Journal of Hazardous Materials 411 (2021) 125046

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Journal of Hazardous Materials

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Transforming wet blue leather and potato peel into an eco-friendly

bio-organic NPK fertilizer for intensifying crop productivity and retrieving
value-added recyclable chromium salts
Subhasish Majee a, Gopinath Halder a, Dalia Dasgupta Mandal b, O.N. Tiwari c, *,
Tamal Mandal a, *, 1
a
Centre for Technological Excellence in Water Purification, Department of Chemical Engineering, National Institute of Technology Durgapur, India
b
Department of Biotechnology, National Institute of Technology Durgapur, India
c
Centre for Conservation and Utilisation of Blue Green Algae, Division of Microbiology, ICAR- Indian Agricultural Research Institute, New Delhi, India

A R T I C L E I N F O A B S T R A C T

Editor: Dr. R. Teresa An attempt has been made to address two important issues, the solid waste management of leather industry and
soil fertility. The SEM images revealed altered surface-morphology.The EDS elemental analysis exhibited pres­
Keywords: ence of about 13.2% nitrogen (N), 50.56% carbon (C), 2.69% phosphorus (P) in the collagenous material of wet
Wet blue leather blue leather (WBL) after chromium removal. In potato peel biochar (PPB) prepared the EDS analysis corrobo­
Collagenous material
rated the presence of N P K in 5%, 1.4% and 21.64% respectively. In the formulated bio-organic NPK fertilizer,
Chromium precipitation
using chromium free WBL and PPB, the percentage of N, P, K, was in 13.10, 2.41, 20.20% respectively which was
Potato peel biochar
Bio-organic NPK fertilizer authenticated by EDS. Its effect on okra (Abelmoschus esculentus) plant showed higher growth (1.11 fold fruit size)
and total chlorophyll content (1.61 fold) than in untreated soil (control) but displayed similar result as in
presence of chemical fertilizer. The released free ammonia in soil with bio-organic NPK was more (37.02%) than
with chemical fertilizer (6.10%). DNA intercalation study showed the non-hazardous impact on soil. The FTIR,
XRD, SEM-EDS, AAS further specified the conversion of the WBL extracted acidic chromium-rich solution by
MgO into crystalline chromium for commercial use.

1. Introduction
Today, effective soil management strategies are one of the most
essential need for increasing soil fertility and crop production as the
dependence on inorganic chemical-based fertilizers, cause a serious
threat to human health and the environment. Intensive use of com­
mercial inorganic fertilizer decrease the organic matter content; decline
soil pH; essential nutrients; minerals; and finally deteriorate soil health
(Lin et al., 2019; Abera et al., 2018; Li et al., 2012). Moreover vegetables
and fruits grown on inorganically over-fertilized soil are prone to pests
and diseases (Karungi et al., 2006; Parmar and Chakraborty, 2016). To
avoid this, bio based-fertilizer can be a choice for better crop production.
The studies have shown that organic fertilizer has higher total nitrogen
(N2) and organic matter content, which can help to improve the soil’s
physical, chemical, and biological property by increasing the population
of useful microbes in the soil (Álvarez et al., 1988). The search of an
effective process for the formulation of an eco-friendly, low cost and
competent organic fertilizer is hence, in demand. Use of solid organic
waste (enriched with proper nutrients) for the preparation of
environment-friendly compost could be an effective alternative. A large
quantity of solid waste generated from leather industries is chromium
(Cr) containing wet blue leather (WBL) which needs special attention for
management. Chromium has potential deleterious impacts on the
environment as it is lethal to living cells and is carcinogenic (Tasca and
Puccini, 2019; Kolomaznik et al., 2008; Dhal et al., 2013), but it also has
few important applications as in; Recently chromium-free specially
synthesized tanning agents have been found which can be utilized in the
tanning industry to solve the problem of chrome pollution and related
issues (Qiang et al., 2016; Gao et al., 2019; Jia et al., 2019) but till date
most of the leather industries use chromium-bearing chemicals as a
tanning agent to protect the leather from microbial-deterioration (Tariq
et al., 2009). Utilization of this solid waste in an alternative manner may

* Corresponding authors.
E-mail addresses: ontiwari1968@gmail.com (O.N. Tiwari), tamal.mandal@che.nitdgp.ac.in (T. Mandal).
1
Orcid ID: 0000–0002-0493–747X.

https://doi.org/10.1016/j.jhazmat.2021.125046
Received 29 August 2020; Received in revised form 1 January 2021; Accepted 3 January 2021
Available online 7 January 2021
0304-3894/© 2021 Elsevier B.V. All rights reserved.
S. Majee et al. Journal of Hazardous Materials 411 (2021) 125046

benefit the industry for its management and economic aspects and will
save the environment as well. Previous investigations have demon­
strated several chromium removal techniques (Vibhatabandhu and Sri­
thongouthai, 2018; Zhao et al., 2019) but acid treatment is the required
one for chromium separation without destroying the protein part. It can
convert the WBL into a precious source for nitrogen-rich collagen and
valuable chromium (Poulopoulou et al., 1998). WBL could be an
ingredient of organic manure and soil conditioner, as the nitrogen
content of WBL is reported to contain about 140 g/kg of N (dry weight),
and thus after chromium separation, it could be utilized as nitrogen rich
manure. It is known that nitrogen, phosphorus and potassium all three
must be present in manure. Conventionally, farmers use inorganic
potassium-phosphorus-rich fertilizer for higher crop yield, which is
expensive. To overcome this a cheap substitute is being searched. The
potato peel biochar (PPB) can be utilized as potassium-phosphorus
source as it is reported to contain a large quantity of potassium and
phosphorus nutrients (Vaitkevičienė, 2019). Potato peel is an abun­
dantly available domestic solid waste and is eco-friendly because of it’s
natural biodegradability (Wazir et al., 2018). Food and Agriculture
Organization of the United Nations (FAO) data reveals that there is a
significant rise in potato crop plantation over the last decades. If used
properly it can act as a cost-curtailing resource of potassium and
phosphorus.
Both chromium-free wet blue leather waste, and potato peel biochar
can act as nitrogen, phosphorus, and potassium source for an environ­
mentally benign bio-organic NPK fertilizer. Till now many different
types of formulations for organic manure have been reported using one
type of waste material as a nutrient source in combination with inor­
ganic chemical compounds (Majee et al., 2019; Nogueira et al., 2010).
Gwenzi et al. (2017) developed a biochar based N–P–K fertilizer by
impregnating ammonium nitrate, potassium dibasic phosphate, and
single super phosphate solution into the biochar. Shi et al. (2020) pre­
pared granular biochar mineral urea composite which is the combina­
tion of blending urea with green waste biochar supplemented with clay
minerals of bentonite and sepiolite. Very few attempts have been made
for the formulation of NPK enriched organic fertilizer by combining two
natural organic solid waste as nutrient source.
In this present study, two organic ingredients, chromium-free
collagen of WBL waste as nitrogen source and potato peel biochar as
potassium-phosphorus source was strategically used for the synthesis of
NPK rich bio-organic manure. Proper measures were taken to remove
chromium from WBL to get another valuable crystalline chromium oxide
for further commercial use. This is the first report where chromium re­
covery in the form of crystalline chromium oxide from wet blue leather
waste was obtained and characterized. Both collagen fraction and potato
peel biochar were characterized using sophisticated analytical tools
SEM, FTIR, EDS along with the amalgamated NPK rich bio-organic
manure. The growth and other parameters of the okra (Abelmoschus
esculentus) plant was examined and simultaneously compared in differ­
ently treated soils. The eco-toxicity of this bio-organic fertilizer formu­
lation on soil was assessed by DNA interaction study using fluorescence
spectroscopy.
purchased from the authorized dealers of IFFCO (Indian Farmers Fer­
tiliser Cooperative Limited), Durgapur, India. The chemical constituents
of NPK 10:26:26 fertilizer are 50.04% diammonium phosphate, 43.98%
muriate of potash, 1.5% urea, 3.6% silica and 0.88% moisture. This
composition with respect to elemental nitrogen, neutral ammonium
citrate soluble phosphate (as P2O5), and water soluble K2O is equivalent
to 10%, 26%, and 26% respectively. The soil used in the pot experiment
was collected from Faridpur (adjacent to NIT Durgapur, WB, India). The
nitrogen (N) and potassium (K) content in the soil was 0.045% and
0.030% respectively, and the phosphorus content was negligible (not at
detectable level).
2.2. Preparation of bio-organic NPK fertilizer
2.2.1. Processing of WBL: chromium extraction and nitrogenous collagen
production
Chromium was extracted from WBL by a unique H3PO4 (1 N) acid
treatment method (Lima de Oliveira et al., 2008). Treatment with acid
was done three times In brief, about 6 g WBL was allowed to react with
300 mL H3PO4 (1 N) acid solution for 24 h at pH 3 and temperature of
30 ◦ C. After filtration, washing was repeated twice with fresh 1 N H3PO4
acid. The treated WBL was properly washed with distilled water and
tested for residual chromium in both extracted solutions and WBL using
atomic absorption spectrophotometer (AAS) (Flame analysis method,
iCE 3000 Series/Thermo Scientific).
2.2.1.1. Optimization of MgO dosage for recovery of chromium from WBL
extract. The extracted and separated chromium fraction from WBL was
treated with MgO at different dosages of 2–20 g/L for 12 h following a
standard protocol (Wang et al., 2016). Obtained precipitate (Green
color) was separated and supernatant was analyzed for chromium by
AAS. Doses of MgO were optimized for chromium recovery. The sepa­
rated chromium precipitated sample (S1) was dried in a hot air oven for
analysis by scanning electron microscopy (SEM), energy dispersive x-ray
spectroscopy (EDS), and atomic absorption spectrophotometer.
2.2.1.2. Granular chromium recovery and characterization. The treat­
ment time with MgO was extended from 24 h to15 days. The precipitate
was transformed into a new type of greenish colored irregular granular
shaped material (Fig. 1). It was dried in a hot air oven at 90 ◦ C tem­
perature for 6 h. The transformed material (S2) was characterized by x-
ray diffraction (XRD) (PAN analytical, B.V., PW3040/60, Netherlands)
and Fourier transform infrared spectrometer (FTIR) (SPECTRUM RX1,

2. Materials and methods

2.1. Materials

The solid waste, wet blue leather as nitrogen source was supplied by
Leder Chemische Private Limited, Kolkata, India. The potato peel for
biochar was collected from a canteen ‘Wonder Café’ in NIT Campus,
Durgapur, West Bengal, India. All analytical grade chemicals such as
H3PO4, HCl, CuSO4, K2SO4, H2SO4, MgO, calf thymus DNA (ctDNA),
ethidium bromide (EtBr), acetone and methyl red indicator were pur­
chased from Sigma-Aldrich, Merck and HiMedia. The model plant okra
(Abelmoschus esculentus) seeds were procured from the local nursery of Fig. 1. Greenish granular chromium obtained by MgO treatment from extrac­
Durgapur, India. NPK 10:26:26 complex grade chemical fertilizer was ted solution of WBL.

2
S. Majee et al.
Perkin Elmer). The pH of the supernatant was close to neutral pH 6.5
that might be due to the addition of a basic chemical agent (MgO).
2.2.2. Preparation of potato peel biochar
For biochar, collected potato peels were properly washed with water
3–4 times to remove any residual dust particles and dried at 80 ◦ C in a
hot air oven for complete removal of moisture. After crushing in a
mixture grinder it was carbonized in an electric furnace at 450 ◦ C for 1 h
(Ehraman, 1994). The temperature selected was based on the previous
work by Novak et al. (2009) which showed that biochar produced at low
temperature contains several functional groups and is suitable for
agricultural application, whereas biochar production at high tempera­
ture reduces its nutritional value and consumes extra energy (Novak
et al., 2009; Sarfaraz et al., 2020). Using SEM, EDS the prepared biochar
was characterized to detect phosphorus, potassium, and other nutrients
in it.
2.2.3. Bio-organic NPK fertilizer preparation
For preparing bio-organic NPK fertilizer, two organic components
such as the chromium separated collagenous ingredient of WBL (as a
nitrogen source) and potato peel biochar (as potassium and phosphorus
source) was amalgamated in the ratio of 1:2.5. These ingredients were
uniformly mixed in a plastic container and dried at ambient temperature
at (30 ± 2 ◦ C) for 24 h (the maturation time). The hard manure was
crushed to get the powder for easy distribution to the agricultural field.
2.2.4. Estimation of total nitrogen present in the bio-organic NPK fertilizer
The total nitrogen content present in the formulated bio-organic NPK
fertilizer was analyzed by following the Kjeldahl method (Bremner,
1960). About 0.5 g of the air-dried bio-organic NPK fertilizer was dis­
solved in 10 mL of 98% H2SO4 into the digestion flask and then added
1 g of digestion mixture (K2SO4: CuSO4 and SeO2 in the ratio of 10:4:1)
to it. The mixture was heated up at a low temperature until the color
became yellowish-green. The digested liquid sample was cooled and
diluted with distilled water to a final volume of 50 mL. Next, 10 mL of
40% sodium hydroxide solution was added into 10 mL of the diluted
sample, in a micro Kjeldahl apparatus for NH3 liberation. The tip of this
apparatus was immersed in a boric acid solution to trap the produced
NH3. The ammoniacal solution was titrated against 0.01 N hydrochloric
acids until the greenish-blue color turned into light pink. The total ni­
trogen content of the bio-organic NPK fertilizer was calculated by
following the Eq. (1) (Bhat et al., 2017)
(A − B) × N × 1.4
Totalnitrogen = (1)
S
Where, N = normality of hydrochloric acid used 1.4 multiplication
factor, S = weight of the sample (g), A = volume of titrant used for
sample (mL), B = volume of titrant used for blank (mL).
2.3. Pot experiment on okra (Abelmoschus esculentus) plant
The soil used in this study was collected from Faridpur area [adjacent
to NIT Durgapur, WB, India]. The soil was characterized and observed to
contain 0.045 nitrogen [N] and 0.030% potassium [K] with negligible
phosphorus content corroborating with the information available in the
web site of the Government of India. To assess the efficacy of this bio-
organic NPK fertilizer as nitrogen, phosphorus, potassium nutrients for
promotion of plant growth, pot experiments were conducted on okra
plant (Abelmoschus esculentus) in soil with and without addition of
formulated bio-organic fertilizer and compared with chemical fertilizer
containing soil. The plant growth study was accomplished following a
modified process of Lima et al. (2010). The overall growth regarding the
stem size, leaf size, and fruit size was examined three times in triplicates.
Three separate pots for each kind of experimental soil were kept and
repeated three times. In the first set, 2 g of chemical fertilizer was added
3
Journal of Hazardous Materials 411 (2021) 125046
with 2 kg of soil and numbered as pot-I. The second set was with 4 g of
bio-organic NPK fertilizer marked as pot-II, and in the pot III only 2 kg of
natural soil (collected from agricultural field) was added for control. The
growth of the potted plants were monitored regularly up to 70 days.
Origin pro 8.5.0 software was used for statistical parametric analysis and
Fisher’s least significant difference (LSD) test to determine the signifi­
cant difference in results of the plant growth.
2.4. Ammonia detection and DNA intercalation study
The detection of ammonia in soil was done on 45th day of pot
experiment. For this investigation, 5 g of soil at each experimental set
was dissolved in 50 mL of deionized water (Nelissen et al., 2012; Abiven
et al., 2005) and agitated for 96 h in the shaking incubator (LSI-3016R).
Liberated ammonia in the aqueous soil extracts was detected using
ammonia analyzer (Orion Dual star, Thermo Scientific) (Banwart et al.,
1972). For DNA intercalation study, from each set, collected 5 g of soil
sample at specified time, dissolved in 50 mL deionized water, agitated
for 96 h and filtered with Whatman-40 filter paper. Supernatant was
collected for ethedium bromide (EtBr) fluorescence quenching study by
intercalation of toxic compounds (released from fertilizer if any) with
the calf thymus DNA (ctDNA). The effect on DNA was examined indi­
rectly by fluorescence spectroscopy (G9800A, Cary eclipse, Agilent
Technologies) (Shahabadi et al., 2009).
2.5. Effect of different fertilizers on chlorophyll content of leaf sample
Chlorophyll content was detected in leaves of experimental plants
treated with prepared bio-organic NPK fertilizer and compared with
their respective control using the standard protocol of Arnon (1949)
(Arnon, 1949). In brief 1 g of the finely cut leaf was crushed and ho­
mogenized in 20 mL of 80% acetone solution for 1 h. After centrifuga­
tion for 10 min at 5000 rpm the supernatant was collected. This step of
extraction process was repeated until the residue became color less. The
absorbance of the chlorophyll extract was measured by UV-Vis spec­
trophotometer at 645 nm, and 663 nm against the 80% acetone solvent
as blank (Raut et al., 2017). The following formulae were applied for
quantification of chlorophyll A (chl A), chlorophyll B (chl B), and total
chlorophyll (total chl)
1.27(A663) − 2.69(A645) × V
Chlorophyll A(mg/gm tissue) =
1000 × W
22.9(A645) − 4.68(A663) × V
Chlorophyll B(mg/gm tissue) =
1000 × W
Total chlorophyll(mg/gm tissue) =
20.2(A645) + 8.02(A663) × V
1000 × W
Where, A645 = absorbance at a wavelength of 645 nm, A663 = absor­
bance at a wavelength of 663 nm, V = final volume of chlorophyll
extract in 80% acetone, W = weight of leaf sample used for chlorophyll
extraction.
2.6. Seed germination test and vigor index
Germination test was conducted with chemical fertilizer, bio-organic
NPK fertilizer and control (no fertilizer) at 28 ± 2 ◦ C temperature in
triplicates. Percentage of germination was calculated by following the
equation at the 7th day of seed sown (ISTA, 1999).
Numberofseedsgerminated
Germination % =
Totalnumberofseedssownforgermination
× 100
Vigor index value was computed using the following formula of
Abdul-Baki and Anderson (1973).
Vigorindex = Germination(%) × Meanofseedlinglength(root + shoot)
S. Majee et al.
2.7. Statistical analysis
Origin pro 8.5 software was used for the statistical analysis of the
experimental data. Analysis of variance (ANOVA) and least significance
difference (LSD) tests were conducted to test the significant difference in
the variables at 5% level of significance.
3. Results and discussion
3.1. WBL waste characterization
Atomic absorption spectrometric Flame analysis method (iCE 3000
series/Thermo Scientific) detected total chromium of about 13,000 mg
per kg of WBL. The morphology of WBL was investigated by SEM (EVO
MA 15 / 18, CARL ZEISS) and represented in Fig. 2a. It showed the
presence of collagen fiber structure stuck to each other with limited
disorientation and looked like a thick bundle. This is probably due to
chrome tanning as reported (Abebaw and Abate, 2018). The
morphology of WBL obtained is quite similar to the reported work of
Oliveira et al. (2008) with indication of presence of chromium into the
collagen fiber of WBL waste (Nogueira et al., 2010; Oliveira et al., 2008).
The elemental analysis using EDS (51N1000 – EDS System, Oxford In­
struments Nano analysis) is shown in Fig. 2a, depicts the presence of
chromium. In this study, as revealed by EDS, WBL contains 3.32%
chromium (Cr), along with 15.69% nitrogen (N), 3.75% sodium (Na),
7.06% chlorine (Cl), 40.25% carbon (C), 0.08% potassium (K).
Fig. 2. SEM and EDS for characterization of (a) WBL and (b) WBL after chromium separation.
4
Journal of Hazardous Materials 411 (2021) 125046
3.2. Chromium separation from WBL and characterization
As mentioned in Section 2, after three consecutive phosphoric acid
treatment (wash) removal of 11750 (±124) mg chromium per kg of WBL
was obtained. As represented in Table 1, about 90.38 ( ± 0.96) %
chromium was removed from WBL after three wash and it is observed
that 100% removal can be possible if acid wash is done for five times.
The chromium content in the collagen (after 3rd wash) was 1238 mg/
kg, as exhibited by AAS analysis. There was no significant difference of
chromium removal between three replicates at 5% level of significance
(P-value> 0.05) as per ANOVA test.
Eq. (2) represents the schematic way of chromium extraction from
wet blue leather by H3PO4 acid treatment (Lima de Oliveira et al., 2008).
Wet blue leather-Cr + H3PO4 → Collagen + CrPO4 (2)
The chromium present in the WBL combines with the phosphate
group of the H3PO4 acid turned into CrPO4 accordingly the collagen
fraction become freed from the bonding of chromium salt (Jiang et al.,
2016). Chromium free WBL was again analyzed using SEM and EDS for
its surface characterization and elemental analysis. In Fig. 2b, the SEM
image of collagenous material showed the altered surface morphology
due to chromium elimination (Nogueira et al., 2010; Lima de Oliveira
et al., 2008). It was noticed that the thick collagen fiber bundle as in raw
WBL was absent which may be resulted due to the chromium separation
by phosphoric acid treatment. Besides the fibrous structure was
collapsed and transformed into undifferentiated nature. In the EDS
spectra (Fig. 2b) related to raw WBL, a smaller peak (decreased by
0.19%) for chromium element was noticed in this case indicating
chromium elimination. EDS elemental analysis also verified the
S. Majee et al.
Table 1
Chromium extraction from WBL by H3PO4 (1 N) acid treatment.
H3PO4 (1 N) acid Initial conc. of chromium Required chemicals
treatment steps in WBL (mg/Kg) and time
1st step 13,000 300 mL H3PO4 (1 N)
for 24 hrs
2nd step 6750 300 mL H3PO4 (1 N)
for 24 hrs
3rd step 3500 300 mL H3PO4 (1 N)
for 24 hrs
Total chromium removal
*The chromium extraction process was performed in three replicates and statistical analysis was done at 0.05 level of significance. The ± values represent standard
error of the mean. The ± values represent standard error of the mean.
presence of other elements such as 13.2% nitrogen (N), 50.56% carbon
(C), and 2.69% phosphorus (P) in this Cr free collagenous material.
3.3. Characterization of potato peel biochar
Biochar of potato peel was prepared as mentioned in Section 2 and its
characterization was done to use it as K and P source. The surface
morphology of the potato peel biochar (PPB) in the SEM image (Fig. 3)
exposed the multiple irregulars and rough surface structure of pyrolyzed
material. Non-spherical particles of various sizes along with small pores
were also witnessed on the surface of the sample probably due to
dehydration and volatilization at high temperature. Chemical constitu­
ents of this was analyzed using energy dispersive x-ray spectroscopy
(EDS). Fig. 3 established the occurrence of various macro and micro­
nutrients such as nitrogen (N) 5%, 1.4% phosphorus (P), 21.64% po­
tassium (K), 1.24% magnesium (Mg), and 27.56% carbon (C). This
finding is substantial for considering the prepared PPB as a nutrient-rich
ingredient for bio-organic NPK fertilizer formulation. Biochars from
plant and animal sources are well investigated and have been well-
characterized. Studies recommended that the preparation of biochar at
450 ◦ C is relevant for its agricultural use (Sarfaraz et al., 2020). Our
analysis correlates well with previous investigation which revealed that
the structural components of different biochar consist of an assemblage
of inorganic elements e.g., Ca, Mg, P, K etc. in its ash fraction along with
aromatic and non-aromatic organic compound. Consequently they are
acknowledged as viable soil amendment to increase C sequestration crop
yields and fertility characteristics (Domingues et al., 2017; Jindo et al.,
2014).
Fig. 3. SEM and EDS for potato peel biochar characterization.
Journal of Hazardous Materials 411 (2021) 125046
Weight of WBL taken for chromium The amount of extracted Total percentage
extraction experiments (mg) chromium from per kg WBL removal
6000 6250 ( ± 32) mg 48.07
5962.5 3250 ( ± 31) mg 73.07 (1st + 2nd)
step
5943 2250 ( ± 62) mg 90.38 (1st + 2nd +
3rd) step
11750 ( ± 124) mg 90.38 ( ± 0.96)
3.4. Characterization of bio-organic NPK fertilizer prepared
3.4.1. SEM and EDS analysis for characterization of bio-organic NPK
fertilizer
The Cr free WBL collagen fraction and biochar of potato peel was
mixed thoroughly in 1:2.5 ratio and characterized using the sophisti­
cated analytical techniques such as SEM, EDS, FTIR, for its proper use in
agricultural field. The SEM image revealed a new texture of the
formulated bio-organic manure (Fig. 4). The dense and sticky nature of
the collagen was converted into an unorganized thread-like structure
which may be due to the presence of potato peel biochar after proper
mixing. Additionally, it was noticed that the biochar particles were
stacked over the thread-like structure of collagenous material. The EDS
analysis (Fig. 4) clarifies the occupancy of a significant percentage of
nitrogen (N), phosphorus (P), potassium (K) and magnesium (Mg), in
13.10%, 2.41%, 20.20% and 1.16% respectively. Also 33.74% carbon
(C), 0.23% chromium (Cr) and other trace elements were displayed in
the prepared bio-organic NPK fertilizer. This support that all major
nutrients were present as required in any commercial fertilizer. More­
over, here addition of potato peel biochar (PPB) enriched the nutritional
value of prepared fertilizer by providing high amount of nitrogen, po­
tassium, phosphorus, and trace amount of magnesium nutrient.
Nogueira et al. (2010) showed similar nitrogen content (14% N that is
1.12 g nitrogen in 8 g collagen) accompanied with phosphorus and
potassium which is very much effective for promising agronomic results.
It is to be discerned that PPB has contributed largely in formulating this
manure that would help to increase the retention of nutrients (P, K, Mg
etc.) in the soil profile (Laird et al., 2010). According to Utomo et al.
(2011), the biochar ingredient has been reported to enhance N, P and K
nutrients uptake by the plant (Utomo et al., 2011). The presence of high
carbon content in this prepared fertilizer would improve the soil organic
carbon and soil fertility as well (Jatav et al., 2017). All the physico­
chemical properties of the bio-organic NPK fertilizer suggested that it
5
S. Majee et al. Journal of Hazardous Materials 411 (2021) 125046

Fig. 4. SEM and EDS for formulated bio-organic NPK fertilizer characterization.

can be used as a new auxiliary of chemical fertilizer for agricultural
application.
3.4.2. Fourier transform infrared spectrometry analysis
The FTIR spectrum in Fig. 5 depicted broad spectra around
3000 cm− 1 to 3800 cm− 1 for the amide A band of collagen, flanked by
N–H stretching vibrations from peptide linkages and O–H stretching
vibration of hydroxyl group (Rosu et al., 2018; Sionkowska, 2006). The
band at 1554 cm− 1 resembled with the N–H bending vibration of amide
II band as reported by Rosu et al. (2018) (Rosu et al., 2018). Besides
there are distinct peaks around 2960 cm− 1, 1640 cm− 1 for aliphatic C–H
stretching (Tinti et al., 2015) and carbonyl stretching vibrations (C˭O) of
amide I band respectively (Hedberg et al., 2014). The O–H stretching
vibration of phenolic compounds present in PPB (Claoston et al., 2014)
has been noticed at around 1400 cm− 1 along with the peak at
1240 cm− 1 C–O stretching of carboxylic acids or C–N stretching by
amide II band similar to the band demonstrated by Smidt et al. (2002)
(Smidt et al., 2002). There was a very large peak at 1050 cm− 1 attrib­
uted to C–O bond stretching frequency of polysaccharides (Lammers
et al., 2009). Due to presence of silica in PPB, small peaks at 776 cm− 1
and 690 cm− 1 was also observed corresponding to asymmetric bending
vibrations of Si–O–Si. Additionally peaks at 560 cm− 1 and 464 cm− 1
showed symmetric stretching vibrations of Si–O minerals as obtained in
previous reports (Shi et al., 2020; Mohan et al., 2018). The FTIR data
presented here exposed characteristics groups contributed from both the
ingredients in the bio-organic fertilizer as a mixture. A range of nutrients
especially the carboxyl and phenol groups in ionized form will influence
the formation of soil charges and can contribute to soil fertility using this
prepared bio-organic fertilizer.
3.4.3. Total nitrogen in formulated bio-organic NPK fertilizer for plant
growth
Total nitrogen content present in the formulated bio-organic NPK
fertilizer was about 130 g/Kg as quantified by micro Kjeldal method.
This amount is close to that found by Nogueira et al. (2010) i.e 140 g/Kg
of Nitrogen in their prepared bio-fertilizer using collagen. It hinted that
it might be used as a good nitrogenous fertilizer in order to minimize the
excessive use of chemical fertilizers (Nogueira et al., 2010). In pot
experiment to study the growth effect on okra the quantity of prepared
bio-organic NPK fertilizer used was 1.08 times more than that of used by
Nogueira et al. (2010) in their study to get the similar nitrogen effect on
plant growth.
3.5. Plant growth study
The effect of indigenously prepared and characterized bio-fertilizer
on the growth of okra plant was examined and compared with that of
chemical fertilizer treated and untreated soil (control). In Table 2,
growth of the okra plant examined on the day 70 represented the plant
height of 48.7( ± 0.73) cm, leaf size 13.4( ± 0.32) cm, diameter 4.2
( ± 0.17) cm, and fruit size 10.5( ± 0.21) cm for bio-organic NPK fer­
tilizer treated soil whereas for control soil and chemical fertilizer used,
the effect on plant height, leaf,diameter and fruit size were 44.8
( ± 0.55) cm, 12.1( ± 0.43) cm, 3.4( ± 0.15) cm, 9.4( ± 0.20) cm and
49.2( ± 0.88) cm, 13.8( ± 0.20) cm, 4.4( ± 0.15) cm, 10.7( ± 0.26) cm
respectively. The effect of formulated NPK organic fertilizer and chem­
ical fertilizer found nearly similar however compared to control both
showed more plant growth. Compared to control, large leaf size, and
proliferation of fruit size are the two most significant outcome of these

Table 2
Effect of different fertilizers on plants.
Fertilizer Plant height Leaf size Diameter Fruit size
mean (±SE) mean (±SE) mean (±SE) mean (±SE)

Control 44.8 (±0.55) 12.1 (±0.43) 3.4 (±0.15) 9.4 (±0.20)

2 g bio- 46.5 (±0.37) 12.4 (±0.15) 3.7 (±0.11) 9.9 (±0.15)
organic
4 g bio- 48.7 (±0.73) 13.4 (±0.32) 4.2 (±0.17) 10.5 (±0.20)
organic
Chemical 49.2 (±0.88) 13.8 (±0.20) 4.4 (±0.15) 10.7 (±0.26)
LSD(0.05) 2.175 0.978 0.489 0.691

*The pot experiment was conducted in three replicates and statistical analysis
was done at 0.05 level of significance. The ± values represent standard error of
Fig. 5. FTIR analysis of prepared bio-organic NPK fertilizer. the mean.

6
S. Majee et al.
two fertilizers. There was about 1.11(10.5 ± 0.21 cm) and 1.13
(10.7 ± 0.26 cm), fold boost in length of the fruits with bio-organic
fertilizer and chemical fertilizer respectively over the control fruit size
of 9.4 ( ± 0.20) cm. In the present study the N & K ratio of formulated
bio-organic fertilizer (13.10:20.20) (using 1:2.5 of WBL and potato peel)
and chemical fertilizer (10:26) were close. The change in growth at
initial (seedling) and final stage (Fig. 6) indicated the valuable role of
the prepared bio-organic fertilizer in this study. The organic manure
showed a better response on plant growth from 45 th day onwards
whereas commercial fertilizer caused rapid plant growth due to early
and fast release of nutrients (NPK) into the soil. Previous reports also
suggest that the initial slow growth in presence of organic manure due to
controlled nutrient release may increase the water holding capacity of
the soil; as a result, the health of the plant remain worthy leading to
better crop yield along with saving in cost of the labor (Sharma and
Mittra, 1991; Itelima et al., 2018). Fast and excessive amounts of
ammonia liberation from chemical fertilizers can harm the seedling and
soil environment (Sharma and Mittra, 1991) which is not the case with
bio-organic NPK fertilizer. In the pot experiment a fixed amount of
chemical fertilizer i.e 2 g was mixed 2 kg soil and compared with
varying amount bio-organic fertilizer to study their efficacy in plant
growth. As observed the growth rate was higher in chemical fertilizer
because of higher release rate of nutrients from chemical fertilizer
(Nogueira et al., 2010) than bio-organic fertilizer. Conversely equivalent
growth was observed in soil with 4 gm of bio-organic fertilizer i.e.
similar growth effect can be obtained by increasing bio-organic fertilizer
amount twice that of chemical fertilizer. As prepared bio organic fer­
tilizer used collagenous fraction after Cr extraction. The role of chro­
mium is important in this study. The symptoms of Cr toxicity in plants
comprise decrease of germination, reduction of growth inhibition of
enzymatic activities, impairment of photosynthesis and oxidative im­
balances. The effect of chromium was insignificant here. It is to be noted
that the soil used in the present study contained 0.055 mg Cr /Kg soil.
Studies showed that Cr level higher than 5.0 mg/kg in plant species are
toxic on a dry-weight basis (Kabata-Pendias and Pendias, 2001). More­
over the permissible limit of chromium for plants is 1.30 mg/kg as
Fig. 6. Plant growth for (1) control plant (2) plant fertilized by bio-organic NPK fertilizer (3) plant fertilized by chemical fertilizer.
7
Journal of Hazardous Materials 411 (2021) 125046
recommended by WHO (Pais and Jones, 1997). The chromium present
in formulated bio organic fertilizer (0.71 mg/kg) enhanced soil’s
maximum Cr content to 0.765 mg/kg when added but it appears
non-hazardous to plant growth because it is within the limit of recom­
mendation by the regulatory authorities. The results also indicated that
growth pattern edible plant okra was not negatively affected state
non-hazardous nature of this bio-organic fertilizers. Moreover it is
already known that in plant tissues, the Cr (VI) is converted to Cr (III)
that has the tendency to bind to the cell walls, which hinders the further
transport of Cr within plant tissues. To avoid any deleterious effect of Cr
to plant, 100% Cr free bio-organic fertilizer could have been produced
by washing WBL with phosphoric acid five times instead of three times
as presented in the Table 1. This two extra wash may enhance the cost of
this formulated fertilizer by 30% so in this study it was formulated with
three time acid wash WBL.
The ANOVA test shows that there was significant difference of plant
growth (plant height, stem size and leaf size) between experimental
plants at 5% level of significance. The LSD comparison at 5% level of
significance between experimental plants for plant growth revealed that
there was significance difference between control plant and organically
fertilized plant, control plant and chemically fertilized plant but there
was no significance difference between organically and chemically
fertilized plants Table 2.
Further the DNA interaction study of aqueous fraction of soils
revealed the non-toxic nature of the bio-organic fertilizer developed.
3.6. Effect of prepared bio-organic fertilizer on chlorophyll
Fig. 7, showed the highest content of chlorophyll A 1.75 (±0.11),
chlorophyll B, 1.19 (±0.08) and total chlorophyll 2.94 (±0.19) (in mg/g
of leaf tissue) for chemical fertilizer compared to 1.03(±0.07), 0.71
(±0.04) and 1.74(±0.11)for control plant and 1.68 (±0.10), 1.13
(±0.07), and 2.81 (±0.17) for bio-organic NPK fertilizer. The results
found for bio-organic fertilizer can well compete with the amount of
chlorophyll found in the plant fertilized by chemical fertilizer. Sen et al.
(2016) reported that applying high nitrogen sources to the soil increases
S. Majee et al. Journal of Hazardous Materials 411 (2021) 125046

Fig. 7. Chlorophyll content (mg/g) in the leaf sample of experimented plants.

the amount of chlorophyll in plant leaves (Sen et al., 2016). The studied
okra plant showed a notable effect in chlorophyll content when grown in
soil treated with formulated bio-organic NPK fertilizer which might be
due to better supply of required nitrogen and other nutrients as
compared to normal soil in control (Attarde et al., 2012).
3.7. Seed germination test and vigor index of okra seed
Okra seed grown in soil untreated, treated with chemical fertilizer,
bio-organic NPK fertilizer showed percentage of germination about
86.6, 76.6, 83.3, and vigor index of 980.3 (±57.54), 696 (±26.40),
809.6 (±15.10) respectively. The results are presented in Table 3.
3.8. Ammonia detection study of aqueous soil extracts
Liberated free ammonia in aqueous soil extracts was measured and
found 1.65 (±0.06) ppm, 2.46 (±0.10) ppm, 2.62 (±0.14) ppm for un­
treated, chemical fertilizer and bio-organic NPK fertilizer treated soil
respectively. The soil fertilized by bio-organic NPK fertilizer had 37.02%
more free ammonia than normal soil and 6.10% more than chemically
fertilized soil. It seems organic nitrogen in formulated fertilizer has
increased the ammonia concentration to the soil by the ammonification
process. Higher nitrification due to high ammonia content may get
converted into (NO–2), and nitrate (NO–3) significantly by soil microor­
ganisms (van Kessel et al., 2015). It is reported that nitrate is the
plant-available form of nitrogen taken up by plants for growth and
development (Amoo and Babalola, 2017) which may have contributed
for better and higher growth of model plant in case of bio-organic NPK
manure than control in this study.
3.9. DNA intercalation study
The DNA intercalation study was performed by molecular fluores­
cence technique using the ethidium bromide (EtBr) probe as depicted in
Fig. 8. It revealed the changes in fluorescence emission spectra during
the interaction between ctDNA and toxic compounds of fertilizer in
sample (Yang et al., 2013). The spectra for chemical fertilizer showed a
significant reduction in emission intensity of the ctDNA–EtBr complex
without shifting the wavelength which indicated that the toxic com­
pounds may have competed with EtBr and intercalated with ctDNA
(Zhang et al., 2013). The intercalation of toxic elements such as sulfur,
zinc, lead, cadmium, chromium, arsenic, vanadium, molybdenum,
mercury,nickel etc. released from chemical fertilizer have possibly
caused the quenching of the fluorescence intensity of the ctDNA–EtBr
complex (Gimeno-García et al., 1996; Savci, 2012). The bio-organic NPK
fertilizer showed less quenching and fluorescence intensity similar to
that of natural soil. Thus it can be inferred that less toxic compound
released from bio-organic NPK fertilizer and it is less harmful to the soil
microbial communities like natural soil. From the EDS study it reveals
that, the elements present in the bio-organic fertilizer are nitrogen,
potassium, phosphorus, magnesium, chromium and silica. In chemical
fertilizer some other trace elements like cadmium (Cd), Lead (Pb)

Table 3
Percentage germination and vigor index.
Treatment % Mean value of seedling Vigor index
Germination length (cm) mean (±SE) mean (±SE)

Control 76.6 9.08 (±0.11) 696 (±26.40)

Bio-organic NPK 83.3 10.70 (±0.24) 809.6 (±15.10)
fertilizer
Chemical 86.6 11.30 (±0.38) 980.3 (±57.54)
fertilizer

*The vigor index experiment was carried out with three replicates and statistical
analysis was done at 0.05 level of significance. The ± values represent standard Fig. 8. Fluorescence emission spectra of EtBr − ctDNA with aqueous soil ex­
error of the mean. tracts without fertilizer and with fertilizer.

8
S. Majee et al.
mercury (Hg), nickel (Ni) may have present in negligible amount other
than NPK. From the literature review it is clear that silica can bind single
strand only and very little effect in double strand DNA. Chromium can
bind to the double strand of DNA and break the strands in reducing
environment. The presence of chromium could be responsible for
quenching in fluorescence intensity to some extent by soil treated
bio-organic fertilizers which is about 0.76 mg/kg of soil. However it is
within the limit of recommendation as the permissible limit of chro­
mium for plants is 1.30 mg/kg as recommended by WHO. However,
effects on quenching in fluorescence intensity by chemical fertilizer was
more due to presence of other trace elements as revealed from EDS
analysis. This clearly indicates that the use of bio-organic fertilizer will
not only be a better option to protect the soil microorganisms, but
helpful for nitrogen and other minerals uptake in the plant; and also
responsible for maintaining the good soil condition for better agricul­
tural productivity.
3.10. Chromium recovery
3.10.1. Chromium recovery from phosphoric acid treated WBL extract and
characterization
For the recovery of chromium from the acid precipitated material,
MgO treatment process was followed as stated in material and method
section. Fig. 9 demonstrates the percentage removal of chromium from
extracted fraction by MgO at different dose. The results showed that
17 g/L of MgO dose is optimum for chromium precipitation with
maximum i.e. 99.85% efficacy. It diminished the chromium concentra­
tion level in WBL from 107.64 ppm to 0.162 ppm, which is within the
permissible discharge limit (Altaf et al., 2008). The SEM image (Fig. 10)
revealed a few numbers of irregular shaped particulate material scat­
tered throughout the sample. Most probably these particles were syn­
thesized by the chromium precipitation process. Fig. 10 demonstrated
about 2.68% (weight percentage) of chromium precipitated from chro­
mium extracted solution using MgO. Further AAS analysis verified about
6322 mg/kg chromium content in the MgO precipitate part. The Cr
extraction was carried out at pH 3 but the pH was found 6.5 after pre­
cipitation with MgO. The percentage of other chemical elements as in
Fig. 10 by EDS studies were: oxygen (O) 50.91%, 6.10% magnesium
(Mg), 11.91% phosphorus (P), 10.11% nitrogen (N), 18.28% carbon (C).
3.10.2. Characterization of the transformed material (S2)
3.10.2.1. X-ray diffraction analysis (XRD) analysis of chromium fraction.
The transformed chromium laden material was characterized by X-ray
Fig. 9. Chromium recovery using MgO at different dose from acid extracted solution of WBL.
9
Journal of Hazardous Materials 411 (2021) 125046
diffraction technique with Cu Kα radiation (λ = 0.15406 nm) at 40 kV.
The XRD pattern of the material (Fig. 11) exhibited fine and intense
peaks characteristic of well-crystallized material. The EDS spectra
showed multiple reflections indicating a multiphase instead of a pure
compound. Chromium oxide (Cr2O3) commonly known as green cin­
nabar, chromium oxide (CrO3), phosphorus oxide (PO2), and magne­
sium (Mg) were identified as indicated in Fig. 11. Crystallite sizes of
multiple compounds were calculated from the XRD data using Sheerer
equation (Klug and Alexander, 1974) in X′ Pert High Score Plus soft­
ware. Green cinnabar (Cr2O3) exhibiting a rhombohedral structure with
an average crystal size of 24.3 nm, while chromium oxide (CrO3)
revealed an orthorhombic structure; the average crystal size was
35.3 nm. Likewise, there was a monoclinic structure for phosphorus
oxide (PO2) and the hexagonal structure of magnesium (Mg) with an
average crystal size of 26.2 nm and 24.1 nm respectively. Peak position,
reference pattern code, and other information related to the crystalline
material were summarized in Table 4.
3.10.2.2. Fourier transform infrared spectrometry analysis. The FTIR
spectra was shown in Fig. 12. The broad spectra around 3400 cm− 1
assigned to the OH− stretching of water molecules (Stefanescu et al.,
2011). The absorption band at 2380 cm− 1 corresponded to the carbon­
ate group that probably included from the atmospheric carbon dioxide
during the transformation of precipitate into crystalline material
(Anandhi et al., 2017). The wide band around 1645 cm− 1 might be
attributed to asymmetrical stretching of the chromium carboxylate of
Cr2O3 (Anandhi et al., 2017). FTIR spectra showed a small band vibra­
tion at 1160 cm− 1, which could be assigned to the P˭O stretching re­
flected from phosphorus oxide of the transformed material (S2) (Trivedi
et al., 2015). The peak at 900 cm− 1 in the material could be attributed to
chromyl (Cr˭O) vibration of CrO3 and/or Cr2O3 also reported by Trivedi
et al. (2015) (Weckhuysen et al., 1996; Trivedi et al., 2015). The spectra
at 560 cm− 1 recognized to Cr–O stretching that was clear evidence for
the presence of metal oxygen bond of Cr2O3 as per the studies by Hen­
derson (2010).
4. Cost estimation of bio-organic NPK fertilizer production
Cost estimation of the bio-organic NPK fertilizer prepared from solid
waste materials is quite important to assess the application potential of
the same towards commercial exploitation. The total expenditure for
bio-organic NPK fertilizer production has been calculated considering
several associated factors such as cost of waste materials, chromium
extraction process, chemicals, electricity charges, overhead charges etc.
S. Majee et al. Journal of Hazardous Materials 411 (2021) 125046

Fig. 10. Chromium salts extracted from WBL.

Fig. 11. XRD analysis of chromium salts extracted from WBL.

Table 4
X-ray diffraction analysis of extracted chromium salts.
Sl Compound name Chemical Crystal Average crystallite size Peak position (2θ degree) with corresponding Reference pattern
no. formula structure (nm) planes (hkl) code

1. Chromium Oxide (Green Cr2O3 Rhombohedral 24.3 41.5392 (113) 00–038–1479

cinnabar) 44.6548 (202)
54.9609 (116)
2. Chromium Oxide CrO3 Orthorhombic 35.3 26.1447 (111) 00–032–0285
52.4159 (311)
66.3145 (251)
3. Phosphorus Oxide PO2 Monoclinic 26.2 17.0613 (− 111) 00–050–1494
19.3411 (111)
29.7260 (221)
35.2232 (− 222)

About 286 g wet blue leather and 714 g potato peel biochar have been
CWM = (CWBL + CPP ) = $(0 + 0) = $0
used for making one kg of bio-organic NPK fertilizer and the total
expenditure of various measures for fertilizer preparation process was
calculated in USD ($) as follows: I. Cost of H3PO4 for chromium extraction of 286 g WBL (CCE) = $
0.185
I. Cost of waste materials (CWM): II. Cost for biochar preparation of dry potato peel (CBC) = CB = cost
of burning (heat) = hours × consumption of unit × per unit cost
• WBL is an industrial waste and is widely available in leather com­ = 1 × 2 × 0.065 = $ 0.13
panies. III. Cost for the preparation of distilled water (CDW):
Thus, cost of WBL waste (CWBL) = $ 0 To prepare 1 L of DW = (electric consumed by the distillation
• Potato peel is an easily available domestic waste. column in unit × cost for per unit) = 0.5 × 0.065 = $ 0.033
IV. Cost for crushing of prepared fertilizer (CFC) = (hours × electric
Therefore, cost of potato peel (CPP) = $ 0 consumption in unit × cost for per unit)
= 0.167 × 0.15 × 0.065 = $ 0.0016

10
S. Majee et al.
Fig. 12. FTIR Spectrum of recovered granular chromium salt from acid
extracted WBL by MgO treatment.
Therefore, the total expenditure for bio-organic NPK fertilizer pro­
duction = (CWM + CCE + CBC + CDW + CFC) = $ 0.35
Overheadcosts = 10%oftotalexpenditure = (0.1 × 0.35) = $0.035
Netcostforproductionofthefertilizer = $(0.35 + 0.035) = $0.38
The cost estimation suggests that the production of this fertilizer is a
low-cost process. Net expenditure for production of bio-organic NPK
fertilizer is only $ 0.38 (In INR 28.21) per kg, The cost of reported bio-
organic fertilizer of Rs. 28.31/kg is acceptable as compared to the fer­
tilizers available in the Indian market, cost varies from Rs.60 [$0.75] to
Rs. 360/kg [$4.5]. Moreover, it is much cheaper if compared with the
best grade because due to the slow release of nutrient for longer period
of time can keep the soil healthy and fertile. So the grade of produced
bio-organic fertilizer can be considered equivalent to higher grade one
with Rs 350/kg [$4.5]. In addition, this is also showing no negative
impact on natural soil microbial population.
5. Conclusion
In this study SEM and EDS characterized the formulated bio-organic
NPK fertilizer made from organic component such as collagenous ma­
terial and potato peel biochar. It was found rich in nitrogen (N), phos­
phorus (P), potassium (K) nutrients and it showed a considerable growth
enhancement on okra plant. The efficacy of bio-organic NPK fertilizer
was evaluated to be close to the chemical fertilizer as both the fertilizers
were able to supply adequate amount of nutrients effecting improved
growth, nearby 12–14% increase in fruit size, and 62–68% extra chlo­
rophyll in the leaves. The nutrients release of the chemical fertilizer was
more rapid with time and caused faster plant growth at initial stage,
whereas bio-organic NPK fertilizer was controlled, that enhanced the
plant growth uniformly. Free ammonia content in organically fertilized
soil was more than 37.02% of natural soil and 6.10% of the chemically
fertilized soil. DNA intercalation study revealed that the bio-organic
NPK fertilizer had a less harmful effect on ctDNA, suggesting that it
can be utilized as an eco-friendly bio-organic NPK fertilizer as an
alternate substitute for chemical fertilizer. This conversion of WBL into
valuable bio-organic NPK formulation would reduce the solid waste
management problem of leather industry. Moreover, the harmful chro­
mium component of wet blue leather waste was also successfully
recovered into two different forms of chromium oxide that are Cr2O3
(green cinnabar) and CrO3. This work converts hazardous waste mate­
rials to an environmentally benign product, and also emphasizes on
recovery strategy to protect the environment from unwanted chromium
11
Journal of Hazardous Materials 411 (2021) 125046
pollution. The cost analysis revealed that the price of prepared bio-
organic NPK fertilizer is significantly lower than the commercially
available chemical fertilizer.
CRediT authorship contribution statement
Subhasish Majee: Contributed in data generation, Set up experi­
ments, Data analysis, Drafting the manuscript and final revision. Gopi­
nath Halder: Contributed in interpretation, Supervised, Editing,
Revision. Dalia Dasgupta Mandal: Contributed in toxicological study
interpretation, Editing and Revising the article. O.N. Tiwari: Helped in
analytical part and Critical revision of the article, Designed part of
experimental work. Tamal Mandal: Conceptualized and designed the
work. Helped in analysis and Interpretation, Supervised, Critical revi­
sion of the article.
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.
Acknowledgment
Authors would like to acknowledge Department of Science and
Technology, Government of India, project number DST/TM/WTI/WIC/
2K17/84(G) and SR/FST/ET1- 405/2015(C)/NITDURGAPUR for the
financial assistance and necessary instrumental facilities. Dr. Tiwari
conveys his gratitude to HoD of Division of Microbiology, ICAR-IARI,
New Delhi for her constant encouragement and help.
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