significantly different (p ≤ 0.05) by LSD’s test)

The camputation of the mobility index
(MI) for Lead on the basis of the following equation:

MI= ×100

The movement of Pb in the various chemical fractions was dependent on the total metal content of the soil samples. The relative immobility of the metals added to the soil is a reflection of the chemical
reactions that occur in the soil.

IJSEIn controRl soil, Pb was mainly occurred

The amount of metal accounted this way is perceived to be available for plants. The mobility index of Lead determined in the treat of CM at 0%, 1% and 5% level within the limits of 50.95%, 50.44% and 49.66%, respectivity.
High concentration of heavy metals in
reducible fraction (bound to Fe and Mn oxides) can be attached to prevalence mechanism (37). These metals can be abandoned into the environment under extremely reducible conditions. Bolan et al., (8) deduce that the addition of biosolid compost was potential in decreasing the concentration of F1 -Cd fraction and increasing the concentration of F4 -Cd fraction that may be because of different attributes of compost and soil used. The writers derived that composting of pig manure for 63 days was more effective in reducing the bioavailability of Cu than Zn (15).

3.3. Distribution chemical fractions of pb in the Lead added to the soil treatment

in the F5 and F2 fractions. whereas Pb was
mainly occurred in the F2 and F3 , after 100 and 200 mg kg-1 pb addition in bulk soil (P
< 0.05) (fig 2A). In the control, the contents of F2 were 2.08 mg kg-1 in bulk soil and drastically increased to 7.78 mg kg-1 after 100 mg kg-1 pb addition and
12.13 mg kg-1, after 200 mg kg-1 Pb addition (fig 2A). Compared with the
control, the application of Pb concentration at 100 and 200 mg kg-1 levels significantly increased the content of F4 (p<0.05); but that at F5 fraction, decreased Pb concentration significantly relative to the control (p<0.05) (fig 2A). The movement
of metals in the various chemical fractions was dependent on the total metal content of the soil samples (23).

3.4. Distribution chemical fractions of Pb in the interactions between cow manure and Lead concentration treatment

The interactions between cow manure and Lead concentration, based on the
tessier method, the different chemical fractions of Pb mainly contained acid-

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extractable, oxidable and organic matter- bond fractions. The application of CM at
1% level led to a concentration of Pb decreased significantly in the F1 fraction, these diminution is significant since this is the fraction that have cretain influence on metal bioavailability and significant increase of F2 fraction when pb addition
was higher than 0 mg kg-1 (p<0.05).
Whereas at 5% level had more effect on
the contents of F1 and F2 (table 3). The presence of Pb in the acid-extractable fraction might introduce a pH proper for metal precipitation. Moreover, calcium carbonate may treat as a strong absorbent for heavy metals and could complex as double salts similar CaCO3 .MCO3 (29). Walker et al., (39) showed that addition of OM, particularly from cow manure, decreased the soluble/exchangeable levels of Cu, Zn, Mn and Pb, as found already for cow manure/Zn in differing soil types
amount of Pb associated with F3 may include metals present in other poorly crystalline compounds (12). Brown et al., (9) also reported that an increase in Pb portioning to Fe_ Mn oxides, along with increased organic complexation was observed exerting different biosolid amendments.
The F4 became predominant after Pb addition. The contents of F4 increased with the increased Pb addition, but the maximum proportion was found at 200 mg kg-1 Pb addition. The application of CM at
5% level resulted in the significant accumulation of F4 in the range of 100-
200 mg kg-1 Pb addition, but that at 1%
level exhibited no significant impact. The preference of Pb for organic matter is supported by the high stability of Pb complexes with organic components (26,34). Lead form relatively strong organic-metal complexes, which can be the

IJSEreason for sRlightly movement of the pb in

Metal species correlated with organic
and oxidable fractions are also not easily bioavailable. They are drastically held and bind. Their abandonment into the soil solution pertains on drastic depletion of minerals amount of the soil solution, decomposition and oxidation of organic matter (36). The concentration of F3 were the low in control soil. However, the contents of F3 increased with elevated Pb addition and CM, among the four fractions. When Pb (NO3 ) was added in
the range of 100–200 mg kg-1, the
proportion increased linearly. The high
the soil. The agents of –COOH and –OH
proffered by OM increased the binding sites and incorporated with pb to form insoluble and immobile complexes, therefore the concentration of free Pb reduced and the potential environmental hazard was diametrically decreased (7)
Results also indicated that Applying different rates of CM, along with increased Pb concentration, decreased Pb concentration significantly relative to the control (C) in the F5 fraction. Attribution F5 pattern increased with CM reduce.

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Table3. Effect of interactions between cow manure and Lead concentration on Pb distribution among different fractions after 4 months of incubation time


A similar distribution pattern was observed for

Pb by Abbaspour and Golchin, (3). They showed that addition vermicompost increased Pb concentration significantly in the F 2, F 3 and F 4 fractions and reduced it in the F1 and F5 fractions relative to the control. usually, different chemical and physical attributes of composts include electrical conductivity, pH, ionic strength, cationexchangeable capacity, elements content and sorptive capacity of the inorganic and organic structures, affect the mobility and availability of heavy metals in soils (5,8). Also related that due to a high percent of humified organic matter, vermicompost can reduce the availability of heavy metals in soil with adsorption and with forming strong complexes with humic matters.

Zheng et al., (43) related changes that occur

to Pb speciation, distribution and bioavailability during the point of composting. The concentration five fractions were increased during the while process of compost. However, the percentage distribution with consideration to total Pb were changed in the following; exchangeable, bound to Fe-Mn oxides and bound to carbonates Pb with consideration to total increased, while the percentages of bound to organic matter and residual Pb with respect to total Pb were declined during composting. The information indicates that the content of Pb in the less toxic

portion, such as consisting of organic matter

and sulphide bound and residual Pb, was increased, and that the contamination and bioavailability of heavy metal Pb in sewage sludge was declined during composting. Haug et al., (15) represented changes in the speciation and DTPA extractable Cu and Zn during humification of organic matter in pig manure composts. Composting of pig manure concluded in a significant decrease in content of water soluble, exchangeable, acid extractable and Fe-Mn oxides- bond fractions of Cu and Zn and increase in organic matter- bond and residual fraction of Cu and Zn.

The metals bioavailability of soil pertains to a large content on their distribution between solution and solid phase, which in turn is dependent on soil processes example OM, CEC and pH. The effect of organic matter amendments on heavy metal solubility depend drastically on the proportion of humification of their organic matter and their effect on soil pH (38). Commonlly, the concentration of an element in the soil solution is depend on the parity between the solid phase and soil solution, with pH playing the definite role (18). The study delineated that in cases of accumulation of heavy metals from continuous application of inorganic fertilizer to soils agronomy, Cow Manure can be used to immobilize the heavy metals in the

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contaminated soil. And extremes under slowly conditions.


The mobility of Pb chemical fractions in

soil was dependent on both Pb addition and CM application levels. The FR5 Rfraction accounted for the largest proportion of Pb to control. When pb addition was higher than 0

FR2 Rfractions increased most rapidly. The FR 2R fractions were most important in controlling the Pb mobility and availability in soil. Also it increased Pb concentration significantly in the FR3R and FR4R fractions. But, the application of CM at 1% and 5% levels, significantly declined the F1 and F2 fractions. As a result, the CM was able to, in the short-term,

mg kg-1, the F , F , F

and F


reducing heavy metal mobility, and could help

R 1R

R 2R



significantly increased but FR5R reduced, relative to the control. The CM application at 1% and

5% levels, suggests that CM can decrease the transformation of Pb from exchangeable to

less mobile fractions, in the soil after 4 months of incubation time. By the addiation CM, the

to facilitate the primary revegetation of soils

contaminated by Lead. It is necessary to show the availability of soil heavy metals following organic matter application, as changes occur with time, due to microbiologically medal transformations of organic structures.


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