TMP 8521

Proceedings of the International Academy of
Ecology and Environmental Sciences

Vol. 4, No. 1, 1 March 2014
Qi Zhang/July-August 2013
International Academy of Ecology and Environmental Sciences

Proceedings of the International Academy of Ecology and
Environmental Sciences
ISSN 2220-8860
Volume 4, Number 1, 1 March 2014
Editor-in-Chief
WenJun Zhang
Sun Yat-sen University, China
International Academy of Ecology and Environmental Sciences, Hong Kong
E-mail: zhwj@mail.sysu.edu.cn, wjzhang@iaees.org
Editorial Board
Taicheng An (Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, China)
Jayanath Ananda (La Trobe University, Australia)
Ronaldo Angelini (The Federal University of Rio Grande do Norte, Brazil)
Nabin Baral (Virginia Polytechnic Institute and State University, USA)
Andre Bianconi (Sao Paulo State University (Unesp), Brazil)
Iris Bohnet (CSIRO, James Cook University, Australia)
Goutam Chandra (Burdwan University, India)
Daniela Cianelli (University of Naples Parthenope, Italy)
Alessandro Ferrarini (University of Parma, Italy)
Marcello Iriti (Milan State University, Italy)
Vladimir Krivtsov (Heriot-Watt University, UK)
Suyash Kumar (Govt. PG Science College, India)
Frank Lemckert (Industry and Investment NSW, Australia)
Bryan F. J. Manly (Western EcoSystems Technology Inc. and University of Wyoming, USA)
T.N. Manohara (Rain Forest Research Institute, India)
Ioannis M. Meliadis (Forest Research Institute, Greece)
Lev V. Nedorezov (University of Nova Gorica, Slovenia)
George P. Petropoulos (Institute of Applied and Computational Mathematics, Greece)
Edoardo Puglisi (Università Cattolica del Sacro Cuore, Italy)
Zeyuan Qiu (New Jersey Institute of Technology, USA)
Mohammad Hossein Sayadi Anari (University of Birjand, Iran)
Mohammed Rafi G. Sayyed (Poona College, India)
R.N. Tiwari (Govt. P.G.Science College, India)
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Proceedings of the International Academy of Ecology and Environmental Sciences, 2014, 4(1): 1-6
Article
Microorganism as a tool of bioremediation technology for cleaning

environment: A review
Ravindra Singh1, Pushpendra Singh1, Rajesh Sharma2

1
Department of Biological Sciences, M.G.C.G. University Chitrakoot Satna, M.P., India
2
Department of Biotechnology, V.B.S. Purvanchal University Jaunpur, U.P., India
E-mail: drrsinghmgcgv@rediffmail.com,rsinghmgcgv@gmail.com
Received 2 May 2013; Accepted 6 June 2013; Published online 1 March 2014
Abstract
The term bioremediation has been introduced to describe the process of using biological agents to
remove toxic waste from environment. Bioremediation is the most effective management tool to manage the
polluted environment and recover contaminated soil. The hazardous wastes generated from the chemical
processes/operations are being treated using physico-chemical and biological methods by the respective
industries to meet the prescribed standard as per the Environmental Protection Act, 1986. The wastes treated
by the respective industries are collected at Common Effluent Treatment Plant, before discharge into the
environment. After the treatment of collected waste at Common Effluent Treatment Plant, the solid and treated
effluents are segregated and disposed of into the soil- water environment. In spite of the present treatment
technology, the organic pollutants are found persisting in the soil-water environment above their acceptable
level. Hence, bioremediation is an innovative technology that has the potential to alleviate the toxic
contamination.
Keywords hazardous waste; bioremediation; microorganism; bioreactor.
Proceedings of the International Academy of Ecology and Environmental Sciences
ISSN 22208860
URL: http://www.iaees.org/publications/journals/piaees/onlineversion.asp
RSS: http://www.iaees.org/publications/journals/piaees/rss.xml
Email: piaees@iaees.org
EditorinChief: WenJun Zhang
1 Introduction
Today, biotechnology is being considered as emerging science for environmental protection. The technology
involves the use of microorganisms for biological treatment of air, water and soil pollutants. Biotechnological
treatment is carried out at lower temperature and pressure which requires less energy than the conventional
physico-chemical treatment technology. The industries generating hazardous wastes have found beneficial
measures from the emerging trend of biotechnological treatment. Biotechnological innovations for treatment
for hazardous waste under controlled environmental conditions have been found cost–effective means of
reducing the pollution potential of waste water, leading to enhanced public acceptance and compliance with
environmental legislation (Fulekar, 2010). Environmental pollution such as contaminated soil or surface /
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2 Proceedings of the International Academy of Ecology and Environmental Sciences, 2014, 4(1): 1-6
ground water can be solved by bioremediation and / or phytoremediation by use of biological living organisms
and green plants.
Bioremediation uses biological agents, mainly microorganisms i.e. yeast, fungi or bacteria to clean up
contaminated soil and water (Strong and Burgess, 2008). This technology relies on promoting the growth of
specific microflora or microbial consortia that are indigenous to the contaminated sites that are able to
perform desired activities (Agarwal, 1998). Establishment of such microbial consortia can be done in several
ways e.g. by promoting growth through addition of nutrients, by adding terminal electron acceptor or by
controlling moisture and temperature conditions (Hess et al., 1997; Agarwal, 1998; Smith et al., 1998). In
bioremediation processes, microorganisms use the contaminants as nutrient or energy sources (Hess et al.,
1997; Agarwal, 1998; Tang et al., 2007).
Bioremediation is defined as the process by which microorganisms are stimulated to rapidly degrade
hazardous organic pollutants to environmentally safe levels in soils, sediments, substances, materials and
ground water. Recently, biological remediation process have also been devised to either precipitate effectively
immobilize inorganic pollutants such as heavy metals. Stimulation of microorganisms is achieved by the
addition of growth substances, nutrients, terminal electron acceptor/donors or some combination thereby
resulting in an increase in organic pollutant degradation and bio-transformation. The energy and carbon are
obtained through the metabolism of organic compounds by the microbes involved in bioremediation processes
(Fulekar et al., 2009).
Bioremediation process involves biotransformation and biodegradation by transforming contaminants to
non–hazardous or less hazardous chemicals. Often, the micro-organisms metabolize the chemicals to produce
carbon dioxide or methane, water and biomass. Biotransformation is any alteration of the molecule or structure
of a compound by micro-organisms. Biodegradation is the breaking down of organic or bioaccumulation and
biotransformation of inorganic compounds into environmental friendly compounds.
2 Microbial Bioremediation
Micro-organisms are now known to be the principal agents, which can clean and modify the complex
lipophilic organic molecules, once considered recalcitrant, to simple water soluble products. They first attack
these organic chemicals by the enzymatic apparatus acquired during the course of enrichment, when they are
exposed to these specific or structurally related compounds. Presence of these contaminants in the environment
either induces or depresses the enzymatic function of microorganisms. This capability largely depends upon
the selective microbial community as well as on the structural and functional groups of toxic compounds.
These water soluble intermediates are usually attacked by primary or secondary groups of organisms to form
inorganic end products, resulting in complete biodegradation. Bioremediation is the use of living organisms,
primarily microorganisms, to degrade the environmental contaminants into less toxic forms. It uses naturally
occurring bacteria and fungi or plants to degrade or detoxify substances hazardous to human health and/or the
environment. The micro-organisms may be indigenous to a contaminated area or they may be isolated from
elsewhere and brought to the contaminated site. Contaminated compounds are transferred by living organisms
through reactions that take place as a part of their metabolic processes. Biodegradation of a compound is often
a result of the actions of multiple organisms. When microorganisms are imported to a contaminated site to
enhance degradation, the process is called as “Bio-augmentation”. The microorganisms with the genetic
capacity to transform compounds of interest must be present in contaminant metabolism to occur in a
bioremediation process. In certain cases, the addition of organisms acclimated to specific contaminants, or bio-
augmentation, may decrease the duration of lag phases. The ability to effectively bio-augment bioremediation
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Proceedings of the International Academy of Ecology and Environmental Sciences, 2014, 4(1): 1-6 3
system is a function of the process used. Bioremediation is an option that offers the possibility to destroy or
render harmless various contaminants using natural biological activity (Gupta, 2003).
3 Bioremediation Organisms
Microorganisms that carry out biodegradation in many different environments are identified as active members
of microbial consortiums. These microorganisms include: Acinethobacter, Actinobacter, Acaligenes,
Arthrobacter, Bacillins, Berijerinckia, Flavobacterium, Methylosinus, Mycrobacterium, Mycococcus,
Nitrosomonas, Nocardia, Penicillium, Phanerochaete, Pseudomonas, Rhizoctomia, Serratio, Trametes and
Xanthofacter.
Microorganisms individually cannot mineralize most hazardous compounds. Complete mineralization
results in a sequential degradation by a consortium of microorganisms and involves synergism and co
metabolism actions. Natural communities of microorganisms in various habitats have an amazing
physiological versatility, they are able to metabolize and often mineralize an enormous number of organic
molecules. Certain communities of bacteria and fungi metabolize a multitude molecules that can be degraded
is not known but thousands are known to be destroyed as a result of microbial activity in one environment or
another. Most bioremediation systems are run under aerobic conditions, but running a system under anaerobic
conditions (Colberg and Young, 1995) may permit microbial organisms to degrade otherwise recalcitrant
molecules.
4 Bioremediation Research Studies Using Designed and Developed Laboratory Bioreactors

4.1 Bioremediation of pesticide in surface soil treatment unit using microbial consortia
The manufacturing and use of pesticides has been rising tremendously in India. The waste generated by the
pesticide industry has become an environmental problem due to the present insufficient and ineffective waste
treatment technology involving physico-chemical and biological treatment. The available data indicates that
pesticide residues remain in surface soil, leading to toxicity in the soil-water environment. The recent advances
in bioremediation technology using microbial consortium has been found effective for treatment of pesticides
in soil. In the present study, a Surface Soil Treatment Unit has been designed wherein bioremediation of
commonly used pesticides namely chlorpyrifos, cypermethrin, fenvalerate, and trichlopyr butoxyethyl ester at
varying concentration viz. 25, 50 and 100 mg/kg have been carried out using cow-dung microbial consortia
under simulated environmental conditions. The bioremediation conditions have been monitored and
maintained during the study. The investigation has been extended till the parent compound was converted into
intermediates and/or less harmful compounds. These then will further mineralize, from part of the microbial
food chain and/or become integrated into the humic fractions. The results presented here highlight the potential
of cow-dung slurry consortia for bioremediation of soil contaminated with pesticides in surface soil treatment
unit (Geetha and Fulekar, 2008).
4.2 Bioremediation of pesticides using scale up process bioreactors
To assess the bioremediation potential of Pseudomonas aeruginosa (NCIM, 2074) by improving its
adaptability to increasing concentration of chlorpyrifos using scale up process. Pseudomonas aeruginosa
isolate NCIM 2074 was adapted by subjecting to varying concentrations of chlorpyrifos, i.e. 10, 20, 50, 75 and
100 mg/l in incubator shaker at 37°C and 150 rpm. An initial 10 mg/l concentration of chlorpyrifos was
supplied in minimal salt medium (MSM) under controlled environmental conditions for 14 days. The culture
was subsequently scaled up to higher concentrations of chlorpyrifos by transferring one milliliter from the
medium with 10mg/L to 25 mg/l of the compound. After every 14 days this process was repeated, each time
using medium with higher chlorpyrifos concentration. The entire scale up process continued for a period of 70
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days. Pseudomonas aeruginosa (NCIM 2074) was adapted to increasing chlorpyrifos up to 50 mg/l, but 75 and
100 mg/l was inhibitory to the organism. The biodegradation of chlorpyrifos, as assessed by GC-MS, showed
that chlorpyrifos at 10, 25, 50 mg/l degraded completely over a period of 1, 5 and 7 days, respectively. The
intermediate 3, 5, 6 trichloro-2-pyridion, 2, 4-bis (1, 1 dimethyiethyl) phenol and 1, 2 zenedicarboxylic acid
persisted during bioremediation, but in the long run these convert to CO2, biomass and nutrients.
Pseudomonas aeruginosa (NCIM 2074) has been of potential use in bioremediation of chlorpyrifos at
concentrations up to 50 mg/l, but the organism is inhibited by higher concentrations (Fulekar and Geetha,
2008).
4.3 Bioremediation of benzene using a designed and developed partitioning bioreactor
A bioreactor has been designed and developed for partitioning of aqueous and organic phases with a provision
for aeration and stirring, a cooling system and a sampling port. The potential of a cow dung microbial
consortium has been assessed for bioremediation of phenol in a single-phase bioreactor and a two-phase
partitioning bioreactor. The advantages of the two-phase partitioning bioreactor are discussed. The
Pseudomonas putida IFO 14671 has been isolated, cultured and identified from the cow dung microbial
consortium as a high-potential phenol degrader. The methods developed in this study present an advance in
bioremediation techniques for the biodegradation of organic compound such as phenol using a bioreactor. We
have also demonstrated the potential of microorganisms from cow dung as a source of biomass (Singh and
Fulekar, 2009).
4.4 Bioremediation of benzene using cow dung microflora in two phase partitioning bioreactor
Bioremediation of benzene has been carried out using cow dung microflora in a bioreactor. The bioremediation
of benzene under the influence of cow dung microflora was found to be 100% and 67.5%, at initial
concentrations of 100 mg/l and 250 mg/l within 72 h and 168 h respectively; whereas at higher concentration
(500 mg/l), benzene was found to be inhibitory. Hence the two phase partitioning bioreactor (TPPB) has been
designed and developed to carryout biodegradation at higher concentration. In TPPB the contaminant found to
be biodegraded at 5000 mg/l concentration up to 50.17% over a period Q1 of 168 h. Further the Pseudomonas
putida MHF 7109 was isolated from cow dung microflora as potential benzene degrader and its ability to
degrade benzene at various concentrations was evaluated. The data indicates 100%, 81% and 65% degradation
at the concentrations of 50 mg/l, 100 mg/l, 250 mg/l within the time period of 24 h, 96 h and 168 h
respectively. The GC-MS data also shows the presence of catechol and 2-hydroxymuconic semialdehyde,
which confirms the established pathway of benzene biodegradation. The present research proves the potential
of cow dung microflora as a source of biomass for benzene biodegradation in TPPB (Singh and Fulekar, 2009).
4.5 Bioremediation of pesticide chlorpyrifos in mycorrhizosphere ecological remediation Unit using
ryegrass
The potential of ryegrass for rhizosphere bioremediation of chlorpyrifos in mycorrhizal soil was investigated
by the green house pot culture experiments. The pot cultured soil amended at initial chlorpyrifos concentration
of 10 mg/kg was observed to be degraded completely within 7 days where the rest amended concentrations
(25–100 mg/kg) decreased rapidly under the influence of ryegrass mycorrhizosphere as the incubation
progressed till 28 days. This bioremediation of chlorpyrifos in soil is attributed to the microorganisms
associated with the roots in the ryegrass rhizosphere, therefore the microorganisms surviving in the
rhizospheric soil spiked at highest concentration (100 mg/kg) was assessed and used for isolation of
chlorpyrifos degrading microorganisms. The potential degrader identified by 16S rDNA analysis using
BLAST technique was Pseudomonas nitroreducens PS-2. Further, bio-augmentation for the enhanced
chlorpyrifos biodegradation was performed using PS-2 as an inoculum in the experimental set up similar to the
earlier. The heterotrophic bacteria and fungi were also enumerated from the inoculated and non-inoculated
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rhizospheric soils. In bio-augmentation experiments, the percentage dissipation of chlorpyrifos was 100% in
the inoculated rhizospheric soil as compared to 76.24, 90.36 and 90.80% in the non-inoculated soil for initial
concentrations of 25, 50 and 100 mg/kg at the 14th, 21st and 28th day intervals respectively (Korade and
Fulekar, 2009).
4.6 Biodegradation of petroleum hydrocarbon compounds toluene and o-xylene (BTX) by Pseudomonas
putida strain MHF 7109
Pseudomonas putida MHF 7109 has been isolated and identified from cow dung microbial consortium for
biodegradation of selected petroleum hydrocarbon compounds – benzene, toluene, and o-xylene (BTX). Each
compound was applied separately at concentrations of 50, 100, 250, and 500mgL-1 in minimal salt medium to
evaluate degradation activity of the identified microbial strain. The results indicated that the strain used has
high potential to degrade BTX at a concentration of 50mgL-1 within a period of 48, 96, and 168 h,
respectively; whereas the concentration of 100mgL-1 of benzene and toluene was found to be completely
degraded within 120 and 168 h, respectively. Sixty-two percent of o-xylene was degraded within 168 h at the
100mgL-1 concentration level. The maximum degradation rates for BTX were 1.35, 1.04, and 0.51mgL-1 h-1,
respectively. At higher concentrations (250 and 500mgL-1) BTX inhibited the activity of microorganisms. The
mass spectrometry analysis identified the intermediates as catechol, 2-hydroxymuconic semialdehyde, 3-
methylcatechol, cis-2- hydroxypenta-2,4-dienoate, 2-methylbenzyl alcohol, and 1,2-dihydroxy-6-
methylcyclohexa- 3,5-dienecarboxylate, for BTX, respectively. P. putida MHF 7109 has been found to have
high potential for biodegradation of volatile petroleum hydrocarbons (Singh and Fulekar, 2010).
5 Genetic Engineering
Scientists are currently looking into certain genetically engineered microorganisms to increase their ability to
metabolize specific chemicals such as hydrocarbons and pesticides. The possibilities of using genetic
engineering for improvement of bioremediation process had an early boost in the late 1980’s. Recombinant
DNA techniques have been studied intensively to improve the degradation of hazardous waste under
laboratory condition. The genetically engineered microorganisms have higher degradative capacity and have
been demonstrated successfully for the degradation of various pollutants under defined conditions. Genetic
modification technology has resulted often in a wide variety of current and potential applications for use in the
process of bioremediation. Bioremediation explores gene diversity and metabolic versatility of
microorganisms (Fulekar, 2009). The genetic architecture of these organisms makes them valuable in
biodegradation, biotransformation, biosorption and bioaccumulation. The necessary blue print of gene
encoding for biodegradative enzymes is present in chromosomal and extra-chromosomal DNA of such
microbes. Recombinant DNA techniques facilitate to evolve the ability of an organism to metabolize a
xenobiotic by detection of such degradative genes and transforming them into appropriate host via suitable
vector under the tight control of appropriate promoters. It depends on susceptibility to alteration and exchange
of genetic information. The recombinant DNA technology explores PCR, anti-sense RNA technique, site
directed mutagenesis, electroporation and particle bombardment techniques. The biotechnology armed with
recombinant DNA technology is now fine tuning the bioremediation technology by improving pollutant–
degrading microbes through strain improvement and genetic modification of specific regulatory and metabolic
genes that are crucial in developing effective, safe and economical techniques for bioremediation.
Bioremediation is not effective only for the degradation of pollutants but it can also be used to clean unwanted
substances from air, soil, water and raw materials form industrial waste. Bioremediation is not effective only
for the degradation of pollutants but it can also be used to clean unwanted substances from air, soil, water and
raw materials form industrial waste.
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References
Agarwal SK. 1998. Environmental Biotechnology (1st ed). 267-289, APH Publishing Corporation, New Delhi,
India
Colberg PJS, Young LY. 1995. Anaerobic degradation of no halogenated homocyclic aromatic compounds
coupled with nitrate, iron, or sulfate reduction. In: Microbial Transformation and Degradation of
Toxic Organic Chemicals. 307–330, Wiley-Liss, New York, USA
Fulekar MH. 2009. Bioremediation of fenvalerate by Pseudomonas aeruginosa in a scale up bioreactor.
Romanian Biotechnological Letters, 14(6): 4900-4905
Fulekar MH. 2010. Bioremediation Technology: Recent Advances. Springer, Netherlands
Fulekar MH. 2010. Environmental Biotechnology. CRC Press and Science Publisher, USA
Fulekar MH. 2010. Nanotechnology- its Importance & Applications. IK International, India
Fulekar MH, Geetha M. 2008. Bioremediation of chlorpyrifos by Pseudomonas aeruginosa using scale up
technique. Journal of Applied Biosciences, 12: 657-660
Fulekar MH, Geetha M, Sharma J. 2009. Bioremediation of Trichlorpyr Butoxyethyl Ester (TBEE) in
bioreactor using adapted Pseudomonas aeruginosa in scale up process technique. Biology and Medicine, 1
(3): 1-6
Geetha M, Fulekar MH. 2008. Bioremediation of pesticides in surface soil treatment unit using microbial
consortia. African Journal of Environmental Science & Technology, 2(2): 36-45
Gupta R, Mahapatra H. 2003. Microbial biomass: An economical alternative for removal of heavy metals
from waste water. Indian Journal of Experimental Biology, 41: 945-966
Hess A, Zarda B, Hahn D, et al. 1997. In situ analysis of denitrifying toluene and m-xylene degrading
bacteria in a diesel fuel contaminated laboratory aquifer column. Applied and Environmental Microbiology,
63: 2136-2141
Korade D, Fulekar MH. 2009. Rhizosphere remediation of chlorpyrifos in mycorhizospheric soil using
ryegrass. Journal of Hazardous Material, 172: 1344-1350
Sharma J, Fulekar MH. 2009. Potential of Citrobacter freundii for bioaccumulation of heavy metal – copper.
Biology and Medicine, 1(3): 7-14
Singh D, Fulekar MH. 2009. Benzene bioremediation using cow dung microflora in two phase partitioning
bioreactor. Journal of Hazardous Material, 175: 336-343
Singh D, Fulekar MH. 2010. Biodegradation of petroleum hydrocarbons by Pseudomonas putida strain MHF
7109 isolated from cow dung microbial consortium. Clean Soil, Air, Water, 38(8): 781-786
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1-6

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Article
Are the sensitive zones degrading? A modelling approach using GIS

and remote sensing
S. Selvalakshmi1, S. Jayakumar2, V.S. Ramachandran1

1
Department of Botany, Bharathiar University, Coimbatore- 641 046, India
2
Department of Ecology and Environmental Sciences, Pondicherry University, Puducherry-605 014, India
E-mail: selvaphd09@gmail.com
Received 14 November 2013; Accepted 20 December 2013; Published online 1 March 2014
Abstract
This present study assesses the sensitive zones and the forest density class prone to degradation using remote
sensing and Geographical Information System (GIS) techniques in the Mudumalai Tiger Reserve (MTR),
Southern Western Ghats (India). For assessing the vulnerability of degradation on different vegetation density,
the drivers responsible for degradation were considered. LANDSAT MSS and IRS-LISS III satellite image
was used to classify the vegetation density by applying Normalised Difference Vegetation Index (NDVI)
technique and to create the sensitive zone maps for two different time periods, 1973 and 2010 using weighted
overlay analysis. About 47% of the present forest area is under a low risk category, 24% is under medium risk
category and about 7% (2517 ha) is under the high-risk category in 2010. The natural disturbances such as
forest fire, wildlife grazing, and expansion of agricultural land induced by anthropogenic pressure over the
decades are the reasons of forest cover change in Mudumalai. The area under no-risk zone has severely
decreased, and medium and high risk zone has drastically increased when compared to 1973 where high
prioritization for conservation planning is ideal.
Keywords weighted overlay analysis; vegetation density; risk zone; anthropogenic pressure; Western Ghats.
ISSN 22208860
1 Introduction
The forest resource is an important bearing on the environmental and ecological security for the country and its
people (Zhang, 2007). Ecologically sensitive areas are identified with a flexible system of management and
embedded with protected areas with an adaptive regime of regulation. The sensitive areas are aimed to attain
more comprehensive than focusing merely on biodiversity richness or ecological fragility (Gadgil, 2011). The
deforestation and degradation of forests arose due to the expansion of hill slope and valley agriculture, fire,
clear-felling, grazing and encroachment, which result in constant stress and exploitation to the forest (Zhang et
al., 2006; Zhang, 2012). Due to these heavy pressures, the forest cover of tribal and hill districts in India has
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lost 548 km2 and 679 km2 respectively in 2011 (FSI, 2011). Formal protection of natural resources in reserves
has tended to be ad hoc favoring the biodiversity of areas that are least valuable for commercial use, in public
tenure but earnest to reserve, most charismatic and with the least need for protection (Margules, 2002) which is
quite important for the protection strategy.
The gross forest cover loss was accounted to be 0.6 % per year during 2000 to 2005 at global level (Hansen
et al., 2010). Forest Survey of India assessed the country’s forest cover as 692,027 km2 (FSI, 2011), which
represents 21% of the total geographical area, whereas Tamil Nadu covers 28,306 km2. The total forest cover
of Nilgiris was 56.3 % in 2009 (State Environment Report of Tamil Nadu, 2009). According to Myers et al.,
(2000), forest cover in India declining at the rate of 2.6 %. However, the rate of deforestation is highly debated.
Few works has been done only in the selected areas of India which are under-going deforestation and
degradation in India (Jha et al., 2000; Giriraj et al., 2008; Joseph et al., 2009; Reddy et al., 2009; Reddy et al.,
2010), but did not specifically focus on sensitive zone mapping and the drivers influencing degradation using
models. Modelling the sensitive zones is important for understanding forest cover dynamics in the reserve
forest. These models can provide a better understanding of the factors that drive forest changes and generate
future forest cover scenarios to support the design of policy responses (Achard, 2002). Therefore, a thorough
research on ecologically sensitive regions would help us formulate better management strategies at the micro
level by understanding the causative factors for the formation, use and maintenance of forests and to undertake
effective management plan, which is known for its role in environmental protection and prevention of damage.
To monitor the forest cover and the degradation pattern, remote-sensing offers consistent observation at a finer
scale with more precision and in a cost-effective manner (Tucker, 2000) and it is considered to be an essential
data source for the appraisal of natural environments as it provides valuable information for interpreting the
landscapes. The study is aimed to produce a sensitive zone maps at different years in order to detect the
changes that have taken place particularly in the settlements nearby to the forest and subsequently to analyse
the trends in degradation pattern occur due to the expansion of agricultural lands over a period of 1973 and
2010.
2 Materials and Methods

2.1 Study area
The study focuses on a segment of Southern Western Ghats, Mudumalai Tiger Reserve (Mudu-Old; Malai- hill
- ancient mountain), which is one of the most popular and the oldest wildlife sanctuaries in India. The
sanctuary lies between 11° 32’ - 11° 43’N latitudes and 76° 22’-76° 45’ E longitudes and covers an area of 321
km2 (Fig. 1). It lies in the tri-junction of the three southern states of India viz., Tamil Nadu, Kerala and
Karnataka. There is a distinct rainfall gradient from east to west varying from 500 mm to 2000 mm.
Mudumalai Tiger Reserve (MTR), primarily dominated by deciduous forests along with tropical semi-
evergreen, moist deciduous, dry deciduous and dry thorn forests (Champion and Seth, 1968). Mudumalai is the
most important for its socio-cultural because most of the tribal settlements and pilgrim sites are located inside
the reserve forest, which results in one of the threatened habitats due to continuous human disturbances.
Moreover, large tracks of forest are degraded and converted into agricultural lands. The objective of this study
is to characterize sensitive zones, its rate of deforestation using remotely sensed data and to check whether the
degradation occurs in the sensitive areas of MTR, Southern Western Ghats, India.
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Fig. 1 Geographical location map of the study area in India.
Satellite images of LANDSAT MSS (1973) and IRS P6 LISS III (2010) are geometrically corrected using
topographic maps in ERDAS Imagine 11.0 software. False colour composite image of the study area was
generated from LANDSAT MSS and IRS LISS III with appropriate band combinations. The common
uniformly distributed Ground Control Points (GCPs) were marked with root mean square error less than a
pixel. For analyzing the degradation prone areas, several thematic layers were created for weighted overlay
analysis. The conversion of forest often relates to physical accessibility variables. Accessibility to a road is a
significant factor of deforestation. The role of road access was highlighted in predicting the location of
deforestation in many areas, such as the Basho Valley, Northern Pakistan (Ali et al., 2005), Northern Thailand
(Cropper et al., 2001) and the Congo Basin (Wilkie et al., 2000). The location of water affects to the location
of cultivation; therefore, the proximity to water is closely related to deforestation. Permanent cultivation in the
area seemed to be concentrated close to water. Bawa et al., (1997) found that deforestation was strongly
correlated with the extension of cropland area in Asia and Latin America. Deforestation occurs due to
important drivers like proximity to road, town and forest/non-forest in southern Cameroon (Merten and
Lambin, 1997). Elevation, slope, proximity to road, settlement and proximity to forest/non-forest edge were
the key factors of forest change in southeast Mexico (Mas et al., 2004). Topography often influences the
spread and extent of forest conversion. For example, a case study in Costa Rica (Sader, 1988) found that as the
slope gradient increased, deforestation decreased. The forest loss and degradation are associated directly with
proximity to roads and villages upto 6 kms (Menon, 2001) that confirm the disturbing effect of roads by
logging, mining, grazing, agriculture and urban development. By applying this technique, six priority
themes have been selected by considering slope, settlements, vegetation, road, water body and pilgrim sites.
Two kilometer buffer maps were created and the areas of highest priority are ranked based on its usage and
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importance. Survey of India toposheets were used to digitize thematic layers such as settlements, road
networks, pilgrim sites and rivers using ArcGIS 10 software. A Digital Elevation Model of Mudumalai was
generated by using Shuttle Radar Topography Mission (SRTM) data set and used for the estimation of slope
factor and classified according to National Natural Resource Management System (NNRMS) slope
classification. To assess the vegetation conditions, Normalised Difference Vegetation Index (NDVI) approach
is used by applying following formula and classified as per Forest Survey of India density classification.
NDVI =NIR-R /NIR+R
where NIR = near infrared; R = visible red.
Fig. 2 a. Buffer zone of pilgrim sites; b. Buffer zone of settlements; c. Buffer zone of Roads; d. Buffer zone of major rivers.
Weighted overlay model with spatial analysis shows promise in its ability to integrate using different
drivers on a complex problem to identify the degradation prone areas. It is a technique for applying a common
measurement scale of values to diverse and dissimilar inputs to create an integrated analysis. The weighted
overlay analysis helps in designing the conservation initiatives which provide in assigning the priority to
macro-level drivers and weightage to each class, which has higher chances of causing deforestation in
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Mudumalai. Though many input layers are used to create a single output layer, it is well known that all the
input layers are not equally significant (Jayakumar et al., 2002). Based on this concept, more important layer
has given high priority than the others. This is the advantage of weighted overlay approach, where one can
assign weightage to each class and layers separately. Knowledge based weight assignment was carried out for
each thematic layer, and they were integrated and analyzed using ERDAS imagine11.0. Weighted overlay only
accepts integer rasters as input so continuous rasters have been reclassified as integer before they can be used.
L=PI*Wt
where L is the layer, Wt is the weightage of each class, and PI is the percentage of importance.
Fig. 3 Conceptual diagram illustrating the preparation of sensitive zones.
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The anthropogenic pressures are likely to be high near the hamlets, road network (metalled, unmetalled)
and pilgrim sites where the festival takes place every year; thereby people might create secondary and tertiary
routes within the forest leads to increasing accessibility and vulnerability of the forest stand in the proximity.
Spatial data of the six factors (road, river, settlements, slope, pilgrim site and forest density) were prepared as a
set of GIS layers and overlaid together for final sensitivity classification for degradation. Two kilometer buffer
for the road network, settlements, pilgrim site and rivers is generated, and high weightage was given to the
zone nearer to the forest cover, accordingly to derive the sensitive zone (Fig. 2).
Each criterion and factor received a weight and a score which represented its relative importance in the
zonation of sensitive areas, which are highly prone to degradation. Further analysis was done in raster-based
format and was then reclassified into five classes using the ‘reclassify’ function. The topographical feature that
mostly influences the deforestation process is the degree of slope, which was generated from SRTM satellite
image. High slope areas is less used by the people, accordingly the weightage was given. Vegetation map of
two years (1973 and 2010) was generated using NDVI, which is used as an indicator of vegetation condition.
Forest density was calculated and classified into the five classes by assigning suitable weightage based on its
usage. The sensitive zone maps generated for different periods are compared with the degraded areas using
NDVI map of the latter period. The overall workflow is given in the Fig. 3.
Fig. 4 Methodology flow chart for the change detection analysis.
Using the sensitive zone maps and forest density class maps for 1973 and 2010, change in the area between
sensitive zones was calculated. Change in the area of forest density was calculated using NDVI of two time
periods. Sensitive zone map of 1973 was compared with the forest density class of 2010 to identify the major
distribution of sensitive zones between the forest density classes in 2010. Finally, a comparison was made
between the combined categories of the sensitive zone of 1973 and forest density classes of 2010 with forest
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cover density of 1973, to quantify the area under each sensitive zone of 1973 in combination with a density
class of 2010 belongs to the area of same density class in 1973 (Fig. 4).
3 Results
Comparison of vegetation density between 1973 and 2010 shows a significant decrease in the area of very
dense forest (2.4 km2) in 2010. Of the total area covered by natural vegetation (321 km2) the moderately dense
and very dense forests together occupied an area of 20.9% in 2010 whereas in 1973, it was 22.2%. Open forest
(deciduous forest), which is the major vegetation type of Mudumalai covered 19950 ha in 2010. The area
under open forest has shown a significant increase of about 1545 ha between 1973 and 2010. However, the
other forest cover classes showed a decreasing trend between 1973 and 2010 (Table 1).
Table 1 Change detection in the vegetation density between 1973 and 2010.
Vegetation Type Area in 1973 Area in 2010 Change in Area
(ha) (ha) (ha)
Non forest 361.08 21.13 -339.94
Scrub forest 6249.24 5414.27 -834.96
Open forest 18405.44 19950.62 1545.189
Moderately dense 4481.33 4289.26 -192.06
Forest
Very dense forest 2670.48 2442.60 -227.87
Negative values indicate decrease in area; Positive values indicate increase in area.
The results of weighted overlay analysis show that the different forest areas of Mudumalai which are prone
to degradation are at different risk levels. In 2010, about 47.36% of the forest area (15212 ha) was under a low
risk category, which includes the forests that are situated far away from the human settlements, with no
accessibility to nearby roads, river systems and pilgrim sites. The medium risk zones occupied an area of about
7981 ha (24.84%) of the total forest cover in 2010 whereas it was 3915 ha (12.17%) in 1973. The high risk
zone has observed large-scale changes in forest cover between 1973 and 2010, and the change in area is about
1567 hectares (Table 2). The occurrence of a high risk zone was mainly around the settlement area.
Table 2 Change detection in the area of sensitive zones of 1973 and 2010.
Class 1973 2010
Area Area Area Area
(ha) (%) (ha) (%)
No risk zone 15494 48.17 6409 19.95
Low risk zone 11804 36.70 15212 47.36
Medium risk zone 3915 12.17 7981 24.84
High risk zone 950 2.95 2517 7.83
The degrees of sensitive areas are classified into four categories namely: no risk zone, low risk zone,
medium risk zone and high risk zone (Fig. 5a and 5b).
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Fig. 5 a. Sensitive zone map of 1973; b. Sensitive zone map of 2010.
Change detection analysis was performed between the risk zones of 1973 and the vegetation density of
2010 (Table 3). It was observed that medium and high risk zones were distributed mainly in the open forest
region (4177.45 and 523.63 ha respectively). Secondly, about 24% of a high risk zone was present in very
dense forest.
Table 3 Change matrix between sensitive zones in 1973 and vegetation density in 2010.
Area of NDVI in 2010
Area of Sensitive (ha)
zone in 1973 Non forest Scrub forest Open forest Moderately Very dense
dense forest forest
No risk zone 0.85 715.95 979.82 262.58 109.31
Low risk zone 0.04 2281.03 14325.7 1971.64 869.93
Medium risk zone 0.02 2120.89 4177.45 1787.76 1057.29
High risk zone 0.05 267.5 523.63 333.43 374.49
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According to 1973 risk zonation, though the area under scrub forest in 1973 was 96.91 ha that has
increased to 267.5 ha in 2010. Similarly, the open forest in 1973 has 277 ha under high risk zone has increased
to 523.63 ha in 2010 (Table 4). These increases in the areas from 1973 to 2010 and this change may be due to
the expansion of the agricultural lands and usage of footpaths and roads for the firewood collection and cattle
grazing.
Table 4 Change detection between sensitive zones and vegetation density class.
Area of forest density in Area of forest density in
2010 1973
Degradation Prone zone
(ha) (ha)
Scrub Open Scrub Open
High risk zone 267.5 523.63 96.91 277.48
Medium risk zone 2120.89 4177.45 974.74 2561.52
The current study deviates from all other prioritization study in the following aspects: a) in this study, two
sensitive zonation maps were prepared for two time periods, 1973 and 2010. b) the sensitive zonation map
prepared for 1973 was compared with the forest cover map of 2010 and the area under different sensitive
zonation, and their corresponding present density classes were assessed. c) The combinations of sensitive
zonation in 1973, and their corresponding forest cover class in 2010 were once again compared with the forest
cover class of 1973, to bring out the contribution of area by various forest cover density classes in 1973 to the
degraded forest in 2010, which belong to highly risk category. d) A comparison of 1973 and 2010 risk
zonation alone reveals that the area under no risk zone has drastically decreased and medium and high-risk
zone has considerably increased.
4 Conclusion
This study investigated the mapping of sensitive zones using remote sensing and the weighted overlay model
in Mudumalai Tiger Reserve. We parameterized the weighted overlay model to analyze the whether the
sensitive zones of the forest are degraded from 1973-2010. The rates and driving factors of forest changes were
identified using remote sensing data. Then, these data were used to calculate the area of risk zones and
vegetation density. The identification of the areas vulnerable to forest changes is fundamental in the
Mudumalai and has important implications for biodiversity conservation in the region. One of the most
important applications would be to relate the spatial patterns of forest changes to the spatial distribution of
species. The loss of the forest is mainly near the settlement area which threatens the survival of many species
in this region. In particular, cultivation within forest around the settlements areas drastically altered the
composition and abundance of plant species and the conversion of forest into cropland indicates increasing
pressure on the steep land areas in the surrounding areas. From a protected area management perspective, the
risk zone and vegetation density maps of Mudumalai can help protected area managers identify where
conservation and forest management efforts should be focused. The monitoring process can be implemented
by regularly updating satellite derived maps of vegetation density and predicting forest change patterns.
Moreover, these sensitive maps can be used to focus biological conservation efforts.
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Acknowledgements
Authors are thankful to Department of Science and Technology-Innovation in Science Pursuit for Inspired
Research (INSPIRE) fellowship program for their funding support. Authors are grateful to Tamil Nadu Forest
Department and field assistants for necessary field support.
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Article
Impact of land use on the distribution of toxic metals in surface soils

in Birjand city, Iran
M. H. Sayadi, M. R. Rezaei
Department of Environmental Sciences, University of Birjand, Birjand, Iran
E-mail: mh_sayadi@yahoo.com
Abstract
Accumulation of toxic metals in the soil is one of the most important issues and treats plants, animals and
humans. The aim of this study was to investigate the impact of land use on the distribution of Pb, Cu, Ni, Cr,
Zn and Cd in the surface soils of Birjand city (Amir Abad Area). Dairy farm, agriculture, roads, residential-
road, residential-agriculture and educational centre land use were studied. The samples were taken from depth
of 0-20 cm at 16 stations, as regular grid soil sampling. After digestion with perchloric acid and nitric acid, the
concentration of toxic elements was measured using atomic absorption. The results show the highest
concentration of Pb (166.64 mg/kg) is found in the station number 10 in residential–road land use. The highest
zinc concentration (346.50mg/kg) is shown at the station number 1 in dairy farm land use. The maximum
concentration of cadmium (8.57 mg/kg) was at station number 10 with residential–road land use. The mean
concentration of cadmium element is greater than the threshold values. While mean concentrations of other
elements when compared to other researches shows high values, indicating that the soil is contaminating and in
the near future the concentration of toxic metal will be beyond the threshold values. So entry of toxic elements
into the human food chain greatly increases. It can suggest, in order to cleanup and reduce the rate of increases
toxic elements in the soil and special management and considerations may apply.
Keywords soil contamination; heavy metals; land use; public health.
ISSN 22208860
1 Introduction
Urban soils were contaminated by different pollutants including heavy elements which are accumulated in long
term. Studies on soil contamination began in earnest from 1960 and in 1966, contamination of heavy metals
into the soil introduced (Purves, 1966). Contamination of soils with heavy metals is expanding and become a
major issue and unavoidably (Meravi and Prajapati, 2013). Many human activities cause the entry of toxic
elements in soils, so that the pollution intensity is higher than natural soils and soon will be out of standard
range and serious threats to human health (WHO, 2003; Wilson and Temple 2001). Urban activities in
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different ways release the heavy metals in urban soils. For example, the transport of passengers and goods
(Tirusha Thambiran and Roseanne, 2011), industries, factories and mining (Sayadi et al, 2009), agriculture
(Wong et al., 2002; Mico et al., 2006), dairy farms (Nicholson et al, 2003), dust (Lin et al., 2005), municipal
waste (Sayadi and Sayyed, 2010), and municipal and industrial wastewater (Mapanda et al., 2005) could be
noted. Among the human activities, vehicles (such as brake and exhaust) and various agricultural activities and
workshops are responsible for most of the contamination of soils (Zehetner et al., 2009). Given the importance
of soil pollution and its negative impact on public health and the environment, many studies have been
conducted in various cities around the world, Like Hong Kong (Li and Liu, 2001), Syria (Moller et al., 2005),
Ireland (Dao et al., 2013) Italy (Manta et al., 2002) Spain (Jose´ Antonio Rodrı´guez et al., 2008) Zimbabwe
(Mapanda et al., 2005) Australia (Tiller, 1992) China (Wu et al., 2011) and also some cities in Iran such as
Tehran (Sayyed and Sayadi, 2011), Isfehan (Amini et al., 2007), Kerman (Noori et al., 2009), Hamadan
(Karami et al., 2011).
Heavy metal contamination of soils in urban researchers has studied a variety of toxic elements. Choose
the heavy elements is based on previous studies of similar areas. Urban soils is strongly influenced by human
activities, especially in parks, roads and surrounding building which soils prepare from other areas. So the soil
geochemical studies makes difficult. The main objective of this study was to assess the toxic elements and
identifying pollution sources of heavy metals in surface soils with different land use.

2.1 Study area
Amir-Abad city at vicinity of Birjand city is a relatively small city with a population of 1,000 and located on
the west side of Birjand city which is from south Khorasan province. Existence of faculty of agriculture along
with numerous Livestock farming and agricultural activities is given special status to this area. Urban activities,
different workshops and small settlements, and also one of the main highways in the region of south Khorasan
province are potential sources of heavy metals pollutants that threaten the environment. Dairy farm, agriculture,
roads, residential- road, residential-agriculture and the educational centre land use are studied. Table 1 shows
the land use along with geographical coordinates of the stations under study.
It should be noted that the stations number 2, 3, 4 and 5 are without a certain user and contains only low-
traffic roads.
Table 1 Geographic coordinates and land use of the sampling stations.

Geographical Geographical
Stations Stations
Land use coordinates Land use coordinates
No: No:
N E N E
1 Dairy farm 32.860 59.141 9 Residential- 32.869 59.145
Road
2 - 32.861 59.143 10 Residential- 32.865 59.143
Road
3 - 32.859 59.153 11 Residential- 32.865 59.151
Agriculture
4 - 32.855 59.158 12 Agriculture 3.868 59.158
5 - 32.865 59.142 13 Residential- 32.878 59.138
Agriculture
6 Educational 32.866 59.143 14 Residential- 32.879 59.142
centre Agriculture
7 Road 32.867 59.143 15 Agriculture 32.880 59.152
8 Road 32.862 59.158 16 Agriculture 32.881 59.155
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2.2 Sampling and analysis of soil samples

Soil sampling were taken from depth of 0-20 cm in a regular network in 16 stations, each stations were
sampled with 5 sub samples in three replicates. Geographical locations of the sampling stations were
determined using a global positioning system tool. After mixing the 5subsamples for each station, 1.5 kg soil
sample is taken (Sayadi and Sayyed, 2011). The soil samples were air-dried and then passed through 2 mm
sieve. After digestion with perchloric acid and nitric acid, the concentration of toxic elements was measured
using atomic absorption (Page et al., 1982; Burt, 2004).
3 Results
Table 2 shows the mean concentrations of toxic elements in surface soils of Amir-Abad city. As Table 2
shown the mean concentrations of toxic elements at the stations are not equal and significantly different
(Pvalue <0.001). As Table 2 shows that the highest concentration of Pb (166.64 mg/kg) relevant to station No.
10 with residential–road land use and the lowest concentration of lead (12.2 mg/kg) relevant to Station No. 3
without any special land use. The highest copper concentration (226.7 mg/kg) was detected at station No. 9
with residential-road land use and the lowest value (11.4 mg/kg) found at station No. 3 without any special
land use. The highest zinc concentration (346.50 mg/kg) is obtained at station no.1 with dairy farm land use
and The lowest value (18.23 mg/kg) were observed at station no. 5 without any special land use. The highest
concentration of nickel (99.48 mg/kg) was found at the station number 9 with residential–road land use and the
lowest value (10.07 mg/kg) is shown at station number 12 in the agriculture area. Station No. 10 in residential-
road area has shown the maximum amount of Cr (192.03 mg/kg) and the lowest value (15.42 mg/kg) found at
station no. 2 without any special land use. The highest concentration of cadmium (8.57 mg/kg) observed at
station no. 10 with Residential-Road land use and The lowest value (0.05 mg/kg) observed at station no. 2
without any special land use.
Table 2 Average concentrations of heavy metals in the soils from different stations (mg/kg).
Station No: Land Use Pb Cu Zn Ni Cr Cd
1 Dairy farm 19.4 165.11 346.50 89.9 81.7 1.3
2 - 13.9 17.1 33 26.31 15.42 0.05
3 - 12.2 11.4 24.79 19.02 20.11 0.6
4 - 15.4 11.7 21.2 20.74 22.63 0.82
5 - 17.33 20.6 18.23 16.66 34.25 0.36
6 Educational Centre 28.17 19.3 56.66 33.85 59.7 1.7
7 Road 62.82 41.58 125.2 41 23.14 0.69
8 Road 65.14 37.5 93.04 37.5 67.45 0.44
9 Residential- Road 109.90 226.7 118.04 99.48 186.34 2.92
10 Residential- Road 166.64 202.61 122.58 72.13 192.03 8.57
11 Residential-Agriculture 45.37 48.03 112.98 32.11 64.67 1.59
12 Agriculture 58.24 30.29 80.10 10.07 56.8 0.46
13 Residential-Agriculture 49.75 36.27 105.39 22.04 36.60 1.01
14 Residential-Agriculture 33.44 42.53 127 35.51 36.92 0.93
15 Agriculture 22.20 24.45 68.55 17.84 88.73 1.46
16 Agriculture 25.57 27.22 52.17 26.27 34.40 1.51
Minimum 12.20 11.40 18.23 10.07 15.42 0.05
Maximum 166.64 226.70 346.50 99.48 192.03 8.57
Mode 30.81 33.28 86.57 29.21 46.86 0.97
Mean 46.59 60.15 94.09 37.53 63.79 1.53
Standard deviation 41.27 70.24 77.99 26.50 53.71 2
PValue 0.000 0.000 0.000 0.000 0.000 0.000
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Table 2 summarizes the overall statistics of the measured parameters in surface soils of the study area.
Table 2 shows that the concentrations of toxic elements in the study area were listed as Zn> Cr> Cu> Pb> Ni>
Cd. Although the maximum concentrations of trace elements like zinc have a significant impact on the mean
concentration.
4 Discussion
Modernization and urbanization phenomenon has become a critical problem for soil pollution. Therefore from
different ways enter to the food chain and can cause serious and sometimes fatal diseases such as cancer. Table
3 shows that comparison between the mean concentrations of heavy metals in the study area with natural
concentrations of these elements in Tehran and China and also the threshold limits of these elements in the soil.
Table 3 Concentrations of heavy metals in the study area and the concentration of these elements (mg\kg).
Pb Cu Zn Ni Cr Cd
Study Area 46.59 60.15 94.90 37.53 63.79 1.53
Tehran 1 5.12 9.62 11.26 11.56 10.36 0.34
China 2 26 22.6 74.2 26.9 61 0.097
Threshold 100 75 200 40 75 1
Limit 3
1
Sayadi and Sayyed 2011; 2CEPA, 1990; 3HGR 2000.
Table 3 shows the average concentration of Cd element is greater than the threshold limit and natural
concentration in Tehran and China. The mean values concentrations of all elements is higher than natural
concentrations of these elements in Tehran and China indicating soil contamination in the study area maybe
beyond the threshold limit in the near future.
Tables 2 and 3 show that the concentrations of Pb, Cu, Ni, Cr, and Cd are higher than the threshold limit at
the stations number 9 and 10 with residential- road land use. The concentration of Cu, Zn, Ni, Cr, and Cd is
higher than the threshold limit at station No. 1 with dairy farm land use. The concentration of Cr and Cd is
higher than the threshold limit at station no. 15 with Agriculture land use. The concentration of Cd is also
higher than the threshold limit at station numbers 6, 13,11and 16.
4.1 Impact of land uses on the concentration of toxic elements
Pb
Fig. 1 shows the mean concentration of Pb in the different land uses. As shown in Fig. 1, the highest
concentration of Pb observed in residential-roads and then road land use, as residential-roads land use is higher
than the threshold limit, the lowest concentration of Pb was found in the stations without any special land use.
Pb is usually added to the environment from activities of mining, metallurgical, sewage systems, and smoke
from burning gasoline vehicles. Use of fertilizers and pesticides for agricultural purpose are releasing Pb to the
soils. Battery industry, munitions, soldering, alloys, pigments are also other sources of Pb in the environment
(Patel et al., 2006).
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Fig. 1 Pb concentrations in various land uses.
Chronic Pb poisoning often occurs and growing children, pregnant women and the elderly are most at risk
for Pb poisoning. The affect of Pb and bone and bone marrow cause anemia. Other symptoms of poisoning are
neurological disorders that often lead to paralysis. Accumulation of Pb in the liver and kidneys are impaired in
their work as well as high levels of Pb concentration in children causes' acute Pb encephalitis (Homdy et al.,
2002).
Table 2 shows that the stations 7, 8, 9, 10 have the higher Pb concentrations. Thus it can be concluded that
the main sources of Pb pollution in the region is transportation system. Similar results have been obtained in
different parts of the world (Zehetner et al., 2009; Wu et al., 2011; Guney et al., 2010).
Cu
Fig. 2 shows the mean concentration of Cu in the different land uses. As shown in Fig. 2, the highest
concentration of Cu found in residential-roads and then dairy farm land uses which are also higher than the
threshold and the lowest concentration of Cu was found in the area without any special land use.
Cu extensively used in electrical cables, various alloys, pigments, cooking equipment, pipelines, chemical
plants, fertilizer, pesticide and melting furnace, that could be environmental pollution sources (Gowd and
Govil, 2008). Although, Cu is an essential element for human health but in higher concentration cause
neurological effects, liver and kidney dysfunction, anemia, intestinal distress, coma and even death (Siegel,
2007). Table 2 shows the stations 1,9,10 have higher copper concentration. So we can conclude that dairy farm
and residential activities along with transportation system are the main sources of Cu pollution in the region.
Similar results have been obtained in different parts of the world (Nicholson et al., 2003).
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Fig. 2 Cu concentrations in various land uses.
Zn
Fig. 3 shows the mean concentration of Zn in the different land uses. As shown in Fig. 3, the higher
concentration of Zn found in dairy farm land use and then residential-roads, roads and residential–agricultural
land uses. The mean concentration of Zn in dairy farm land use is more than threshold values and the lowest
concentration of Zn was found in the area without any special land use.
Zinc is necessary element for humans, animals and plants and often be found in the limestone soils.
Chemical fertilizers and pesticides and pigments are added Zn to the environment and Zn is also used in a
variety of alloys such as bronze and brass (Romic and Romic, 2003). Anemia, muscle pain, stroke, blood
diseases and even death are from complications of zinc excess (Roa et al., 2001; Pais and Benton, 1997).
Fig. 3 shows that the soil around dairy farm contains considerable amounts of zinc, which is even higher
than the threshold limit. In a study conducted in agricultural soils in England and Wales showed 37-40% of Zn
concentration on agricultural soils is due to animal manure (Nicholson et al., 2003).
Ni
Fig. 4 shows the mean concentration of Ni in the different land uses. As shown in Fig. 4, the highest
concentration of Ni found in dairy farm and then residential-roads land uses which are beyond the threshold
limit. While the lowest concentration of Ni found in the area without any special land use.
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Fig. 3 Zn concentrations in various land uses.
Fig. 4 Ni concentrations in various land uses.
Nickel metal used by humans, mainly in batteries and storage tanks of petroleum products and natural
weathering of rocks, especially basalt stones releasing Ni into the environment (Demirel, 2006; Sayadi and
Torabi, 2009). Nickel can replace the essential element in the structure of enzymes and breaks down the
metabolism pathway (Vincoli, 2003). It is carcinogenic in high concentrations, increasing the threat of heart
and liver, as well as weight-reducers (Homady et al., 2002). Table 2 and Fig. 5 show that the soils surrounding
residential-road and dairy farm areas containing considerable amounts of nickel element which is accumulate
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in the soils by different ways. Other studies have reached similar results (Mapanda et al., 2005; Sayadi et al.,
2010).
Cr
Fig. 5 shows the mean concentration of Cr in the different land uses. As shown in Fig. 5, the higher
concentration of Cr found in residential-roads and then dairy farm land use in the study area and beyond the
threshold limit. While the lowest concentration of Cr found in the area without any special land use.
Fig. 5 Cr concentrations in various land uses.
The possible sources of Cr are textile, leather, pharmaceuticals and metals activities. Pigments containing
chromium, oil and grease compounds also contain some Cr (Kumar et al., 2008). Cr is an essential component
for the metabolism of fats and protein but high concentrations of Cr (VI) may damage the liver and kidneys
and can cause cancer (Krishna and Govil, 2004).
Table 2 and Figure 5 show that the soil surrounding residential-roads and dairy farm areas contain high
amounts of Cr that these results are obtained by other researcher. (Moller et al., 2005; Sayadi et al., 2009).
Cd
Fig. 6 shows the mean concentration of Cd in the different land uses. As shown in Fig. 6, the higher
concentration of Cd found in residential-roads and then educational centre land uses in the study area while the
lowest concentration of Cr found in the area without any special land use. It is interesting to note that, the
concentration of Cd in soils under studied are exceeded the thresholds limit in various land use with the
exception of road land use and the area without any special land use.
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Fig. 6 Cd concentrations in various land uses.
Fig. 7 percentage distributions of heavy metals concentrations in various land uses.
Batteries factories, pigments, plastics, ceramics, glass and plating jewelry materials will enter cadmium
into the soil and are sources of cadmium pollution in the environment (Gowd and Govil, 2008).
Chronic kidney disease; cardiovascular disease; anemia; respiratory system; cancer of the prostate, lung
and skin; rickets and dental caries cadmium pollution are some of the Cd complications (Siegel, 2007).
Cadmium threshold limit is 1 mg/kg (Table 3). Table 2 and Figure 6 show that Cd concentration of the most
of the surface soil is exceeded threshold limit and seriously threatens its residents' health. Suggesting further
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studies carry out on human disease with its relationship to the distribution of Cd concentration in soil in the
study area.
4.2 Percentage distribution of toxic elements in different land uses
Fig. 7 shows the mean concentration of the toxic metals in the different land uses. As shown in Fig. 7,
Residential-road land use has the highest percentage distribution of toxic metals, chromium, cadmium and
dairy farm lands has the highest percentage of zinc and nickel.
5 Conclusion
The main sources of entry of heavy metals in the soil of by human activities arise by the modernization and
urbanization phenomenon. According the obtained results it can be concluded that among different land use,
the soils in the Residential-Road land use accumulate higher rate of Pb, Cd, Cr and Cu concentrations. Ni and
Zn is more accumulated in dairy farm land use, however residential-road land use is also having a significant
amount of these elements.
Considering that the mean Cd concentration and Cd concentrations in most of the stations is greater than the
threshold value, so can easily enter the human food chain. More specifically, the study area is agriculture and
Dairy farm area and their production of this area transport even to other city, so can cause a variety of illnesses
and diseases. With a few exceptions the average concentration of toxic elements (except Cd) is a lower
threshold limit but in comparison with other urban areas is high, indicating the toxic elements are increasing
into the soil and with the gradual increase of toxic elements in the soil and enter to the food chain of human
beings and expected to exceed from the threshold limit in near future. Suggesting to reduce the increasing rate
of these elements into soils and the reduction of toxic elements in the soil must be special management and
largely considerations be provided to community health and the relationship between the concentration of toxic
metals (particular Cd) in surface soils and chronic disease in the study area is assessed.
Acknowledgments
This study was funded by the Research Council of University of Birjand which as a Research Project was
conducted in 2012. Authors are appreciated the authorities of Research Council and Faculty of Natural
Resources and Environment, University of Birjand, due to their sincere cooperation.
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Article
Examination of nitrification inhibition by sorghum (Sorghum bicolor)

in soil around its roots
Adel Ghoneim1,2, Abdulla Al-Modaihsh1, Saied Naeem2, Tamer Metwally2, Elsaied Gewailly2, Azza
Ebid3
1
Soil Science Department, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh, 11451,
Saudi Arabia
2
Agricultural Research Center, Rice Research and Training Center (RRTC), Sakha, 33717, Kafr El-Sheikh, Egypt
3
Department of Biology, College of Science, Princess Nora Bint Abdul Rahman University, P. O. Box 376114 Riyadh 11335,
Saudi Arabia
E-mail: aghoneim@ksu.edu.sa
Received 2 September 2013; Accepted 18 October 2013; Published online 1 March 2014
Abstract
Biological nitrification inhibition refers to release some chemical substances from plant root that
suppresses/slowdown soil nitrification. This study was conducted to clarify whether sorghum (Sorghum
bicolor) inhibit nitrification in soil around the root. Sorghum cultivated in pots filled with a brown lowland soil
and examined nitrification rate in the soil around its root comparing with bare soil. Two experiments were
conducted. In the first experiment, sorghum was cultivated in a growth chamber and soil sample were collected
four times during the growth period. Nitrogen in the form of (NH4)2SO4 (120 mg N kg-1 soil) was mixed with
the soil samples and incubated at 30C, for 21 days. Nitrification rates were estimated based on NO2-+NO3--N
accumulation per unit time. Results showed that in sometime nitrification rate in the soil around sorghum root
was lower than that in the bare soil however, in other times there were no difference between them. Second
experiment was conducted by using soil samples collected from the pots in which sorghum was cultivated in a
greenhouse. The results showed nitrification in the soil around sorghum root was lower than that in the bare
soil. Nitrification was inhibited in soil around the sorghum roots, however, this inhibition varied with
incubation period. The differences of N application showed a little effect of nitrification inhibition rates.
Keywords sorghum; rhizosphere soil; nitrification inhibition.
ISSN 22208860
1 Introduction
Nitrification is oxidation of ammonium (NH4+) to nitrate (NO3-) by specific soil microorganisms. It changes
relatively immobile NH4+-N to highly mobile NO3--N, which can result in nitrogen losses from nitrification-
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denitrification and NO3- leaching. Problems of low N use efficiency due to N losses through nitrification-
denitrification, leaching constantly being reported (Glass, 2003; Giles, 2005). About 70% of 100Tg of N
applied as N fertilizer is lost through nitrification and other transformation, which translates to about 17 billon
US dollars, in addition the unknown costs from environmental impact such as NO3- pollution and
eutrophication of surface water and gas emission (Apr et al., 2002; Giles, 2005). Considerable effort has been
invested in the identification of chemicals that can inhibit, or at least slow the nitrification process (Slangen
and Kerkhoff, 1984; Prasad and Power, 1995; McCarty, 1999). Some plants have been reported to have the
ability to release some biological active substances from their roots that suppresses nitrification. These
compounds are called as biological nitrification inhibition (BNI). Biological nitrification inhibition refers to
the release of root exudates capable of inhibiting bacteria responsible for the nitrification process. Subbarao et
al. (2006a), Subbarao et al. (2006b), Subbarao et al. (2007a) and Subbarao et al. (2007b) showed import new
findings of the role of plants in moderating nitrification in soil. However, it was not fully clarified whether
these plants which showed strong nitrification inhibition in the assay really inhibit nitrification in soil where
the plants grow. Sorghum (Sorghum bicolor) had been reported to possess inherent ability to release some
biochemical compounds from their roots that could inhibit nitrification in soils, a phenomenon known as
biological nitrification inhibition (Subbarao et al., 2007a; Hossain et al., 2008). The study was conducted to
clarify evidence of the existence of biological nitrification inhibition function and whether sorghum inhibit
nitrification in soil around its root.

2.1 Soil used for the experiment
Fine-textured brown lowland soil (Haplic Brown Lowland Soils, USDA Soil Taxonomy) was collected from
Crops and Field Laboratory, Agriculture and Forestry Research Center, Saitama Prefecture, Japan between 0-
30cm depth, sieved with 2.0 mm sieve, mixed thoroughly and kept in plastic bags in a greenhouse. The soil
properties were as follows: pH (H2O) 6.33, total N 0.69 g kg-1 dry soil, total C 6.0 g kg-1 dry soil, C/N ratio 8.7,
NH4+-N 0.98 mg kg-1and NO3--N 0.80 mg kg-1, clay 20%, silt 33% and sand 47%.
2.2 Treatments
2.2.1 Growth chamber experiment
Seeds of sorghum (hybrid sorgo) was germinated at 25C in trays containing sand and watered with distilled
water. Eight seedlings, one-week old were sown in each pot (0.0884 m2), and the seedlings were thinned to
four per pot 7 days after sowing. The pots were prepared with three replications. Four treatments were as
follows: (1) soil cultivated with sorghum and N fertilizer was applied @ 50 kg N ha-1 as urea one week after
planting. (2) Soil cultivated with sorghum without N application. (3) Bare soil with N applied @ 50 kg N ha-1
as urea. (4) Bare soil without N application. Plants were grown in a growth chamber with day/night
temperature regimes of 25/30C and 70% relative humidity.
2.2.2 Greenhouse experiment
Seeds of sorghum (PVK-801) were geminated same as in the growth chamber experiment. Four seedlings were
sown in each pot (0.0884 m2), and the plants were thinned to four per pot, one week after sowing. Four
treatments were established as follows: 1. Soil cultivated with sorghum without NPK fertilizers (Plant –N). 2.
Soil cultivated with sorghum and fertilized with the rate of 100-200-40 Kg NPK ha-1, respectively (Plant +N).
3. Bare soil without sorghum and NPK application (no plant –N) and 4. Bare soil with NPK @100-200-40 Kg
NPK ha-1 (no plant + N). Phosphorus and potassium applied as basal application before planting in the form of
KCl and Na2HPO4, respectively while N fertilizer was applied in two splits at planting and one month after
sowing. The pots were prepared with 3 replications. Plants were grown in a green house.
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2.2.3 Soil incubation to test nitrification inhibition

Represented soil samples were collected from each treatment at 43, 60, 68 and 133 days after sowing in the
growth chamber experiment and at 63 and 75 days after sowing in the greenhouse experiment. The soil
samples were brought to the laboratory and only the soil adhered to the roots (rhizosphere soil) was removed
and used for the following experiment. The soil samples were mixed thoroughly one by one and the initial
moisture content was determined. The initial pH (H2O) of each soil sample was measured at a 1:2.5 soil: water
volume ratio with a glass electrode. The soil samples weighed at equivalent weight of 5.0 g dry soil per tube
were mixed with 0.6 ml of mixing solution of (NH4)2SO4 at the rate of 120 mg N kg-1 dry soil, sealed with
parafilm and incubated at 30C. At 0, 3, 7, 14 and 21 days after the incubation, the soil samples were extracted
by shaking with 50 ml of CaSO4.2H2O for 60 min, and then filtered through Wattman No. 5 filter paper. The
filtered solution was then analyzed calorimetrically for NO2-+NO3--N content (sulfanilamide--naphthlamine
method) using an Auto Ion analyzer (model AAII, Brant+Luebbe, Germany) (Anonymous, 1974; Litchfield,
1967; Varley, 1966). Accumulated NO2-+NO3--N was calculated as NO2-+ NO3--N (at 3, 7, 14 or 21 days after
the incubation) - NO2- + NO3--N (at the beginning of the incubation). Nitrification rates were determined based
on nitrite and nitrate accumulation per unit time. Nitrification inhibition rates were calculated using the
following equations
Nitrification inhibition rate (%) in the first 7 days:
(T 2  T 1)
100- * 100
(C 2  C1)
where T2 and T1 denotes amount of NO2-+ NO3--N of treatment accumulated at 7 and 0 day of soil incubation,
respectively, C2 and C1 denotes amount of NO2-+ NO3--N of control (no plant) accumulated at 7 and 0 day of
soil incubation, respectively.
Nitrification inhibition rate (%) from 7th day to 14th day:
(T 3  T 2 )
100- * 100
(C 3  C1)
where T3 and T2denotes amount of NO2-+ NO3--N of treatment accumulated at 14 and 7 day after incubation,
respectively, while C3 and C1 denotes amount of NO2-+ NO3--N of control (no plant) accumulated at 14 and 7
day after incubation, respectively.
2.3 Statistical analysis
The experimental data were subjected to analysis of variance using the IRRISTAT statistical package for
Windows and the differences among the means were analyzed by the least significant differences (LSD).
3 Results
3.1 Growth chamber experiment
The accumulated amount of NO2-+NO3--N after 3 days of incubation for the soil samples taken at 43 days after
sowing showed that presence of sorghum plant resulted to between 2.87-4.80 mg kg-1 of NO2-+NO3-N
accumulation compared with 5.47-18.63 mg kg-1 for control (no plant). The changes in NO2-+NO3--N
accumulation increased with increase the incubation time in a similar trend as observed for the first 3 days (Fig.
1 and 2). After 14 days, about 48.93-67.61 mg kg-1 of NO2- + NO3- has been accumulated under plant treatment
compared with 71.0-72.03 mg kg-1 in bare soil without plant. The NO2- + NO3--N accumulation was not
significantly different (P=0.05) among the treatments for the soil sampled at 43 and 60 days after sowing. In
the third and fourth soil sampling at 68 and 133 days after sowing (Fig. 3 and 4) showed significant differences
among the treatments. Bare soil without plant showed higher level of NO2- + NO3--N accumulation than that
soil around the sorghum roots during the different incubation time. With increasing the incubation time, the
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changes in NO2- + NO3--N accumulation increased in a similar trend. The lower NO2- + NO3--N accumulated in
the soil sampled from the around of sorghum roots at 68 and 133 days after sowing suggested that nitrification
in the soil was less active than that of the bare soil sample (Fig. 3 and 4). Results in Fig. 5 and 6 from the
rhizosphere soil incubation test, bare soil showed that higher rates of nitrification than the values obtained
from sorghum plot. The rate of nitrification of bare soil was more than six fold greater than the value obtained
for the presence of sorghum for the soil sampled at 68 days after sowing (Fig. 5). The rates on nitrification
over the incubation period showed a range of 1.6-4.8 mg kg-1 day-1 of NO2- + NO3- N, however, the
nitrification rates value were higher after 7 days of incubation compared with 14 days after incubation (Fig. 6).
Also at 133 days after sowing, soil nitrification rate was higher in bare soil (ranged from 2.45-5.4 mg kg-1 day-
1
) and the lowest in plant without N (1.8-2.7 mg kg-1 day-1) as shown in (Fig. 2). There was a narrow change in
soil pH between the pre-cropping soil pH (H2O:6.33) and post-cropping values (Table 1). The bare soil (no-
plant) without the N application showed a lower soil pH compared with other treatments. The variation in soil
pH values before incubation test across the N application and plant growth were significant. Root exudates of
sorghum inhibited soil nitrification by 6-85% in fresh soil at 7-14 days after incubation (Fig. 7). Its seems that
the biological nitrification inhibition activity from sorghum root exudates once released, is stable in inhibiting
soil nitrification up to two weeks.
Table 1 pH (H2O) values (mean ± standard deviation) of soil used for the incubation experiment.
Days after sowing
Treatments 43day 60day 68day 133day
Plant – N 6.54b ± 0.05 6.44a ± 0.04 6.42a ± 0.01 6.52a ± 0.04
Plant + N 6.44c ± 0.05 6.52a ± 0.01 6.35b ± 0.01 6.48ab ± 0.14
Bare soil – N 6.51bc ± 0.03 6.38a ± 0.01 6.41a ± 0.01 6.36b ± 0.07
Bare soil + N 6.76a ± 0.06 6.52a ± 0.62 5.85c ± 0.04 5.71c ± 0.19
Plant–N; cultivated sorghum without N application, Plant+N; cultivated sorghum with N application, Bare soil – N; no sorghum
without N application, Bare soil +N; no sorghum with N application; Different letters in each column denote significant
differences (LSD, P<0.05, n=3).
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3.2 Green house experiment

Results obtained from green house experiment are shown in Fig. 8 and 9. The amounts of NO2- + NO3--N
accumulation were significantly different among the treatments for the soil sampled at 63 and 75 days after
sowing. Presence of sorghum showed lower level of NO2- + NO3--N accumulation over the different incubation
time compared with the no plant treatment. At 63 and 75 days after sowing, the inhibition of soil nitrification
was varied with incubation period and ranged from 29-69% (Fig. 10). The pH values were significant different
(P=0.01) among the treatments (data not shown). Soil without plant showed a lower pH value compared with
other treatment.
4 Discussion
In this study, it was shown that nitrification rate in a soil around sorghum root was lower than that the bare soil,
although it was not always. This supports the idea that more NH4+-N was retained in the soil as a result of
nitrification inhibition due to the root exudates of sorghum. Sorghum had been reported to possess inherent
ability to release some biochemical compounds from their roots that could inhibit nitrification in soils, a
phenomenon known as biological nitrification inhibition (Subbarao et al, 2007a; Hossain et al., 2008). The
results of our study showed that the duration of lower nitrification fluctuated from a few days to 3 weeks. Root
exudates of sorghum inhibited soil nitrification by 6-85% in fresh soil at 7-14 days after incubation (Figure 7).
Using an activity-guided fractionation and purification approach Hossain et al.,2008 isolated one of the active
compounds called methyl 3-(4-hydroxyphenyl) propionate, which responsible for inhibitory activity from
sorghum root exudates. One of the possible mechanisms through which sorghum may suppress/slowdown
nitrifying bacterial populations could be through its root exudates. The suppressive/slowdown effect of soil
nitrification would enhance the nitrogen use efficiency, therefore reducing the negative environmental impacts
in most agricultural systems due to NO3- leaching. Subbarao et al. (2007b) and Subbarao et al. (2007c)
reported that NH4+-N ions has a stimulatory role in release of biological nitrification inhibition (BNI) from
Brachiaria humidicola root exudates, however, the exact effects of NH4+-N and/or the associated acidic pH
that follows after its uptake (i.e. the secondary effect of NH4+-N uptake) in release of BNI compounds from
plant root have not yet studied
Ammonium application shows promote a significant increase in the levels of accumulated NO2-+ NO3--N.
When nitrification occurs at low pH values, this may be due to the ability of acid-adapted strains of autotrophic
nitrifying bacteria to function under this condition.
The brown lowland soil with lower total carbon (6.0 g kg-1) and clay content (20%) has been endowed with
less buffer capacity. The buffer capacity play major role in the adsorptive-desorptive soils behavior which
might affect the effect of nitrification inhibition by the sorghum. The nitrification inhibitors by the sorghum
may be a useful tool for increases N use efficiency, decreasing the N emission and N leaching from soil.
5 Conclusion
Production in situ of nitrification inhibitors, either by plant roots or microorganisms within the rhizosphere of
growing crops or pastures, has more appeal as a low-cost alternative to the highly cost synthetic nitrification
inhibitors. The results described here showed that nitrification activity in soil around the sorghum root was
lower than the bare soil. This suggest that by inhibiting the conversion of ammonium to nitrate, losses
associated with leaching and denitrification will be mitigated and economic and environmental benefits will
result. This phenomenon and the effectiveness in soils with different chemical/physical properties require more
investigation in future.
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Acknowledgement
The authors wish to thank Deanship of Scientific Research, King Saud University, College of Food &
Agriculture Sciences, and Research Center for supporting this research study.
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Article
Physiological tolerance and cation accumulation of different

genotypes of Capsicum annum under varying salinity stress
Muhammad Afzal1, Awais Ahmad1, Ali Abdullah Alderfasi1, Adel Ghoneim2, Mohammad Saqib3
1
Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, 2460, Riyadh, Saudi Arabia
2
Department of Soil Sciences, College of Food and Agricultural Sciences, King Saud University, 2460, Riyadh, Saudi Arabia
3
Department of Agronomy, The Islamia University of Bahawalpur, Pakistan
E-mail: Afzal_uaf@hotmail.com
Abstract
A greenhouse experiment was demonstrated to compare tolerance ability of four genotypes (Desi, Sanam,
Kundri, Asia Bok) of Capsicum annum. L. under different levels of saline stress (i.e., control, 40, 80 and 120
mM NaCl). Growth parameters (root, shoot; fresh and dry weight) and physiological (Na+, K+, Ca++,
concentration, stomatal conductance, transpiration rate, photosynthetic rate, chlorophyll a, b contents)
indicators were analyzed to determine tolerability of genotypes. The results indicated that, all genotypes
tolerated only under low level of salinity stress (40 mM NaCl) while a severe growth suppress in general was
observed at higher levels (80 and 120 mM NaCl). Asia Bok was found more sensitive to salinity with 0.626g
shoot fresh weight whereas Desi (1.103g) is comparatively salt tolerant under 120mMNaCl. Chlorophyll a and
b contents and transpiration rate decreases with increases in salinity level in all genotypes with almost similar
trend. Na+ accumulation increase with increase in salinity level but found maximum (14 mg g-1DW) in Asia
Bok while minimum (10.8 mg g-1DW) in Desi. However K+ contents behave reversely to salt concentration
and was recorded maximum in Desi (33 mg g-1DW) at maximum (120 mM NaCl concentration).Stomata
conductance and transpiration rate was found maximum in Desi as compare to the all other three genotypes
under all salinity levels except control. For all above physiological determinants Sanam and Asia Bok have
similar behavior while Desi and Kundri have diversified under all salinity levels. Correlation between varieties
and salinity resulted that continuous increase in salinity affected growth, physiological aspects and cation
accumulation in chilies.
Keywords salinity; genotypes; growth; physiology; tolerance.
ISSN 22208860
1 Introduction
Almost 20% of the world’s agricultural land (FAO, 2010) is affected by soil salinity (Munns, 2002) which
limits crop productivity (Zadeh and Naeini, 2007) and reduces the yield up to 50% (Bray et al., 2000). Soil
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salinity is the major stress that adversely affects the overall metabolism of plant (Roychoudhury et al., 2008)
and leads to agricultural land deterioration and production reduction (Chinnusamy et al., 2005). Salinity affects
soil plant available water (Munns, 2002; Ashraf, 2004), physiological, morphological and metabolic process,
including germination, plant growth and nutritional imbalance (Willenborg et al., 2004). These effects are due
to low soil osmotic potential and ion accumulation (Marschner, 1995) where NaCl is the major salt (Munns
and Tester, 2008) and plants have been induced to switch on mechanism to regulate its uptake and cellular
adjustment (Munns and Tester, 2008). Higher NaCl concentration results in osmotic stress and ionic imbalance
(Maggio et al., 2000) membrane instability, mineral distribution, increased respiration rate, decreased
photosynthesis and Ca++ ion replacement (Muhammad et al., 2011). NaCl concentrations 100-200 mM reduce
plant growth rate and photosynthetic pigments in chilies (Chookhampaeng, 2011). A significant growth
reduction (60 mM NaCl) (Silva et al., 2008) and photosynthetic pigments (100 to 200 mM NaCl) of chilies
occurs due to Na+ and Cl_ accumulation has been reported (Chookhampaeng, 2011). Salt tress results in
suppressed germination and seedling growth (Zeng and Shannon, 2000; Ashraf, 2010), reduced leaf expansion
which cause low photosynthetic area and dry matter production (Mansour and Salama, 2004). Reduction in
plant K+ concentration affects the stomatal conductance, xylem stream flow and transpiration rate (Basu et al.,
2002). Salinity stress indirectly causes oxidative breakdown of peroxisomes, mitochondria and chloroplast
which produces activated oxygen species (AOS) which damage DNA, chlorophyll, cell proteins (Mittova,
2002) and membrane destabilization (Hasegawa et al., 2000).Chilies are a well-known food used as spice, crop
defense and irritant weapon all over the world, enriched with pro-vitamin A, B1, B2, B3, E and P, vitamin C
and antioxidants (Bosland and Votava, 1999). Chilies have various antioxidant compounds (Chuah et al., 2008)
which prevents oxidative damage of human body and prevents it from cardiovascular diseases and cancer
(Oboth and Rocha, 2007). It can dilates blood vessels, prevent heart diseases, cause hotness, relieve pain, faster
digestion, stop bleeding against frostbite, reduces inflammation and arthritis (Zia, 2006). Pakistan is not only
fulfilling its country demands but also exporting a huge amount of chilies to Gulf States, Canada, Sri Lanka,
United Kingdom and USA to earn foreign exchange (Zia, 2006) having 21.8 thousand hectors with 37.2
thousand tons annual production (Anonymous, 2011-12). Due to water shortage and salinity its production has
been decreased (78.3%) (Qureshi, 2012) in Sindh and Punjab, the leading production states (Anonymous,
2005-06).
Chilies have been graded moderately salt sensitive (Kanber et al., 1992) to salt sensitive crop (Haman,
2000) respond differently at different growth stages (Chartzoulakis and Klapaki, 2000). Salinity, if not
controlled or managed properly, affected the chilies seed germination and emergence (Demir and Okcu, 2004)
which ultimately resulted in yield reduction up to 14% and economic losses in addition (Rhoades et al., 1992)
specific ionic imbalance, toxicity water deficit, salt accumulation damage (Zhu, 2002).Pakistan has 6.3 million
hectors of salinity affected land (14% of the total irrigated area) (Anonymous, 2005) which causes 64% yield
reduction on average annually (Afzal et al., 2005). Chilies are adding millions of dollar in GDP of Pakistan
annually (Anonymous, 2006) but yield (1-2 tons ha-1) is much lower as compare to other producers (20 tons
ha-1). Saline irrigation water and soil salinity are the fundamental factors remarkably reducing chilies
production. Introduction of the salt tolerant crops is one of the most effective and faster method to overcome
the salinity problems. Keeping in view the extent of soil salinity and its effects on growth and physiological
parameters of chilies following objectives are formulated for the proposed study, Identification of available
Chillies (Capsicum annum L.) germplasm for salinity tolerance.Determining morphological and physiological
indicators for salinity tolerance in Chillies (Capsicum annum L.).
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2 Material and Methods

The proposed study was carried out at vegetable area, Institute of Horticultural Sciences, University of
Agriculture, Faisalabad. The experiment was laid out in Completely Randomized Design (CRD) with three
replications. Four chilies varieties are Desi, Sanam, Kundri, Asia Bok cultivars and 4 salt (NaCl) treatments i.e.
(Control, 40, 80 and 120 mM NaCl) for inducing salinity in chilies cultivars were used as experimental
material. Seeds were sown in 9L, bottom perforated, plastic pots containing sand rinsed with distilled water.
After emergence of first true leaves (15 days after germination), the number of seedlings per pot was thinned
to three and they were irrigated according to seedlings need. After twenty days of sowing, half strength (0.5)
Hoagland’s nutrient solution was given to plants. Salt treatments were started one month after sowing. To
avoid the osmotic shock NaCl concentrations was adjusted, by gradually increasing 20 mM every two days
until desired concentration reached. Each pot (three plants) was considered as one replicate and there were
three pots per treatment. Agronomic observations like root length, shoot length, root fresh and dry weight,
shoot fresh and dry weight was observed. Chlorophyll contents, photosynthetic rate, transpiration rate,
stomatal conductance, K+, Ca++ and Na+ concentration from leaf sap, was determined. Chlorophyll contents
were determined by using the method of Arnon (1949) and Davies (1976). Fresh leaves (0.5 g) were chopped
into segments of 0.5 cm and extracted with 5 mL acetone (80%) placed over night at 100C. The material was
centrifuged at 14000rpmfor 5 minutes and the absorbance of the supernatant was measured at 480, 645 and
663 nm by spectrophotometer. Then a, b contents were measured by using the following formulae:
Chl a = [12.7 (OD 663) -2.69 (OD 645)] × V/1000 × W,
Chl b = [22.9 (OD 645) -4.68 (OD 663)] × V/1000 × W.
where V isvolume of the solution cm3, W is Fresh weight of the leaf sample g.
Gas exchange characteristics measured by instantaneous net CO2 assimilation rate (A), Transpiration (E)
stomatal conductance (gs) and photosynthetic rate were made on a fully youngest leaf of each plant using an
open system LCA-4 ADC portable Infrared Gas Analyzer (IRGA) (Analytical Development company,
Huddleston, England). Measurement were performed from 9.00 to 11.00 a.m. with the following
specifications/adjustment, molar flow of air per unit leaf area 403.3 mM m-2 s-1, atmospheric pressure 99.9 kPa,
water vapor pressure into chamber ranged from 6.0-8.9 mbar, PAR at leaf surface was maximum up to 1711
(mol m-2, s-1) temperature of leaf ranged from 28.4 to 32.4 0C, ambient temperature ranged from 22.4 to 27.9
0
C, concentration was 352 mol mol-1. Plants collected and washed with detergent for surface disinfection and
rinsed under tab water. Oven dried plants were ground into fine powder by electric grinder. After grinding
digestion took place for Na+, K+, and Ca++ was done according to method described by Yoshida et al. (1984).
One gram oven dried plant samples were transferred to a 100 ml beaker and 10 ml of tri-acid mixture (HNO3,
HCLO4, in a ratio of 2:1) was added and covered it with watch glass. The samples were allowed to settle for
about half an hour till the initial reaction subsided. The sample was heated gently till the solid material
disappeared. Later on vigorous heating was given by peacing over a hot plate till a colorless solution resulted.
When the volume remained 1.5 ml samples were removed and cooled. Then added distilled water and volume
was made 100 ml in volumetric flasks. The analysis of Na+ and K+ used Flame Photometer (Sherwood Flame
Photometer Model-410).
The experiment was laid out according to Factorial Completely Randomized design (CRD- Factorial). Data
were analyzed statistically using ANOVA techniques (Steel et al., 1997) and means were compared by using
LSD.
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3 Results
Recordeddata presented in Table 1 showed that salinity has significant effects on variety. As the salinity level
increased shoot fresh weight decrease. Shoot Fresh weight (SFW), Shoot dry weight (SDW), root fresh weight
(RFS), root dry weight (RWD) have significant effect while in case of variety SFW, RDW has significant
effect while RFW and SDW have non-significant effect on yield. Maximum yield was recorded at T1 (control)
where SFW is 1.9483 g while minimum in T4 at which 120 mmol-1 salinity were applied have SFW is 0.8475
g. Comparative effect was also significant except SDW. Interactive study Variety cum salinity presented in
Table 1. Statistical data represented that maximum SFW was observed in S1 × V4 (control x variety) which
was followed by the treatment (S1 × V1) and (S1 × V2) having SFW1.93 and 1.89 respectively. At higher
salinity level (S4) where 100 m mol-1 NaCl applied variety desi performed better which is high resistant
against salinity having SFW.1.103 Which was followed by variety Kundri (V3) having 0.930 at par with
sanam (V2) and Asia Bok (V1) having 0.730 and 0.626 g SFW respectively. Data regarding RFW at higher
salinity S4 (120 m mol-1) variety desi is more resistant then rest of all. This was statistically similar with
Kundri having SFW 0.276 g. Least significant variety is Asia Bok having SFW 0.153 g at higher salinity. Data
regarding RDW and SDW presented in table. RDW against salinity level is highly significant as salinity level
increased root dry weight decreased while SDW, control treatment has maximum dry weight while other
treatments are statistically alike. Effect of varieties in case of RDW is significant while SDW is non-significant.
Interactive effect of salinity and variety against RDW is significant while against SDW is non-significant
presented in Table 1. Biomass production with reference to salinity is more important aspects in crop plants.
Maximum RDW was 0.243 g observed in variety Desi at control (0 m mol-1) is similar with variety Sanam are
0.233 g which was at par with variety Asia bok having 0.213 g biomass production in root. At higher salinity
level more resistance was desi variety having 0.123 g more bio mass production in roots which was at par with
S3 × V2 (80 m mol-1 × Kundri) having 0.073 g while minimum RDW was observed in Asia bok is 0.023 g.
Osmotic potential decreased (more negative) significantly with increasing concentration of NaCl. The decrease
was also significantly different within the levels of salinity. NaCl induced osmotic stress. Na+ content of the
chilies varieties increased with increase in the intensity of stressful environment. Varieties and salinity means
showed highly significant differences. Interaction study among salinity and variety showed significant effect
among each other presented in (Fig. 1). Chilies variety Asia Bok at maximum salinity level has maximum Na+
accumulation followed by Sanam at same salinity level. While minimum sodium accumulation was observed
named desi variety. K+ contents in chilies varieties decreased with increasing intensity of NaCl osmotic stress.
Data regarding varieties, salinity level and interaction among each other was significant in (Fig. 2). Maximum
K+ concentration was observed in variety Sanam at control (0 m mol-1) while minimum was observed at high
salinity level where 120 m mol-1 NaCl in Asia Bok was observed. Higher levels of osmotic stress seemed to be
more effective in reducing K+ contents. Data regarding chlorophyll (a and b) contents among four chilies
varieties at four different salinity level were presented in Fig. 3 and 4, respectively. Varieties mean showed
highly significant effect against salinity and treatments mean also have significant effect on chlorophyll a
contents. Fig. 3 shows that with the increasing salinity level chlorophyll a contents were decreased. Maximum
chlorophyll a contents were observed at treatment where 0 NaCl was applied in desi chili variety. While
minimum chlorophyll contents was observed in Asia Bok where 120 m mol-1 NaCl applied.
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Table 1 Effect of NaCl on growth (shoot, root) fresh and dry weight in chilies.
A. Salinity Shoot fresh Root fresh weight Root Dry Shoot Dry
level weight (g) (g) weight (g) weight (g)
1 1.9483 A 0.8083 A 0.2133 A 0.5517 A

2 1.2675 B 0.4275 B 0.1225 B 0.2675 B
3 1.0192 C 0.3342 C 0.1058 C 0.2100 B
4 0.8475 D 0.2358 D 0.0583 D 0.2475 B
LSD 0.0913 0.055 0.0119 0.136
B. Variety
1 1.4258 A 0.4950 0.1833 A 0.3542
2 1.2575 B 0.4733 0.1308 B 0.3450
3 1.2125 BC 0.4308 0.0933 C 0.2925
4 1.1867 C 0.4067 0.0925 C 0.2850
LSD 0.058 NS 0.88 NS
C. Interaction
S1V1 1.93 b 0.74 b 0.243 a 0.556
S1V2 1.89 b 0.82 ab 0.233 a 0.540
S1V3 1.74 c 0.74 b 0.163 d 0.470
S1V4 2.23 a 0.92 a 0.213 b 0.640
S2V1 1.43 d 0.50 c 0.193 b 0.340
S2V2 1.21 efg 0.39 cdef 0.130 e 0.250
S2V3 1.27 e 0.46 cd 0.086 f 0.280
S2V4 1.15 fg 0.34defg 0.080 f 0.200
S3V1 1.24 ef 0.42 cde 0.173 a 0.280
S3V2 0.916 h 0.30 efgh 0.123 f 0.160
S3V3 1.08 g 0.40 cdef 0.073 f 0.230
S3V4 0.916 h 0.20 hi 0.053 g 0.170
S4V1 1.103 g 0.31 efgh 0.123 e 0.240
S4V2 0.730 ij 0.20 ghi 0.036 gh 0.340
S4V3 0.930 h 0.27 fghi 0.050 g 0.190
S4V4 0.626 j 0.15 i 0.023 h 0.130
LSD 0.117 0.15 0.0178 NS
SFW = Shoot fresh weight, RFW = Root fresh weight, SDW= Shoot dry weight, RFW= Root fresh weight.
T1; (Control), T2 (40 mM), T3 (80 mM), T4 (120mM) =V1 (Desi), V2 (Sanam), V3 (Kundri), V4 (Asia Bok).
Means not sharing a common letter at LSD 5%.
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Table 2 Coeﬃcients of correlation estimated between salinity on physiological aspects of different chilies varieties.
Ca+ K+ Na+ SC TRA Chla Chlb Photo
K+ 0.9470**
Na+ -0.9577** -0.9979**
SC 0.9788** 0.9242** -0.9383**
TRA 0.9663** 0.9401** -0.9505** 0.9863**
Chl a 0.8224** 0.7804** -0.8102** 0.8283** 0.7951**
Chl b 0.8804** 0.8232** -0.8499** 0.9417** 0.9135** 0.8689**
Photo 0.9391** 0.9133** -0.9340** 0.9547** 0.9453** 0.9326** 0.9444**
Salinity -0.9651** -0.9407** 0.9587** -0.9738** -0.9683** -0.8937** -0.9440** -0.9895**

Ca+ = calcium concentration, K+ = Potassium concentration, Na+= Sodium concentration, SC= stomatal conductance, TRA,
Transpiration rate, Chl a= Chlorophyll a contents, Chl b= Chlorophyll b contents, Photo= Photosynthesis.
Fig. 1 Effect of salinity on Na+ accumulation. Fig. 2 Effect of Salinity on K+accumulation.
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Fig. 3 Effect of salinity on chlorophyll a contents in chilies Fig. 4 Effect of salinity on chlorophyll b contents in chilies.
Fig. 5 Effect of salinity on photosynthesis in chilies. Fig. 6 Effect of salinity on transpiration rate.
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Fig. 7 Effect of salinity on stomatal conductance in chilies.
Effect of salinity on photosynthesis in chilies has a significant effect. With increasing salinity levels
photosynthesis activity reduced in chilies. Treatments and varieties mean have highly significant effect on
photosynthesis. Interaction between varieties and salinity also has highly significant effect presented in (Fig. 5).
Maximum photosynthetic activity was observed in Asia Bok at treatment applied control (0 m mol-1) followed
by the Kundri at same level of salinity. Within increase osmotic potential by increasing sodium and chloride
contents, photosynthetic activity decreased. Minimum photosynthetic activity was observed in Asia Bok where
120 m mol-1 NaCl was applied. While in case of transpiration rate trend also same. As salinity increased
transpiration decreased. Treatment and varieties means have significant effect. Desi variety have maximum
transpiration rate followed by Kundri. Effect of salinity is significant for transpiration rate in chilies varieties.
Mean comparison between salinity and chilies varieties shown in (Fig. 6). Maximum transpiration was
observed in Desi variety at control (0 m mol-1) followed by Asia Bok and minimum was observed in Sanam at
treatment 100 m mol-1 applied. Data regarding stomatal conductance were presented in Fig. 7. Treatments
means have highly significant effect on stomatal conductance while variety have significant effect on stomatal
conductance. Interaction between variety and salinity also has significance on stomatal conductance. Reported
data in Fig. 7 showed that maximum stomatal conductance was observed in Desi variety at control where 0 m
mol-1 salinity applied. As the concentration of salinity was increased stomatal conductance was reduced among
varieties. Minimum stomatal reduction was observed in Sanam at 120 m mol-1 NaCl was applied. The
following (Table 2) is used to estimate the correlation between salinity and physiological effects on chilies
different varieties. In Table 2, data represents about chilies varieties showed that salinity have negative highly
significant effect on Ca+ concentrations that as salinity increased Ca+ concentration decreased gradually.
Almost all physiological parameters have same trends i.e. K+ concentration, stomatal conductance,
transpiration rate, chlorophyll a and b contents and photosynthetic activity have negative highly significant
effects except Na+. Results showed that these parameters in four varieties have inverse relation as
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concentration of salinity increased they have negatively correlate with each other except Na+ its concentration
increased as salinity level increased.
4 Discussion
NaCl reduced growth by reducing cell division and cell enlargement. Shoot and root growth was observed of
stressed chilies plants with 120 m mol-1 treatment have negative effect on it by reducing cell elongation and
division. Salinity reduced shoot and root elongation as shown in (Table.1). Chilies plants are sensitive to
salinity (Chartzoulakis and Klapaki, 2000; Lycoskoufis et al., 2005). Reduction in shoot and root growth due
to reduced water absorption, osmotic effect, mineral deficiency account ionic imbalance and reduced
metabolic activities (Kumar et al., 2005).The physiological effects were also observed in rice under salt stress
(Ozdemir et al., 2004). High salt stress reduced leaf area (Chartzoulakis and Klapaki, 2000); reduction in leaf
growth rate is the preliminary sign response of glycophytes exposed to salt stress (Houimli et al., 2008).
Reduced leaf area available for transpiration, photosynthetic activity and assimilate production might be a
result of osmotic effect (Fig. 1). Water potential and cell volume in cell abruptly decreased due to osmotic
dehydration caused by salt stress (Clouse, 1998; Yu et al., 2004). Effect of salinity to the rooting medium had
an inhibitory effect on root and shoot growth of the pepper plants (Shahbaz and Ashraf, 2007). Reduction in
chlorophyll contents with increase in salinity level in (Fig. 1) this is might be due to excess salinity is often
related to reduction in photosynthetic activity. Reduction in photosynthetic activity, due to stomatal or non-
stomatal factors or both. Chlorophyll contents give quantitative information about photosynthetic activity
(Maxwell et al., 2000).
5 Conclusion
In capsicum annum torabaility against salinity stress varies in different genotypes. Desi variety performed as
more tolerant while Asia Bok is relative is more sensitive to saline stress. All genotypes in general have
suppressed growth, high sodium accumulation and reduced chlorophyll contents, photosynthesis and
transpiration rate. Further molecular study is suggested to identify the tolerance mechanism and optimum level
of different genotype under varying saline concentration.
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ISSN 2220-8860
Volume 4, Number 1, 1 March 2014
Articles
Microorganism as a tool of bioremediation technology for cleaning

environment: A review
Ravindra Singh, Pushpendra Singh, Rajesh Sharma 1-6
Are the sensitive zones degrading? A modelling approach using GIS and
remote sensing
S. Selvalakshmi, S. Jayakumar, V.S. Ramachandran 7-17
Impact of land use on the distribution of toxic metals in surface soils in Birjand
city, Iran
M. H. Sayadi, M. R. Rezaei 18-29
Examination of nitrification inhibition by sorghum (Sorghum bicolor) in soil

around its roots
Adel Ghoneim, Abdulla Al-Modaihsh, Saied Naeem, et al. 30-38
Physiological tolerance and cation accumulation of different genotypes of

Capsicum annum under varying salinity stress
Muhammad Afzal, Awais Ahmad, Ali Abdullah Alderfasi, et al. 39-49
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Proceedings of the International Academy of

Ecology and Environmental Sciences

International Academy of Ecology and Environmental Sciences

Editorial Office: piaees@iaees.org

Publisher: International Academy of Ecology and Environmental Sciences

Microorganism as a tool of bioremediation technology for cleaning

Ravindra Singh1, Pushpendra Singh1, Rajesh Sharma2

Keywords hazardous waste; bioremediation; microorganism; bioreactor.

4 Bioremediation Research Studies Using Designed and Developed Laboratory Bioreactors

Are the sensitive zones degrading? A modelling approach using GIS

S. Selvalakshmi1, S. Jayakumar2, V.S. Ramachandran1

2 Materials and Methods

Fig. 1 Geographical location map of the study area in India.

Fig. 3 Conceptual diagram illustrating the preparation of sensitive zones.

Fig. 4 Methodology flow chart for the change detection analysis.

Fig. 5 a. Sensitive zone map of 1973; b. Sensitive zone map of 2010.

Impact of land use on the distribution of toxic metals in surface soils

Keywords soil contamination; heavy metals; land use; public health.

2 Materials and Methods

Table 1 Geographic coordinates and land use of the sampling stations.

2.2 Sampling and analysis of soil samples

Fig. 1 Pb concentrations in various land uses.

Fig. 2 Cu concentrations in various land uses.

Fig. 3 Zn concentrations in various land uses.

Fig. 4 Ni concentrations in various land uses.

Fig. 5 Cr concentrations in various land uses.

Fig. 6 Cd concentrations in various land uses.

Fig. 7 percentage distributions of heavy metals concentrations in various land uses.

Examination of nitrification inhibition by sorghum (Sorghum bicolor)

Keywords sorghum; rhizosphere soil; nitrification inhibition.

2 Materials and Methods

2.2.3 Soil incubation to test nitrification inhibition

3.2 Green house experiment

Physiological tolerance and cation accumulation of different

Keywords salinity; genotypes; growth; physiology; tolerance.

2 Material and Methods

1 1.9483 A 0.8083 A 0.2133 A 0.5517 A

Ca+ K+ Na+ SC TRA Chla Chlb Photo

Na+ -0.9577** -0.9979**

SC 0.9788** 0.9242** -0.9383**

TRA 0.9663** 0.9401** -0.9505** 0.9863**

Chl a 0.8224** 0.7804** -0.8102** 0.8283** 0.7951**

Chl b 0.8804** 0.8232** -0.8499** 0.9417** 0.9135** 0.8689**

Photo 0.9391** 0.9133** -0.9340** 0.9547** 0.9453** 0.9326** 0.9444**

Salinity -0.9651** -0.9407** 0.9587** -0.9738** -0.9683** -0.8937** -0.9440** -0.9895**

Fig. 1 Effect of salinity on Na+ accumulation. Fig. 2 Effect of Salinity on K+accumulation.

Fig. 7 Effect of salinity on stomatal conductance in chilies.

All manuscripts submitted to Proceedings of the International Academy of Ecology and

 Animal ecology, plant/microbe ecology, wetland ecology, farmland ecology,

Editorial Office: piaees@iaees.org

Publisher: International Academy of Ecology and Environmental Sciences

Volume 4, Number 1, 1 March 2014

Microorganism as a tool of bioremediation technology for cleaning

Examination of nitrification inhibition by sorghum (Sorghum bicolor) in soil

Physiological tolerance and cation accumulation of different genotypes of

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Na+ -0.9577 -0.9979

SC 0.9788 0.9242 -0.9383**

TRA 0.9663 0.9401 -0.9505 0.9863

Chl a 0.8224 0.7804 -0.8102 0.8283 0.7951**

Chl b 0.8804 0.8232 -0.8499 0.9417 0.9135 0.8689

Photo 0.9391 0.9133 -0.9340 0.9547 0.9453 0.9326 0.9444**

Salinity -0.9651 -0.9407 0.9587 -0.9738 -0.9683 -0.8937 -0.9440 -0.9895