Environmental Technology & Innovation 17 (2020) 100636

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Environmental Technology & Innovation

journal homepage: www.elsevier.com/locate/eti

Sulfur, Na2 -EDTA and their mixture effects on phosphorus

release from cow bone char in P-poor sandy soil
Abu El-Eyuoon Abu Zied Amin
Assiut University, Soils and Water Department, Faculty of Agriculture, Assiut, P.O. Box: 71526, Egypt

article info a b s t r a c t

Article history: The high cost and environmental problems related to the production of phosphate fer-
Received 15 September 2019 tilizers as well as their excessive and continuous applications represent great challenges
Received in revised form 15 January 2020 in modern agriculture. Therefore, we conducted an incubation experiment using sandy
Accepted 15 January 2020
soil to evaluate the effects of elemental sulfur, disodium ethylene diamine tetra-acetic
Available online 18 January 2020
acid (Na2 -EDTA) and their mixture with cow bone char as well as incubation periods on
Keywords: changes of phosphorus (P) availability, water soluble P, soil pH, electrical conductivity
Incubation periods (EC), soluble calcium (Ca) and soluble sulfate (SO4 ). Applying sulfur (S), Na2 EDTA and
Phosphorus availability their mixture with cow bone char improved significantly phosphorus availability in soil,
Pyrolysis Olsen-P value increased from 7.65 mg kg−1 (control) to 12.20 mg kg−1 (cow bone char +
Renewable sulfur), 12.80 mg kg−1 (cow bone char + Na2 -EDTA) and 13.07 mg kg−1 (cow bone char +
Soluble sulfate sulfur + Na2 -EDTA) at the end of incubation periods While, cow bone char + sulfur + Na2 -
EDTA treatment lead Olsen-P to decline significantly with increasing incubation periods.
Amending soil by sulfur with cow bone char treatment caused significant increase in
the Olsen-P with increasing incubation periods. Cow bone char + sulfur and cow bone
char + Na2 -EDTA showed non-significant differences in Olsen-P. Because of the increase
in phosphate fertilizer prices, the results of this study demonstrated the great potential
of cow bone char usage which is a promising way to improve phosphorus availability
in P-poor soil as well as being a clean alternative and renewable source of phosphate
fertilizer. Consequently, we recommend utilizing cow bone char with sulfur as slow
release fertilizer with time due to their low cost and it is a safe source of phosphorus
in sustainable agriculture, rather than using Na2 -EDTA due to its high price.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction

More than 80% of the phosphate fertilizers added to the soils turn into virtually insoluble forms, so it usually becomes
unavailable to the plants (Schnug and S.H., 2016). Additionally, the continuous and overuse of phosphate fertilizers in
modern agriculture produced changes in phosphorus cycle and soil biodiversity as well as ecological balance; they also
result in water eutrophication and affects food health (Vassilev et al., 2013). Moreover, these fertilizers increase heavy
metals and radionuclides existence in the soil and transfer them to food chain (Attallah et al., 2019). Consequently, bone
char amendment is a promising alternative source of phosphate fertilizers which has many benefits; high content of P,
renewable source, cheap as well as avoiding soil contamination with heavy metals and radionuclides, all these benefits
help it to play role in sustainable agriculture (Siebers and Leinweber, 2013; Vassilev et al., 2013). Bone mostly consists
of Ca-phosphates which are known as biological apatite. The biological apatite differs from geological apatite in some

E-mail address: abueleyuoon.amin@aun.edu.eg.

https://doi.org/10.1016/j.eti.2020.100636
2352-1864/© 2020 Elsevier B.V. All rights reserved.
2 A.A. Amin / Environmental Technology & Innovation 17 (2020) 100636

Table 1
Some chemical properties of the cow bone char.
Property Unit Value ± SD
pH(1:2.5) 8.25 ± 0.01
EC(1:5) (dS m−1 ) 3.12 ± 0.007
Water soluble Ca(1:5) mg kg−1 101.67 ± 3.51
Water soluble P(1:5) mg kg−1 4.72 ± 0.01
Olsen-P mg kg−1 1552.42 ± 33.8
Total P g kg−1 140.78 ± 0.05
Total Ca g kg−1 288.45 ± 2.66

EC: electrical conductivity.

properties: small crystal size, high carbonates substitutions, and significant high OH− deficiency; these properties enhance
biological apatite solubility (Wopenka and Pasteris, 2005; Boskey, 2007). Bone char is produced by pyrolysis of de-fatted
and de-gelatinized animal bones which was collected from slaughterhouses (Glæsner et al., 2019). The features of P
derived from bone char is controlled by pyrolysis temperature and biomass additions which re-shape Ca-P crystals this
in turn leads to enhance phosphorus availability for plants (Zwetsloot et al., 2015). Amending some soils by bone char
caused an increment of phosphorus availability in comparison with rock phosphate, moreover, the rate and amount of
dissolved phosphorus from bone char are influenced by soil properties as in phosphate rock (Warren et al., 2009). Available
phosphorus provided by bone char is two to five folds more than rock phosphate, while it is 24% lower than triple
superphosphate (Zwetsloot et al., 2015). Sulfur-enriched bone char increased releasing phosphorus from soils at different
pH range and applying bone char at low levels decreased uptake of some heavy metals by plants (Morshedizad et al.,
2016). The increase in nutrients availability released from bone char enriched with sulfur particularly phosphorus was
because of sulfur oxidation by microorganisms decreased soil pH which led to the dissolution of phosphate compounds
(Morshedizad et al., 2018; Zimmer et al., 2018). The addition of bone char to the soil improved the yield of wheat and
potato tubers more than the triple superphosphate and diammonium phosphate (Siebers et al., 2014). Many mechanisms
occurs in soil when adding sulfur as (1) lowering soil pH may led to dissolving phosphorus from phosphate compounds
such as Ca-phosphates (2) replacing phosphate ions (PO4 ) by sulfate ions (SO4 ) produced from sulfur oxidation which
caused releasing phosphorus from exchange sites all these mechanisms affect the availability of the phosphorus (Jaggi
et al., 2005). The ability of organic manure to chelate some cations such as aluminum, calcium, and iron in high P-
fixing capacity soils increases phosphorus availability (Toor, 2009). In the research, I interested to follow two-way. Firstly,
adding chelating agent which is disodium ethylene diamine tetra-acetic acid (Na2 -EDTA) to chelate Ca, secondly, adding
sulfur to increase the dissolution of phosphate compounds in order to improve the efficiency of phosphorus released
from cow bone char because it decreases soil pH. As a result, the aims of the present experiment examine the effects of
applying elemental sulfur, (Na2 -EDTA) and their mixture with cow bone char as well as incubation periods on changes
of phosphorus availability, water soluble phosphorus, soil pH, electrical conductivity, soluble Ca and soluble sulfate in
P-poor sandy soil under incubation condition.

2. Materials and methods

2.1. Cow bone char production

Cow bone char was made by collecting cow bone, de-fatting and de-gelatinizing the bones through boiling them several
times, scratching them with a knife, and The bones were broken into pieces, where their length ranged between 15 and
25 cm. Then the pyrolysis occurred in the absence or reduced supply of oxygen at temperature about 650 ◦ C for 2 h, this
process called charring or pyrolysis. After the pyrolysis process, they turn into bone char and the product was crushed
as powder by a stainless steel mill and then passed through a 1 mm sieve. The chemical properties of cow bone char are
presented in Table 1.

2.2. Incubation experiment

The soil used in this experiment was collected from the surface soil layer (0–20 cm) at Bani Ghalib, Assiut, Egypt.
According to FAO-WRB-classification, the soil was classified as Haplic Arenosols and some soil properties are displayed
in Table 2. Soil Chemistry Laboratory, Soils and Water Department, Faculty of Agriculture, Assiut University, Assiut, Egypt
was the location where the experiment took place. Then, 100 g air-dried soil was placed in each airtight plastic jar (330
ml), and farmyard manure was added at level of 0.4 g to each jar as carbon and nitrogen resource. This experiment consists
of four treatments: control is soil without additions (CK), soil + 0.35 g of cow bone char + 0.06 g sulfur (BCS) jar−1 , soil
+ 0.35 g of cow bone char + 0.2 g Na2 -EDTA (BCE) jar−1 , and soil + 0.35 g of cow bone char + 0.06 g sulfur + 0.2 g
Na2 -EDTA (BCSE) jar−1 . The experiment was organized in a completely randomized design and replicated three times.
The relative rate of 0.35% of cow bone char corresponds to about 8.4 Mg ha−1 , 0.06 g sulfur pot−1 (equivalent to 1440 kg
ha−1 ) 0.2 g Na2 -EDTA, which corresponds to 5.4 mM kg−1 . The moisture of the soil in the jars was maintained at field
 A.A. Amin / Environmental Technology & Innovation 17 (2020) 100636 3

Table 2
Some physical and chemical properties of the soil used in this experiment.
Property Unit Value ± SD
Sand (g kg−1 ) 962
Silt (g kg−1 ) 8
Clay (g kg−1 ) 30
Texture Sand
OM (g kg−1 ) 0.79 ± 0.12
CaCO3 (g kg−1 ) 70.46 ± 2.18
pH 8.73 ± 0.02
EC (dS m−1 ) 2.54 ± 0.04
Soluble Na (mmol kg−1 ) 28.36 ± 0.11
Soluble K (mmol kg−1 ) 3.47 ± 0.08
Soluble Ca (mmol kg−1 ) 17.69 ± 0.17
Soluble SO4 = (mmol kg−1 ) 18.71 ± 2.21
Olsen-P (mg kg−1 ) 5.94 ± 0.37

OC: organic carbon; EC: electrical conductivity.

capacity through adding distilled water to all treatments, and incubated in the dark at the laboratory room temperature
ranged between 24–33 ◦ C for 22, 56 and 78 days, which is the average temperature in Assiut City during the spring season
2019. All jars were well covered, but opened every now and then to preserve the aerobic condition of the soils and water
was added for moisture loss supplement. Eventually, soil samples were taken, air-dried, and crushed for chemical analysis
after each incubation period.

2.3. Chemical analysis

Available phosphorus (Olsen-P) in soil and cow bone char was extracted by 0.5 M NaHCO3 (pH 8.5) at ratio of 1:20 soil
or cow bone char (g)/solution (ml) according to Olsen et al. (1954). The mixture is then filtered by filter paper. The soluble
phosphorus in soil samples under study was extracted by adding distilled water at ratio 1:20 soil (g)/distilled water (ml)
after 30 min end-over-end shaking; the mixtures are then filtered using filter paper. The phosphorus in the all filtered
extracts is determined colorimetrically using the chlorostanous phosphomolybdic acid method (Jackson, 1973). The total
phosphorus and calcium in cow bone char were digested with concentrated H2 SO4 , HNO3 and HClO4 (Grimshaw, 1989),
phosphorus in extracts was measured colorimetrically by chlorostannous phosphomolybdic acid method in sulfuric acid
system (Jackson, 1973). The pH of soil samples and bone char was measured in 1:2.5 of a soil or bone char distilled
water suspension by a glass electrode (Jackson, 1973). Soil extracts were prepared at ratio 1:2.5 soil (g)/distilled water
(ml) after 30 min end-over-end shaking and 2 h standing. While, cow bone char was extracted at ratio 1:5 cow bone
char (g)/distilled water (ml) after 30 min end-over-end shaking and 1 h standing. The mixtures are then filtered using
filter paper. Electrical conductivity (EC) was measured in the extracts using electrical conductivity meter. The soluble Ca
in all extracts was estimated by titration using Na2 EDTA solution. Soluble Na in the extracts was determined using the
flame photometry method. Soluble sulfate was determined in extracts by the turbidimetry method using barium chloride
(Baruah and Barthakur, 1997).

2.4. Statistical analysis

Analysis of variance (ANOVA) used to analyze the data and Duncan test was used for multiple comparisons to perform
significant differences between treatments (p ≤ 0.01). All statistical analyses were carried out using the MSTAT program.

3. Results and discussion

3.1. Available phosphorus

The results indicated that the application of sulfur, Na2 EDTA and their mixture with cow bone char enhanced
significantly (P ≤ 0.01) phosphorus availability (Olsen-P) in P-poor sandy soil compared to the control treatment
(unfertilized soil). Soil available phosphorus content is incremented from 7.99 mg kg−1 (control) to 10.59 mg kg−1 (cow
bone char + sulfur), 13.10 mg kg−1 (cow bone char + Na2 -EDTA) and 13.98 mg kg−1 (cow bone char + sulfur + Na2 -EDTA)
after 22 days of incubation (Fig. 1). The largest value of Olsen-P was observed just after 22 days of adding the mixture of
cow bone char, sulfur and Na2 -EDTA, with maximum Olsen-P amounting to 13.98 mg kg−1 (Fig. 1). For sulfur with cow
bone char treatment, we detected a significant increase in Olsen-P with increasing incubation periods, where it increased
from 10.59 mg kg−1 (22 days) to 12.48 mg kg−1 (56 days). However, cow bone char + sulfur + Na2 -EDTA treatment
caused a significant decline of Olsen-P with increasing incubation periods. The values of Olsen-P at first increased and
then decreased with increasing incubation periods. We also observed that Olsen-P decreased from 13.98 (at day 22) to
4 A.A. Amin / Environmental Technology & Innovation 17 (2020) 100636

Fig. 1. Available phosphorus and soluble phosphorus for different treatments during the incubation periods. Each value represents the mean of three
replicates with the standard error shown by the vertical bars.

12.77 mg kg−1 (at day 56) for cow bone char + sulfur + Na2 -EDTA treatment (Fig. 1). In the current study, the results
indicated that there was a non-significant difference in Olsen-P between the cow bone char + sulfur, cow bone char +
Na2 -EDTA and cow bone char + sulfur + Na2 -EDTA treatments after 56 days of incubation (Table 3). While, at the end
of incubation period, the differences in Olsen-P among the treatments of cow bone char + sulfur and cow bone char +
Na2 -EDTA was non-significant (Table 3). According to Olsen and Sommers (1982) the soils which contain the Olsen-P < 5
mg kg−1 respond, 5–10 mg kg−1 probably respond and >10 mg kg−1 unlikely respond to added P fertilizers. Cow bone char
treatments obtained effective tendency as follows: cow bone char + sulfur + Na2 -EDTA > cow bone char + Na2 -EDTA >
cow bone char + sulfur > control during all incubation periods. Gradual increases in the amounts of available phosphorus
with incubation periods were observed in this study that could possibly be attributed to the release of phosphorus from
these amendments rich in phosphorus. There are three main factors which play important role controlling the equilibria of
P concentrations in soil solution which are: firstly, soil pH value, secondly, the concentration of Ca which co-precipitates
with P ions, and thirdly, the concentration of sulfate and bicarbonate as well as their competition with P on exchange sites
(Hinsinger, 2001). Efficiency of calcium phosphate precipitation is highly affected by Ca/P ratio at constant pH (Mekmene
et al., 2009). The combined application of cow bone char and elemental sulfur may be beneficial to increase P availability
in P-poor sandy soil. The application of elemental sulfur increased releasing phosphorus from cow bone char because of
the increase in the calcium phosphates solubility this caused by oxidizing elemental sulfur producing H+ and SO24− ions,
 A.A. Amin / Environmental Technology & Innovation 17 (2020) 100636 5

Table 3
The effects of applying sulfur, Na2 EDTA and their mixture with cow bone char on available phosphorus, soluble phosphorus and soil pH in soil under
study.
Incubation period Treatment Soil properties (value ± standard deviation)
Available phosphorus (mg kg−1 ) Soluble phosphorus (mg kg−1 ) pH
Control 7.99 ± 0.11 e 0.32 ± 0.03 de 8.66 ± 0.03 ab
22 days CBCa + sulfur 10.59 ± 0.51 d 0.25 ± 0.00 e 8.52 ± 0.01 e
CBC + Na2 EDTA 13.10 ± 0.05 b 0.41 ± 0.02 c 8.65 ± 0.04 b
CBC + sulfur + Na2 EDTA 13.98 ± 0.33 a 0.24 ± 0.01 e 8.56 ± 0.01 d
Control 7.05 ± 0.08 f 0.35 ± 0.04 cd 8.67 ± 0.01 ab
56 days CBC + sulfur 12.48 ± 0.17 bc 0.27 ± 0.07 e 8.52 ± 0.01 e
CBC + Na2 EDTA 12.64 ± 0.56 bc 0.42 ± 0.02 c 8.68 ± 0.01 a
CBC + sulfur + Na2 EDTA 12.77 ± 0.02 bc 0.40 ± 0.01 c 8.54 ± 0.02 de
Control 7.65 ± 0.24 ef 0.27 ± 0.04 e 8.62 ± 0.00 c
78 days CBC + sulfur 12.20 ± 0.40 c 0.30 ± 0.02 de 8.52 ± 0.00 e
CBC + Na2 EDTA 12.80 ± 0.34 bc 0.65 ± 0.01 a 8.67 ± 0.01 ab
CBC + sulfur + Na2 EDTA 13.07 ± 0.03 b 0.52 ± 0.05 b 8.53 ± 0.01 e

Different letters within the same column indicates that the mean significantly differs according to Duncan multiple range test at P < 0.01.
a
CBC: cow bone char.

which decline soil pH (Zhi-Hui et al., 2010). The richness of bone char enriched with sulfur in the amounts of phosphorus,
calcium and sulfur enhances direct biological activity as a result of elemental sulfur oxidation, consequently improving
the availability of nutrients released from bone char (Zimmer et al., 2018). The release of phosphorus from fixation sites
was due to desorption from exchange sites by sulfate ions with applying sulfur in calcareous soil (Jaggi et al., 2005).
Enhancement of phosphorus availability in soil with applying cow bone char + EDTA and cow bone char + sulfur + EDTA
may be a reason of calcium chelation by EDTA. Phosphorus availability in noncalcareous and calcareous soils increased
after adding organic matter with phosphate fertilizer this increase is due to high ability of organic matter to chelate Ca
in P fixing soils (Toor, 2009). The highest values of Olsen-P were found at the beginning of the experiment as a result of
the high ability of Na2 -EDTA to chelate calcium and then tended to decline with incubation periods for all treatments of
EDTA. This is might be because of about 14% of the applied EDTA in some soils was degraded after 20 days (Wen et al.,
2009) and approximately 20% of added EDTA to the soil for remediation can be adsorbed on exchange sites (Zeng et al.,
2005). In addition, 1% and up to the 22% of applied EDTA was retained after remediating calcareous soils (Jez and Lestan,
2016).

3.2. Soluble phosphorus

The soluble phosphorus in the soil increased significantly with the application of cow bone char + Na2 -EDTA treatment
compared to the control at all incubation periods (Table 3). While, cow bone char + sulfur + Na2 -EDTA application leads
to a significant increase in soluble phosphorus compared with the control at the end of incubation period (Table 3). The
soluble phosphorus increased from 0.32 mg kg−1 for control to 0.41 mg kg−1 for cow bone char + Na2 -EDTA treatment
after 22 days of incubation. However, at the end of incubation the soluble phosphorus increased from 0.27 mg kg−1 for
control to 0.65 mg kg−1 for cow bone char + Na2 -EDTA treatment. Compared to the unamended treatment, applying
cow bone char + sulfur to the soil significantly decreased the soluble phosphorus after 56 days of incubation (Table 3).
The incubation periods led to significant increase in the soluble phosphorus for most treatments (Table 3 and Fig. 1).
Generally, the results obtained of this study showed that the soluble phosphorus was higher in the treatments which the
calcium concentration is low with adding Na2 -EDTA treatments (Fig. 1). The maximum value of soluble phosphorus was
observed when adding cow bone char + Na2 -EDTA after 78 days of incubation (Fig. 1). The dissolution of phosphorus from
compounds such as bone depends on supersaturation/undersaturation, pH, ionic strength and the molar ratio of calcium
to phosphate (Wang and Nancollas, 2008). Water soluble P in soil increased with the addition of sulfur-enriched bone
char more than bone char alone (Zimmer et al., 2019).

3.3. Soil pH

Relative to the unamended treatment, cow bone char + sulfur and cow bone char + sulfur + Na2 -EDTA treatments
significantly decreased the soil pH at all incubation periods. While, cow bone char + Na2 -EDTA treatment did not change
soil pH significantly after 22 and 56 days from incubations, except for at the end of incubation it increased significantly
(Table 3). Soil pH values showed a slight declined from 8.66 (control) to 8.52 (cow bone char + sulfur) and 8.56 (cow
bone char + Na2 -EDTA) after 22 days of incubation (Fig. 2). At the end of incubation time (i.e., after 78 days) the soil
pH values decreased from 8.62 (control) to 8.52 (cow bone char + sulfur) and 8.53 (cow bone char + Na2 -EDTA). The
values of soil pH with the additions of cow bone char + sulfur and cow bone char + sulfur + Na2 -EDTA treatments
slightly decreased by 0.09–0.15 units compared to the control treatment at all the incubation periods. Compared to the
6 A.A. Amin / Environmental Technology & Innovation 17 (2020) 100636

Fig. 2. Changes of soil pH and electrical conductivity under different treatments during the incubation periods. Each value represents the mean of
three replicates with the standard error shown by the vertical bars.

control treatment, applying cow bone char + Na2 -EDTA treatment slightly increased soil pH from 8.62–8.67 (Table 3).
Many studies found that the additions of elemental sulfur to alkaline soil decreased soil pH (Amin, 2018; Soaud et al.,
2011) which can be attributed to producing H2 SO4 resulted from oxidizing sulfur by microorganisms (Jaggi et al., 2005;
Soaud et al., 2011). Elemental sulfur applications to some alkaline soils increase soil pH by 0.08 to 0.8 units relative to
the unamended treatment (Soaud et al., 2011). The little changes of soil pH in this study may be explained by the high
buffering capacity of this soil which resists changing in soil pH. Generally, the soil pH is one of soil chemical properties
that play an important role in nutrient availability and microbial activity.

3.4. Electrical conductivity, soluble calcium and soluble sulfate

In the present study, electrical conductivity (EC) in sulfur, Na2 -EDTA and their mixture with cow bone char amended
soil was increased significantly (P ≤ 0.01) compared with the control treatment (Table 4). The soil EC value increased
significantly with increasing incubation periods. The highest value was noticed at sulfur + Na2 -EDTA with cow bone char
treatment at 56 days amounting to 3.45 dS m−1 (Fig. 2). Across the treatments, EC values varied from 2.77 dS m−1 (control)
to 3.08, 2.95, 3.06 dS m−1 in the treatments of sulfur, Na2 -EDTA, sulfur + Na2 -EDTA with cow bone char, respectively
after 22 days (Fig. 2). However, after 78 days the EC values increased from 2.80 dS m−1 (control) to 3.21, 3.03 and 3.44 dS
m−1 in the treatments of sulfur, Na2 -EDTA, sulfur + Na2 -EDTA with cow bone char, respectively (Fig. 2). The application
 A.A. Amin / Environmental Technology & Innovation 17 (2020) 100636 7

Table 4
The effects of applying sulfur, Na2 EDTA and their mixture with cow bone char on some soil properties in soil under study.
Incubation period Treatment Soil properties (value ± standard deviation)
EC Soluble Ca Soluble Na Soluble SO4
Control 2.77 ± 0.02 f 18.92 ± 0.76 c 30.77 ± 0.03 b 18.56 ± 0.27 d
22 days CBCa + sulfur 3.08 ± 0.01 c 23.43 ± 0.76 b 30.14 ± 0.18 bc 26.92 ± 1.01 c
CBC + Na2 EDTA 2.95 ± 0.02 e 18.21 ± 0.60 c 36.06 ± 0.26 a 18.35 ± 0.96 d
CBC + sulfur + Na2 EDTA 3.06 ± 0.01 cd 22.09 ± 1.23 b 35.80 ± 0.32 a 30.51 ± 0.93 bc
Control 2.77 ± 0.06 f 19.16 ± 0.27 c 28.91 ± 0.10 c 17.23 ± 0.48 d
56 days CBC + sulfur 3.20 ± 0.01 b 27.47 ± 0.49 a 30.01 ± 0.32 bc 31.33 ± 1.27 b
CBC + Na2 EDTA 2.99 ± 0.01 de 19.08 ± 0.14 c 36.48 ± 0.73 a 21.16 ± 1.60 d
CBC + sulfur + Na2 EDTA 3.45 ± 0.02 a 26.84 ± 0.71 a 35.36 ± 0.39 a 36.54 ± 1.23 a
Control 2.80 ± 0.02 f 19.36 ± 0.12 c 28.86 ± 0.94 c 19.68 ± 3.23 d
78 days CBC + sulfur 3.21 ± 0.07 b 26.72 ± 1.31 a 28.86 ± 0.72 c 33.23 ± 1.80 ab
CBC + Na2 EDTA 3.03 ± 0.01 cd 19.24 ± 0.41 c 36.16 ± 0.39 a 18.72 ± 0.93 d
CBC + sulfur + Na2 EDTA 3.44 ± 0.0.5 a 27.39 ± 0.36 a 36.27 ± 1.10 a 35.79 ± 3.61 a

Different letters within the same column indicates that the mean significantly differs according to Duncan multiple range test at P < 0.01.
a
CBC: cow bone char.

of elemental sulfur increased the electrical conductivity due to the oxidation of elemental sulfur producing H+ and SO24−
ions, which increase the salt content of the soil (Zhi-Hui et al., 2010). In P-poor sandy soil, when cow bone char amended
with Na2 -EDTA alone results in decreasing soluble calcium significantly (P ≤ 0.01) in comparison with sulfur and sulfur +
Na2 -EDTA treatments (Table 4). The addition of cow bone char amended with Na2 -EDTA decreased none significantly the
soluble calcium compared to control, although the control was unamended by cow bone char (Table 4). On the other hand,
cow bone char amended with sulfur and sulfur + Na2 -EDTA enhanced significantly soluble Ca compared to the Na2 -EDTA
and control treatments (Table 4). At the beginning of 22 days, the soluble calcium in soil extract was found incremented
from 18.92 mmol kg−1 for control (unamended soil) to 23.43 and 22.09 mmol kg−1 for cow bone char with sulfur and
sulfur + Na2 -EDTA treatments, respectively, while adding cow bone char with Na2 -EDTA reduced soluble calcium from
18.92 mmol kg−1 for control to 18.21 mmol kg−1 (Fig. 3). Soluble calcium showed a significant increase with increasing
incubating duration for cow bone char with sulfur and sulfur + Na2 -EDTA treatments (Table 4). While, cow bone char
with Na2 -EDTA alone and control had no significant effect on soluble calcium compared with other treatments. Increased
calcium concentration in soil solution with the addition of elemental sulfur in calcareous sandy soil is due to partial
dissolution of biological apatite from cow bone char and calcium carbonate from soil to supply Ca. Although cow bone
char is added as a source of calcium, soluble calcium content decreased at amending cow bone char with Na2 -EDTA alone
this is attributed to chelating calcium by EDTA compound. Elemental sulfur application in alkaline calcareous soils is
biochemically oxidized producing H2 SO4 , that results in lowering pH and solubilizing CaCO3 which in turn improves the
availability of nutrients in soils such as phosphorus and some micronutrients (Soaud et al., 2011). The cow bone char
+ sulfur application had no significant effect on soluble sodium compared with the unfertilized soil (Table 4). However,
adding cow bone char + Na2 -EDTA and cow bone char + sulfur + Na2 -EDTA to sandy soil caused significant increases
in soluble sodium compared with the control and cow bone char + sulfur treatments at all incubation periods (Table 4).
The soluble sodium at day 22 from incubation increased from 30.77 mmol kg (control) to 36.06 and 35.80 mmol kg for
cow bone char + Na2 -EDTA and cow bone char + sulfur + Na2 -EDTA, respectively, as well as at the end of the incubation
period, the soluble sodium increased from 28.86 mmol kg (control) to 36.16 and 36.27 mmol kg for cow bone char + Na2 -
EDTA and cow bone char + sulfur + Na2 -EDTA, respectively (Fig. 3). Soluble sodium decreased with increasing incubating
duration for control and cow bone char + sulfur treatments. While, cow bone char + Na2 -EDTA and cow bone char +
sulfur + Na2 -EDTA treatments were not significantly different with increasing incubation periods (Table 4). Where, the
soluble sodium decreased from 30.77 (at day 22) to 28.86 mmol kg (at day 78) for control treatment and the cow bone char
+ sulfur treatment declined soluble sodium from 30.14 at day 22 to 28.85 mmol kg at day 78 (Fig. 3). The soluble sodium
increased with cow bone char + Na2 -EDTA and cow bone char + sulfur + Na2 -EDTA because the sodium includes Na2-
EDTA. The application of cow bone char + sulfur and cow bone char + sulfur + Na2 -EDTA to calcareous sandy soil caused
significant increases (p ≤ 0.01) in soluble SO4 = compared to the control (unfertilized soil) and cow bone char + Na2 -EDTA
treatments at all incubation periods (Table 4). However, adding cow bone char + Na2 -EDTA had no significant effect on
soluble SO4 = compared with the unfertilized soil (Table 4). The soluble sulfate at day 22 from incubation improved from
18.56 mmol kg (control) to 26.92 and 30.51 mmol kg for cow bone char + sulfur and cow bone char + sulfur + Na2 -EDTA,
respectively, as well as at the end of the incubation period, the soluble sulfate increased from 19.68 mmol kg (control)
to 33.23 and 35.79 mmol kg for cow bone char + sulfur and cow bone char + sulfur + Na2 -EDTA, respectively (Fig. 4).
Soluble sulfate increased with increasing incubating duration for cow bone char + sulfur and cow bone char + sulfur
+ Na2 -EDTA treatments. While, control and cow bone char + Na2 -EDTA treatments were not significantly different with
increasing incubation periods (Table 4). Moreover, the soluble sulfate increased from 26.92 (at day 22) to 33.23 mmol kg
(at day 78) for bone char + sulfur treatment and the cow bone char + sulfur + Na2 -EDTA treatment enhanced soluble
sulfate from 30.51 at day 22 to 35.79 mmol kg at day 78 (Fig. 4). Increased sulfate concentration in soil solution during this
8 A.A. Amin / Environmental Technology & Innovation 17 (2020) 100636

Fig. 3. Effect of different treatments and incubation periods on soluble calcium and sodium. Each value represents the mean of three replicates with
the standard error shown by the vertical bars.

study is attributed to oxidation of sulfur by sulfur oxidizing bacteria. Furthermore, every one gram of soil contains about
1 billion bacteria cells as well as 200 million fungal hyphae (Zhang et al., 2014). Sulfur application to some calcareous
sandy soils led to significant increase of sulfate concentration (Soaud et al., 2011). In treatments for which no sulfur was
added there was an increase in the concentration of sulfate in soil solution. This is due to the hydrolysis of insoluble
sulfate minerals in these soils (Soaud et al., 2011).

4. Conclusions

The results of the present study revealed that the application of elemental sulfur and Na2 EDTA with cow bone char
represents a feasible and sustainable strategy to enhance the availability of phosphorus released from cow bone char.
The addition of elemental sulfur, Na2 EDTA and their mixture improved the release of unavailable P (biological apatite)
from cow bone char to available P in P-poor sandy soil. It was evident from this study that applying cow bone char with
elemental sulfur, Na2 EDTA and their mixture resulted in the improvement of phosphorus availability in P-poor sandy
soil. Enhancing phosphorus availability was due to chelating calcium by Na2 EDTA and sulfur addition decreased soil pH
causing phosphorus release from cow bone char. However, non-significant differences in Olsen-P were observed when
applying cow bone char + sulfur and cow bone char + Na2 -EDTA. Therefore, we recommend utilizing cow bone char with
sulfur in soil as slow release fertilizer to alternate phosphorus sources which helps ameliorating available phosphorus for
 A.A. Amin / Environmental Technology & Innovation 17 (2020) 100636 9

Fig. 4. The effect of different treatments during the incubation periods on soluble sulfate. Each value represents the mean of three replicates with
the standard error shown by the vertical bars.

many reasons: low cost price of cow bone char with sulfur and it prevents contaminating soils by heavy metals and
radionuclides. Applying the outcome of this research in modern agriculture can be useful because bone char with sulfur
is a safe and cheap amendment. We also do not recommend using Na2 EDTA in the field because of its expensive price.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

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