Journal of Research in Biology Volume 3 Issue 1

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Table of Contents (Volume 3 - Issue 1)

Serial No Accession No Title of the article Page No

1 RA0304 Anatomical variation in the olfactory apparatus of marine teleosts.
Biswas S, Datta NC, Sarkar SK and De SK.

742-746
2 RA0306 The use of purple yam (Dioscorea trifida) as a health-promoting
ingredient in bread making.
Teixeira AP, Oliveira IMA, Lima ES and Matsuura T.
747-758

3

RA0305

Bioefficacy of Novaluron, a chitin synthesis inhibitor against the
tropical warehouse moth, Ephestia cautella.
Sackey I, Eziah VY and Obeng-Ofori D.

759-767

4

RA0308

A Checklist of Butterflies of Meenachil River Basin, Kerala, India.
Vincy MV, Brilliant R and Pradeepkumar AP.

768-774
5 RA0325 Microbial production of glutaminase enzyme.
Mario Khalil Habeeb.

775-779
6 RA0307 A review on the role of nutrients in development and organization of
periphyton.
Saikia SK, Nandi S, Majumder S.

780-788
7 RA0187 Assessing heavy metal contamination of road side soil in urban area.
Sarala Thambavani D and Vidya Vathana M.

789-796

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Anatomical variation in the olfactory apparatus of marine teleosts
Keywords:
Olfactory, Rosette, Scombridae, Carangidae, Platycephalidae, etc.
ABSTRACT:

The olfactory apparatus of marine teleosts viz., Rastrelliger kanagurta,
Scomberoides commersonianus and Platycephalus scaber belonging to the family
of Scombridae, Carangidae and Platycephalidae respectively has been fixed in
10% formaldehyde solution for 24 h and anatomically examined under light
microscope (LM). Anatomical variation regarding the nostrils, olfactory rosette,
occurrence of accessory nasal sacs, olfactory lobes, length of the olfactory nerve
tracts, etc. are observed. These morphological variations may denote species specific
and may decisive for several biological functions.
742-748 | JRB | 2013 | Vol 3 | No 1

This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution and reproduction in all medium, provided the original work is properly
cited.

www.jresearchbiology.com
Journal of Research in Biology
An International Open Access
Research Journal
Authors:
Biswas S
1
, Datta NC
2
,
Sarkar SK
1
and De SK
1
.

Institution:
1. Department of Zoology,
Vidyasagar University,
Midnapore (West) - 721102,
West Bengal, India.

2. 110/20 B. T. Road,
Kolkata - 700108, West
Bengal, India.

Corresponding author:
Subrata Kumar De.

Email:
skdvu@yahoo.co.in

Phone No:
+91 03222 275329

Web Address:
http://www.jresearchbiology.com/
documents/RA0304pdf.
Dates:
Received: 08 Nov 2012 Accepted: 20 Nov 2012 Published: 09 Jan 2013
Article Citation:
Biswas S, Datta NC, Sarkar SK and De SK.
Anatomical variation in the olfactory apparatus of marine teleosts.
Journal of Research in Biology (2013) 3(1): 742-748
An International Open Access Research Journal
Original Research

INTRODUCTION
Olfaction is an important type of chemoreception
which is mediated through well developed, complex and
organized olfactory system in vertebrates (Freitag et al.,
1999). This system generally develops from the
specialized tissue, called olfactory placode (von Kupffer,
1894). In the early stage of development, placodes are
formed from the preplacodal ectoderm of the anterior
region of the embryo, medial to epidermis and lateral to
both neural crest and neural plate (Knouff, 1935).
The developmental process leading to the formation of
fish olfactory organ, is little diverse (Hansen and
Zielinski, 2005). The peripheral olfactory organ is the
first chemosensory organ to develop in fish preceding the
systems of solitary chemosensory cells (Kotrschal et al.,
1997) and taste (Hansen et al., 2003). Fish generally
possesses a paired olfactory organ located at the anterior
part of the head (Derivot, 1984; Hansen and Zeiske,
1998; Dving, 2003; De and Sarkar, 2009). The olfactory
chambers, olfactory rosette, accessory nasal sacs,
olfactory bulbs, olfactory nerve tracts and brain are the
major components of fish olfactory apparatus
(Hamdani and Dving, 2007). The anatomy of the
olfactory apparatus shows wide range of structural
diversity regarding the shape and size of the olfactory
rosette, number of the olfactory lamellae, occurrence of
accessory nasal sacs, etc. among the teleostean groups
(Kleerekoper, 1969). The anatomical details of the
olfactory apparatus in several species belonging to the
diverse teleostean taxa with respect to their habitat are
still an obscure part in sensory biology. The sensory
systems of fishes show notable adaptations according to
habitat and mode of life in comparison with the higher
vertebrates (Bone and Moore, 2008). The present study
focused on the true anatomy of the olfactory apparatus in
three species of different ecological habitat viz.,
Rastrelliger kanagurta (Cuvier, 1816), a marine
oceanodromous fish; Scomberoides commersonianus
(Lacepede, 1801), a brackish water amphidromous fish
and Platycephalus scaber (Linnaeus, 1758), an estuarine
amphidromous fish to unfold their structural components
and functional significance in olfaction.

MATERIALS AND METHODS
Adult, sex-independent specimens of
R. kanagurta, S. commersonianus and P. scaber were
collected from the coastal regions of Bengal, India.
Specimens were preserved in 10% formaldehyde
solution for 24 h and brought to the laboratory. The
olfactory apparatus of these species were dissected out
and separately mount on grease free slide by using
glycerine. The olfactory apparatus of respective species
was examined under light microscope (LM).

RESULTS
R. kanagurta (Figure 1A) possesses two pairs of
nostril viz., anterior nostril and posterior nostril
(Figure 1B). The anterior nostril is an oval shaped
structure, encircled by a thick lip like ridge of skin and
located at the dorsal region of the snout where as the
posterior nostril is situated in front of the eye.
R. kanagurta shows a slit like structure i.e., posterior
nostril which is located at a moderate distance from the
anterior one (Figure 1B). The olfactory apparatus of the
said species is present at the antero-dorsal side of the
snout in between the anterior and posterior nostril
(Figure 1C). The multilamellar olfactory rosette is oval
in shape and is situated at the floor of the olfactory
chamber (Figures 1D and 1E). The triangular olfactory
lamellae vary from 60 to 70 in number per rosette and
are arranged pinnately on the raphe (Figure 1E). The
accessory nasal sacs are clearly marked in the olfactory
apparatus of R. kanagurta. The lacrimal sac is conical in
shape and located at the antero-medial region of
lachrymal bone in association with olfactory rosette
(Figures 1C and 1D). The ethmoidal sac is connected at
the posterior part of the olfactory rosette and situated
within a groove at the antero-lateral extension of the
Biswas et al., 2013
743 Journal of Research in Biology (2013) 3(1): 742-748
frontal bone (Figures 1C and 1D). The olfactory nerve
tracts are arised from the base of olfactory rosette and the
length varies from 16 mm to 18 mm respectively. The
distal part of the olfactory nerve tracts are connected
with olfactory lobes of the brain. The size of the
olfactory lobes is comparatively small in R. kanagurta
(Figure 1C).
In S. commersonianus (Figure 2A), the nostrils
are closely associated. The anterior nostril is an oval
shaped aperture and partly guarded by a nasal flap where
as the posterior nostril is crescentric in shape
(Figure 2B). The olfactory rosette is circular in shape and
consisting of two pairs of olfactory lamellae
(Figures 2C and 2D). The number of the olfactory
lamellae ranges from 100 to 140 per rosette (Figure 2D).
The accessory nasal sacs are not distinct in the olfactory
apparatus. The olfactory nerve tracts are arised from the
base of the olfactory rosette and the length is ranges from
10 mm to 12 mm. The olfactory lobe is comparatively
large in size (Figure 2C).
Interestingly, the nostrils of P. scaber
(Figure 3A) are situated at the antero-dorsal region to the
Biswas et al., 2013
Journal of Research in Biology (2013) 3(1): 742-748 744
Figure 1A - Schematic representation of Rastrelliger kanagurta.

Figure1B - The diagram shows oval shaped anterior nostril and transverse slit like posterior nostrils of
R. kanagurta.

Figure 1C - The olfactory apparatus of R. kanagurta shows olfactory rosette (OLR), lacrimal sac (LS),
ethmoidal sac (ES), olfactory nerve (OLN), olfactory lobe (OL), cerebral hemisphere (CHS),
optic lobe (OPL), cerebellum (CBL), medulla oblongata (MO), etc.

Figure 1D - The olfactory rosette (OLR) along with conical accessory nasal sacs viz., lacrimal sac (LS) and
ethmoidal sac (ETS).

Figure 1E - The dorsal view of oval shaped olfactory rosette shows central raphe (r), two rows of
lamella (l). The olfactory lamella is triangular in shape with prominent dorsal end (de), proximal end (pe)
and ventral margin (vm).
Plate - 1
OLR
LS
ETS

eye and lying far apart from each other. The anterior
nostril is oval in shape and has a tongue like nasal flap
where as the posterior nostril is valvular (Figure 3B).
The olfactory rosette is relatively large in size and oval
in shape (Figures 3C and 3D). The number of the
olfactory lamellae varies from 50 to 76 in number per
rosette (Figure 3D). The absence of accessory nasal sacs
is noted in the olfactory apparatus of P. scaber. The
length of the olfactory nerve tracts ranges from
22 mm - 24 mm. and it is well connected with the
olfactory lobe of the brain (Figure 3C).

DISCUSSION
Olfactory systems of fish are among the most
highly developed olfactory senses of vertebrates
(Kleerekoper, 1969). The sense of olfaction is mediated
through olfactory apparatus associated with nostrils. The
nostrils are responsible for incurrent and excurrent of
water during water ventilation (Nevitt, 1991). The
structure of the anterior and posterior nostril varies
among the teleostean fishes (Kapoor and Ojha, 1972).
The presence of nasal flaps in between the both nostrils
is almost common when they are closely associated
(Teichmann, 1954). The anterior and posterior nostril
probably acts as an avenue for water ventilation through
the olfactory rosette (Cox, 2008). The multilamellar
Biswas et al., 2013
Figure 2A - Schematic representation of Scomberoides commersonianus.

Figure 2B - The diagram shows anterior and posterior nostrils of S. commersonianus. Nasal flap (FL) is
distinct.

Figure 2C - The olfactory apparatus of S. commersonianus is comprises of olfactory rosette (OLR),
olfactory nerve (OLN), olfactory lobe (OL), cerebral hemisphere (CHS), optic lobe (OPL),
cerebellum (CBL), medulla oblongata (MO), etc.

Figure 2D - The circular olfactory rosette shows central raphe (r), two rows of lamella (l). The olfactory
lamella is large, triangular in shape with prominent dorsal margin (dm), dorsal end (de),
ventral margin (vm) and lingual process (lp).
Plate - 2
olfactory rosette perhaps adopt several type of
arrangement pattern (Holl, 1965). This lamellar
arrangement may help in the particular sensitivity to
certain components like amino acids, steroids,
prostaglandins, etc. (Theisen et al., 1991). Anatomically
the lamellar surface in S. commersonianus is much closer
due to the short distance between anterior and posterior
nostril than R. kanagurta and P. scaber. Therefore, the
water soluble odorants may travel short distance to
interact with comparatively greater olfactory lamellar
surface of S. commersonianus. The water ventilation is
assisted by the pumping mechanism of accessory nasal
sacs (Theisen et al., 1991) and may provide the ability
to sniff (Nevitt, 1991; Cox, 2008). R. kanagurta
possesses well developed accessory nasal sacs but
S. commersonianus and P. scaber has no accessory nasal
sacs. The movement of jaws and its associated muscles
are also very significant for the water ventilation
(Nevitt, 1991). Accessory nasal sacs may be found in the
olfactory organs of fishes with widely variable life styles
and habitats, both marine and fresh water which are not
confined to one particular situation (Cox, 2008).
The olfactory nerve may convey the chemical cues
during water ventilation to the brain (Hamdani and
Dving, 2007). The olfactory information plays an
important role in different behaviour of fish such as
Biswas et al., 2013
Plate - 3
Figure 3A - Schematic representation of Platycephalus scaber.

Figure 3B - The diagram shows anterior and posterior nostrils of P. scaber situated at a distance from each
other. Long nasal flap (FL) is also marked.

Figure 3C - The olfactory apparatus of P. scaber is comprised of olfactory rosette (OLR), olfactory nerve
(OLN), olfactory lobe (OL), cerebral hemisphere (CHS), optic lobe (OPL), cerebellum (CBL), medulla
oblongata (MO), etc.

Figure 3D - The circular olfactory rosette indicates central raphe (r) and two rows of lamella (l).
The olfactory lamella is triangular in shape with prominent dorsal margin (dm), dorsal end (de) and
proximal end (pe).

searching of foods, avoidance of predators,
discrimination between individuals of the same and
different, parental care, orientation in migration, etc.
(Hara, 1971). The olfactory system of teleosts is highly a
specialized structure for the recognition of various water
soluble chemical cues, so this may serve as a biological
model to monitor environmental health as well
as specific meagerness of the pollutants. The
xenotoxification of ocean especially the acidification
may also impair the ability of olfactory discrimination of
coastal and marine species (Munday et al., 2009). Thus,
it is necessary to examine the effect of specific toxic
agent at a subcellular level of olfactory structures in
marine teleolsts.

CONCLUSION
This comparative anatomical study on the
olfactory apparatus along with brain in three different
marine teleost belonging to the diverse ecological habitat
shows much structural variation according to the
changing environment which may be significant for
ecomorphology and evolutionary aspects of
neurobiology (Kotrschal et al., 1998). However, the
olfactory system of marine, estuarine and coastal or
migratory species may experience rapid fluctuations of
environmental inorganic ions (Hubbard et al., 2000), so
it could be an interesting part to identify the cellular
components that are involved in the ion regulation of the
olfactory apparatus in these migratory teleosts.

ACKNOWLEDGEMENTS
Authors are thankful to the Head, Department of
Zoology, Vidyasagar University, West Bengal, for
providing the necessary laboratory facilities.

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The use of purple yam (Dioscorea trifida) as a health-promoting
ingredient in bread making
Keywords:
Purple yam (Dioscorea trifida); antioxidant activity; health-promoting food;
Amazon region.
ABSTRACT:

The use of purple yam (Dioscorea trifida) was evaluated as possible
health-promoting ingredient in bread making in the state of Amazonas, Brazil. The
centesimal composition, energy, and antioxidant activity of purple yam and its
incorporated bread formulations (0%, 10%, 15% and 20%) were determined. An
acceptance test and microbiological analysis of the formulations 10%, 15% and 20%
were also performed. Except for lipids, the centesimal composition and caloric values
revealed no statistically significant differences. An addition of purple yam in natura up
to 20%, instead of wheat flour in ordinary bread (0%), can be made with no effect on
the diets energy. The free radical scavenging, 2.2-diphenyl-1-picryl-hydrazyl (DPPH)
and lipid per oxidation (LPO) methods revealed that the greater the percentage of
purple yam being added into the breads the higher the antioxidant activity detected.
The acceptance test applied to compare the three formulations of purple yam breads
revealed a significant difference only in the attribute colour. Purple yam breads
showed no preferable differences. Results highlight the feasibility of purple yam bread
as a health-promoting food in the Amazon region.
747-758 | JRB | 2013 | Vol 3 | No 1

cited.

Research Journal
Authors:
Teixeira AP
1
,
Oliveira IMA
1
, Lima ES
1

and Matsuura T
2
.

Institution:
1. Faculdade de Cincias
Farmacuticas (FCF),
Universidade Federal do
Amazonas (UFAM), Rua
Alexandre Amorim, 330,
Aparecida, CEP: 69010-330,
Manaus, AM, Brasil.

2. Instituto de Cincias
Biolgicas (ICB),
Universidade Federal do
Amazonas (UFAM), Av.
General Rodrigo Octvio
Jordo Ramos, 3000,
Campus Universitrio,
Coroado I CEP: 69077-000,
Manaus, AM, Brasil.

Antonia Paiva Teixeira.

Email:
nietapt@yahoo.com.br

Web Address:
http://www.jresearchbiology.com
document/ RA0306.pdf.
Dates:
Article Citation:
Teixeira AP, Oliveira IMA, Lima ES and Matsuura T.

The use of purple yam (Dioscorea trifida) as a health-promoting ingredient in bread
making.
Journal of Research in Biology (2013) 3(1): 747-758.
Original Research
INTRODUCTION
Yams belong to the family Dioscoreaceae, genus
Dioscorea (Pedralli, 1988; 1997; Pedralli et al., 2002;
Pedralli, 2004). This family is made up by 6 to 9 genera
comprising over 600 species distributed throughout the
Worlds tropical, subtropical and temperate regions
(Barroso et al., 1974; Pedralli, 1988; 1997; Melo
Filho et al., 2000; Pedralli et al., 2002; Pedralli, 2004).
The yams (Dioscorea spp.) yield tubers, which are very
important as staple, nutritional and healthy food, and are
still used as an ingredient in traditional Chinese herbal
medicine. They show a worldwide distribution, and are
found in many tropical countries, in South-Eastern Asia
and Western Africa, where the species were introduced
by cultivators (Rasper and Coursey, 1967; Akanbi et al.,
1996; Omonigho and Ikenebomeh, 2000; Lin et al.,
2005). They can also be found in some American
countries, particularly in Brazil, where one can find them
in all regions, from the Amazon down to the Southern
part of the country (Chu and Figueiredo-Ribeiro, 1991;
Pedralli, 1997; 2004).
Purple yam (Dioscorea trifida) is an American
native species, which was domesticated by Amerindians,
with the cultivar distribution possibly pointing out its
domestication in Brazilian and Guyana border areas,
followed by dissemination throughout the Caribbean
islands (Pedralli, 1988; Pedralli et al., 2002; Pedralli,
2004). D. trifida shows a wide distribution in Central and
South America, from the Caribbean to Peru. In Brazil it
is found all the way from the Amazon right down to the
Southern region. The species is associated to forest
environments-Amazonian highland tropical rainforests,
Coastal Atlantic Forest in Southeastern Brazil and,
mesophytic (seasonable) and gallery forests (Pedralli,
1997).
Here in the Amazonian region, purple yam
(D. trifida) may be consumed in the following ways:
baked, boiled, mashed, as ingredients for soups and meat
stews, and in the formulation of flour for making cakes,
pies and porridges. Nevertheless, this species has
undergone little scientific investigation, so little is known
about its management techniques, genetic improvement,
nutritional potential, industrial use, storage procedures,
characterization, uses as natural dye, as well as its use as
a health-promoting ingredient, among others.
By and large, the bread consumed throughout the
world is made mostly of wheat flour, salt and yeast.
Many other ingredients, have been incorporated into
bread formulation, so as to increase its diversity and
product appeals (Hsu et al., 2004).
A few studies have highlighted the great
potential of purple yam in bread making. In this case,
yam flour may replace part of the wheat flour, improving
bread quality, as well as adding economical advantages
to it (Abramo, 1990; Hurtado et al., 1997; Litvin et al.,
1998; Omonigho and Ikenebomeh, 2000; Ratti, 2001).
Hsu et al., (2004) demonstrated the presence
of antioxidants in the flour of purple yam
(Dioscorea purpurea), in five formulations of breads
prepared with this tubers flour, with excellent
acceptance in Taiwan supermarkets. Contado et al.,
(2009) showed yam (Dioscorea spp.) mucilage-based
loaf to present good public acceptance as to flavor,
aroma and texture with sensory attributes, demonstrating
the use of this tuber to be feasible as improvers in bread
making.
The following aspects motivated the use of
purple yam (Dioscorea trifida) in natura as a bread
manufacturing health-promoting ingredient, in the
present work: 1) its significant world consumption,
presenting a considerable, expanding tillage alternative
(Rasper and Coursey, 1967; Abramo, 1990; IITA, 2007);
2) although, as yet incipient, an increase on the
production of this tuber in the State of Amazonas, Brazil,
especially in Caapiranga and Careiro Castanho
municipalities is being observed. According to the
Instituto de Desenvolvimento Agropecurio do Estado
do Amazonas (IDAM) in 2008, 110 families of the
Teixeira et al., 2013
Caapiranga municipality yielded 2,475 tonnes in an area
of 165 ha; and 3) the presence of antioxidants in purple
yam, which increases the nutritional capacity in breads
made from this tuber (Hsu et al., 2004).
The main aim of the present study was to
evaluate the potential of purple yam yield in the State of
Amazonas, Brazil as a health-promoting ingredient in
bread making. On this context, it determined the
centesimal composition, caloric value, and antioxidant
properties of purple yam as well as of breads made from
this tuber in natura. Then, it undertook an organoleptic
characteristic assessment of the breads, following tasters
panel acceptance criteria. This purple yam species is, for
the very first time, being used in the Amazonian region,
as a feasible alternative for bread making.

Species identification and purple yam tuber
(Dioscorea trifida) collection
Identification of the species Dioscorea trifida
was accomplished by comparisons with a voucher
herbarium specimen (Exsicata number 1353) deposited
at the National Research Institute of Amazonia (INPA)
Herbarium. It is very common to find the purple yam
(D. trifida) exhibiting several color hues of its flesh
(edible portion), in Amazonas State Townships. The
types most easily identified are: roxinho (light purple
flesh); roxo (mid purple flesh); roxo (dark purple flesh);
branco (white flesh); and misto (white-purple flesh)
(Figure 1).
Purple yam samples were collected at two
Amazonas Townships: Caapiranga and Careiro
Castanho. Due to the seasonality and availability of these
tubers in the region, the centesimal composition analyses
of yams and breads were performed with Caapiranga
samples. Yam and bread antioxidant and bread sensory
and microbiological analyses were carried out with
Careiro Castanho samples.
Purple yam bread elaboration
On account of the probability of getting breads
with higher antioxidant concentration (Hsu et al., 2004),
roxo (dark purple flesh) type samples were used in the
present study (Figure 1C). Yams in natura, for replacing
wheat flour, were washed, peeled, weighed, ground
in the liquidizer together with yeast, oil and water.
Then, this mixture was added to the previously mixed
dry ingredients (wheat flour, powdered milk, sugar and
salt). Bread manufacturing formulations can be seen at
(Table 1). Homogenization (30 min.), dough underwent
initial fermentation (60 min.), intermediate time for
Teixeira et al ., 2013
B
A
C
D
E
Figure 1. Flesh color varieties of the kinds of purple yam (Dioscorea trifida)
commonly found in fairs and markets of Manaus-AM. A) roxinho (light-purple
flesh); B) roxo (mid-purple flesh); C) roxo (dark purple flesh);
D) branco (white flesh); and E) misto (white-purple flesh).

bread shaping (25 min.), final fermentation (60 min.),
time for baking (30 min.) are followed for making yam
bread. After being prepared the breads were cooled to
room temperature and packed in polyethylene bags
displaying the products labeling.
Centesimal composition analyses of purple yam and
its incorporated breads
Centesimal composition analyses of purple yam
(Dioscorea trifida), and purple yam incorporated breads
in four formulations: 0%, 10%, 15% and 20%, were done
in triplicate. Moisture, ashes, lipid, proteins and crude
fiber contents were determined according to procedures
described by the Instituto Adolfo Lutz-IAL (2008).
Carbohydrate and caloric values were determined
according to the method of (AOAC, 2005).
Findings obtained on the bread formulation
centesimal composition analyses were subjected to
Table 1. Purple yam (D. trifida) incorporated
bread formulations.
Ingredients Type of bread
*

0% 10% 15% 20%
Wheat flour (g) 500 450 425 400
Purple yam (g) 0 50 75 100
Sugar (g) 10 10 10 10
Salt (g) 5 5 5 5
Yeast (g) 10 10 10 10
Milk powder (g) 10 10 10 10
Oil (g) 10 10 10 10
Water (mL) 250 250 250 250
Total (g)

545 545 545 545
*
Percentage of wheat flour replaced by purple yam.
Total amount of ingredients used for preparing the

breads.

Parameters
Yam
(D. spp.)
*

Yam
(D. alata)
**

Purple Yam
(D.trifida)
***

Moisture (%) 72.60 73.70 76.43 0.50
Lipid (%) 0.20 0.10 1.13 0.69
Protein (%) 2.00 2.30 1.83 0.13
Crude Fiber
(%)
0.60 7.30 1.80 0.05
Ash (%) 0.90 0.90 0.78 0.02
Carbohydrate
(%)
24.30 23.00 18.04 0.66
Caloric value
(Kcal/100g)
100.00 96.00 89.64 4.52
Table 2. Yam centesimal composition.
*
(Montaldo, 1977),
**
(TACO 2006),
***
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statistical analysis through the statistical software
package (Statsoft STATISTICA 8.0 2007). Given to the
number of sampled observations (n=3), Kruskal-Wallis
ANOVA and post hoc tests were applied as a
non-parametric alternative to Fisher ANOVA, for
independent data, in the comparison among the bread
formulations.
Findings showing significance level of (P<0.05)
were considered as statistically significant.
Preparation of purple yam and its incorporated
breads methanolic extract
Samples of purple yam (Dioscorea trifida),
were peeled and ground with the aid of a knife. They
were then dehydrated in a laboratory oven at 60C for
24 h. Purple yam incorporated breads of four
formulations: 0%, 10%, 15%, and 20%, were cut into
1 cm thick slices, and dehydrated in a laboratory oven at
40C for 24 h (Hsu et al., 2004). Dehydrated yams and
breads were ground with pestle and mortar, weighed at
0.125, 0.25, 0.5 and 1.00 g (40, 80, 160 and 330 mg/mL,
respectively). They were placed into small test tubes
added with 5 mL of methanol and left in a rotary shaker
for 24 h. The material was centrifuged at 2,500 RPM
for 10 min so as to obtain the supernatant (methanolic
extract). The antioxidant activity of the samples was
determined by the free radical scavenging, 2.2-diphenyl-
1-picryl-hydrazyl (DPPH) and lipid peroxidation (LPO)
methods. The latter method evaluates the inhibition of
free radicals generated during the linoleic acid
peroxidation, and is based on spectrophotometric
measurements of discoloration (oxidation) of -carotene,
induced by linoleic acid oxidative degradation products
(Marco, 1968; Miller, 1971; Duarte-Almeida et al.,
2006).
Antioxidant activity determination through free
radicals scavenging methods (DPPH) in purple yam
and its incorporated breads
DPPH method, following the methodologies
described by Shimada et al., (1992) and Hsu et al.,
(2004), with some modifications, where 2 mg of DPPH
were dissolved into 15 mL of methanol, and applied so
as to determine the antioxidant activity of samples of
purple yam and its incorporated breads in the four
aforementioned formulations. A micro plate bearing
96 well was used. Thirty microliters (30 L) of the
methanolic extract, plus 170 L of methanol (used as the
blank) were placed in the wells. The reading was
performed on an Elisa reader (DXL 800-BECKMAN
COULTER) at a wavelength of 492 nm, using triplicate
samples. Then, 100 L of the DPPH solution were
added, and the material was stored in a dark place for
30 min, and the reading was repeated as soon as this time
was over. Two hundred microliters (200 L) of methanol
added to 100 l of the DPPH solution were used as
the control. Thirty microliters (30 L) of quercetin
(10 g/mL), 170 L of methanol and 100 l of the
DPPH solution, were used as the standard. The following
formula was used so as to calculate the antioxidant
activity percentage
Antioxidant activity determination through the lipid
peroxidation (LPO) method in purple yam and its
incorporated breads
The determination of the antioxidant activity of
the samples through the LPO method was carried out
according to the method reported by Duarte-Almeida
et al., (2006), based on the methodology originally
described by Marco (1968), and later modified by Miller
(1971). The reactive mixture was prepared in
an Erlenmeyer flask, containing 50 L of linoleic acid,
200 L of tween 80 (emulsifying agent), 150 L of
-carotene solution at 2 mg/mL in chloroform, and
500 L of chloroform. The mixture was then subjected to
evaporation in nitrogen till there was no more
chloroform left. Later, the mixture of 25 mL of
previously oxygen saturated water was added, and during
A sample - A blank
% AA = 100 - x 100
A control

a period of 30 min it was homogenized through vigorous
shaking.
The reactive mixture showed to be clear with
absorbency ranging from 0.6 to 0.7 at a wavelength of
492 nm. A 96 well bearing micro plate was used. Two
hundred forty microliters (240 L) of the reactive
mixture and 10 L of the methanolic extract samples
were placed in the wells. Ten microliters (10 L) of
methanol and an equal volume of butylhydroxytoluene
(BHT) at a concentration of 40 g/mL were used as
control and standard, respectively. The micro plate was
incubated at 50C to speed up the oxidation reactions and
start -carotene discoloration. Discoloration slope
readings of samples, control and BHT (in triplicate) were
performed readily, in an Elisa reader at a wavelength of
492 nm every 15 min for 135 min. The following
formula was used so as to calculate the oxidation
inhibition percentage:
Sensory analysis of purple yam incorporated breads
The acceptance test of purple yam in natura
incorporated breads counted with the participation of 78
non-trained volunteer judges. Each one of them was
provided with an answering card bearing a 9 point
hedonic scale (9-like extremely to 1-dislike extremely),
adapted from Stone et al., (1993) and Silva et al., (2005).
The judges were provided with three purple yam
incorporated bread samples, produced from three
formulations (10%, 15% and 20%) (Table 1). Samples
were served in white, disposable plastic plates; encoded
with three randomly chosen numbers. Samples were
evaluated according to their sensory qualities: global
feel, aroma, flavor, color and texture. Judges were
advised to always rinse their mouth with water before
testing the next sample.
The findings obtained on the acceptance test
were submitted to statistical analysis through statistical
software package (Statsoft STATISTICA 8.0 2007). The
Shapiro-Wilk test rejected the frequency distribution
normality of the three tested bread formulations, in all
their sensory attributes. However, the Levene test
accepted the homocedasticity (homogeneity of variances)
among the formulations for all sensory attributes. As
frequency distribution normality and variance
homogeneity are basic assumptions made for the
application of parametric tests, such as Fishers
ANOVA, and as these assumptions were not attended to,
the Friedman ANOVA followed by post hoc tests were
applied as a non-parametric alternative for paired data in
bread comparisons. Findings presenting significance
level of (P<0.05) were considered as statistically
significant.
Microbiological analysis of purple yam breads
Following the recommendation of the
Brazilian National Health Surveillance Agency
(in Portuguese, Agncia Nacional de Vigilncia
Sanitria, ANVISA), based on Ruling Number 12 (RDC,
2001), we carried out the microbiological analysis so as
to verify Coliforms and Salmonella in samples of the
three purple yam bread formulation samples through the
membrane filtration method (APHA, 2001).

RESULTS AND DISCUSSION
Centesimal composition and caloric value of purple
yam
Moisture (76.430.50), protein (1.830.13) and
ash (0.780.02) contents, as well as the caloric value
(89.644.52) of purple yam (D. trifida) samples analyzed
in the present study (Table 2) show to be near
those presented by Montaldo (1991) for yam
(Dioscorea spp.) and those found in the Brazilian
Food Composition Table TACO (2006), for the yam
(D. alata). Lipid content (1.130.69) stayed well above
that presented by Montaldo (1991) and TACO (2006).
Crude fiber content (1.800.05) is above the value
observed by Montaldo (1991), and well below that
A2 sample - A1 sample
% I = 100 - x 100
A2 control - A1 control
presented in TACO (2006). The high fiber content
presented by TACO (2006) might be due to the
enzymatic gravimetric method employed in the analyses.
That method warrants a higher precision for determining
the dietary fiber as compared to the acid digestion
methodology used in the present study as well as
by Montaldo (1991). Total carbohydrate content
(18.040.66) is well below Montaldo (1991) and TACO
(2006) values. The remaining differences in centesimal
composition values presented by Montaldo (1991) and in
the present study might be related to the different soil
types being employed on planting the tubers and/or to the
different species being utilized. Nevertheless, the
different values presented in TACO (2006) may be
related to the different yam species being analyzed.
Centesimal composition and caloric value of purple
yam incorporated breads
Based on data from Kruskal-Wallis (ANOVA)
followed by post hoc tests (Table 3), it may be asserted
that, except for the lipids (P<0.05), all other centesimal
composition and caloric values of the four purple yam
incorporated bread formulations (0%, 10%, 15% and
20%) showed to be statistically similar (P>0.05). That is,
replacing wheat flour by purple yam in natura in up to
20% neither modifies bread centesimal composition nor
caloric value. As for lipid, statistically significant
difference was only observed for 10% and 20%
formulations; this negligible 0.5% difference may be
neglected in technological applications.
Purple yam incorporated breads centesimal
composition and caloric value were compared to those of
ordinary bread loaf (OBL) (Anton et al., 2006) and
whole bread loaf (WBL) (TACO, 2006) (Table 4). One
notices, a high fiber content (6.90%) in the whole bread
loaf (WBL) (TACO, 2006), relative to the remaining
breads. It can be highlighted that in whole bread
composition, we have the presence of grain-composed
whole flour, almost wholly made up of bran, germ and
endosperm (FDA, 2006). By and large, all other values
show to be approximate. All differences found may be
related to formulations employed in the preparation of
those breads.
Antioxidant activity determination through the free
radical scavenging method (DPPH) in purple yam
and its incorporated breads
Antioxidant activity (% AA) of the methanolic
extract pertaining to purple yam (Dioscorea trifida)
Bread
Moisture
(%)
Lipid
(%)
Protein
(%)
Crude Fiber
(%)
Ash
(%)
Carbohydrate
(%)
Caloric value
(kcal/100 g)
0% 29.79 4.49 11.62 1.95 1.18 50.95 290.73
10% 31.03 4.24 10.67 1.91 1.41 53.41 294.45
15% 32.65 4.87 9.82 1.84 1.38 49.45 280.64
20% 35.09 4.74 10.06 2.34 1.20 53.43 269.17
OBL 34.46 1.93 9.42 2.57 2.09 52.10 247.50
WBL 34.70 3.70 9.40 6.90 2.30 49.90 253.00
Table 4. Centesimal composition and caloric value of ordinary bread loaf (OBL) (Anton et al., 2006),
whole bread loaf (WBL) (TACO, 2006) and purple yam (D. trifida) incorporated breads at 0%, 10%,
15% and 20% (present study).
0
10
20
30
40
50
60
70
80
90
100
40 80 160 330
Concentration (mg/mL)
A
n
t
i
o
x
i
d
a
n
t

a
c
t
i
v
i
t
y

(
%
)

Purple yam
0%Bread
10%Bread
15%Bread
20%Bread
Quercetin
Figure 2. Antioxidant activity expressed by free
radical scavenging percentage, of samples of purple
yam (Dioscorea trifida) and its incorporated bread
extracts in four formulations: 0%, 10%, 15% and
20%, as determined by DPPH method. The quercetin
was used as standard control. Bars indicate standard
deviation.

samples in the concentrations of 330, 160, 80 and
40 mg/mL, as determined by the DPPH method, were
higher than 70%, reaching a maximum of 88.130.12.
This plainly shows this species to exert DPPH radical
scavenging activity (Figure 2). This same figure reveals
purple yam incorporated breads prepared in 10%, 15%
and 20% formulations, to also present a certain
antioxidant activity, reaching 43.321.18; 48.131.17
and 53.711.01 maximum percentile values,
respectively. Those findings are above the values
presented by Hsu et al., (2004) (20-40% approximately),
who used breads of several formulations prepared with
flour from the purple yam tuber (Dioscorea purpurea)
representing the one with the widest variety in Taiwan,
for substituting part of the wheat flour. Bread prepared
with no purple yam at all (0%) showed certain
antioxidant activity, as well, probably due to Maillard
reaction products, where, some hot processed foods,
present free radical scavenging activity (Kim et al.,
2007; Jing and Kitts, 2000; Hsu et al., 2004; Michalska
et al., 2008). Corroborating data from Hsu et al., (2004),
it was confirmed that the antioxidant activity rose as the
percentage of purple yam substituting wheat flour
increased. The high free radical scavenging activity
observed by Hsu et al., (2004) in flour of Taiwan purple
yam (D. purpurea), was also detected in the Amazonian
regions purple yam (D. trifida).
Antioxidant activity determination through the lipid
per oxidation (LPO) method in purple yam and its
incorporated breads
Discoloration slope (Figure 3) and free radical
inhibition activity (Figure 4) determined through the
LPO method confirmed the antioxidant activity (% I) in
purple yam (55.804.85) and its breads from the three
formulations (10%, 15% and 20%), with the values of
46.164.90; 48.203.72 and 49.132.79, respectively.
Tests Bread% Color Aroma Flavor Texture Overall impression
Shapiro-Wilk P
10 0.0009 < 0.0001 0.0023 0.0044 0.0001
15 < 0.0001 0.0009 0.0008 0.0019 < 0.0001
20 < 0.0001 0.0001 0.0002 0.0090 0.0005
Levene P 0.0519 0.5580 0.2306 0.8415 0.5184
Table 5. Probability (P) values calculated from Shapiro-Wilk and Levene tests for evaluating frequency
normality and homogeneity of variances, respectively, of the data obtained in the sensory analysis of the
three tested purple yam incorporated bread formulations.
Values were considered statistically significant at (P< 0.05).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 15 30 45 60 75 90 105 120 135
Time (min)
A
b
s

4
9
2
Blank
Purple yam
0%Bread
10%Bread
15%Bread
20%Bread
BHT
Figure 3. Discoloration slope of purple yam
(Dioscorea trifida) and its incorporated bread
extracts in four formulations: 0%, 10%, 15%, 20%,
blank and BHT, as determined through the LPO
method.
0
10
20
30
40
50
60
70
80
90
Purple yam 0%Bread 10%Bread 15%Bread 20%Bread BHT
I
n
h
i
b
i
t
i
o
n

(
%
)
Figure 4. Inhibition percentage of free radicals of
purple yam (Dioscorea trifida) and its incorporated
bread extracts in four formulations: 0%, 10%, 15%,
20%, and BHT as determined by LPO method. Bars
indicate the standard deviations.
As it was observed by the DPPH method, the
antioxidant activity rose as the percentage of purple yam
substituting wheat flour in the breads increased.
Moreover, bread with no addition of purple yam (0%)
presented some antioxidant ability which might have
resulted from the development of Maillard reaction
products (Kim et al., 2007; Jing and Kitts, 2000; Hsu
et al., 2004; Michalska et al., 2008). Anthocyanins might
be partly responsible for the antioxidant activities
detected in the purple yam (D. trifida) and its
incorporated breads analyzed in the present study, since
these pigments were detected in purple yams, D. alata
(Rasper and Coursey, 1967) and D. trifida L.
(Carreno-Diaz and Grau, 1977; Escudero et al., 2010).
In fact, polyphenols and anthocyanins, usually detected
in plants, might be the active components for this
antioxidant activity in yams (Hou et al., 2001; Hsu et al.,
2004).
Sensory analysis of purple yam incorporated breads
Table 5 shows the rejection of the frequency
distribution normality of the three tested purple yam
incorporated bread formulations (10%, 15% e 20%)
through the Shapiro-Wilk test, and the acceptance of the
homocedasticity among the formulations through the
Levene test on all sensory attributes evaluated in the
acceptance test (i.e. statistically significant values at
(P <0.05).
Friedman ANOVA followed by post hoc tests
applied for comparing the three purple yam incorporated
bread formulations revealed a significant difference
(P<0.05), only for the colour attribute (Table 6). The
bread at 20% presented a better evaluation regarding the
remaining ones, probably due to the higher purple yam
concentration, which gives the final product a more
attractive kind of color. It was observed that the larger
the purple yam amount being added to the bread the
higher the mean score obtained (values ranging from
6.15 to 6.97).
Choosing a determined food should depend
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mainly on its nutritional value. Nevertheless, color,
aroma and texture are the factors usually guiding the
consumers preference rate. Of these three factors, color
interferes the most on the products preference (Bobbio
and Bobbio, 2001).
Given that there were no preferential differences
among the other sensory attributes, the three breads
evaluated can be considered approved.
Microbiological analysis
Considering that the microbiological analysis
was negative for Coliforms and Salmonella (Table 7), the
purple yam incorporated breads may be considered
proper for human consumption, as long as they have
been properly handled.

CONCLUSIONS
Through such findings, one concludes that any
of the purple yam incorporated breads tested in the
present study (10%, 15% and 20%), can substitute
ordinary bread (0%), with no effect on the diets caloric
value, since their centesimal compositions are similar.
Due to the presence of antioxidants in purple yam
incorporated bread, and to their ability to fight free
radicals, those breads can be considered as
health-promoting food. Furthermore, these findings point
out the feasibility of the consumption of purple yam
incorporated bread, as an alternative in the local bread
making industry, and an incentive to a larger production
of this tuber in the Amazonian region.

ACKNOWLEDGEMENTS
The authors are indebted to the Fundao de
Amparo Pesquisa do Estado do Amazonas (FAPEAM)
for the master scholarship granted to Antonia Paiva
Teixeira. To Dr. Antonio Jos Inhamuns da Silva and
MSc. Cynthia Tereza Corra da Silva of Universidade
Federal do Amazonas (UFAM) for kindly having
allowed to carry out the centesimal composition analyses
in their laboratories. To MSc. Antonio Fbio Lopes de
Sousa, Arleilson de Sousa Lima and Ana Cludia dos
Santos for their invaluable aid in undertaking of the
laboratory analyses. To Misters Claudio Adriano
Cardoso Amanajs and Francisco de Oliveira Batista for
their logistical support in the collection of purple yam
samples.

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Bioefficacy of Novaluron
, a chitin synthesis inhibitor against the tropical

warehouse moth, Ephestia cautella.
Keywords:
Novaluron, Hocklicombi, Ephestia cautella, warehouse moth, chitin,
loss assessment.
ABSTRACT:
The tropical warehouse moth, Ephestia cautella (Lepidoptera: Pyralidae) is a
major pest of stored maize in Ghana. It is controlled mainly by the use of synthetic
insecticides which has become a major challenge in the stored product industry in
Ghana. Both laboratory and field trials were conducted to evaluate the efficacy of
novaluron, a chitin synthesis inhibitor against E. cautella. Five concentrations of
Novaluron (0.1, 0.2, 0.3, 0.4 and 0.5 mL/L of water) were prepared and each
concentration was topically applied on the notal regions of 10 fifth instar larvae of
E. cautella per concentration. At 0.4 mL/L and 0.5 mL/L treatments, larval mortality
ranged between 50-80% after 96 h of exposure. Also, Novaluron (0.5 mL/L) was used
to treat four surfaces (concrete, wood, glass and plastic) usually encountered in
structural insect pest management systems and the larvae exposed to these surfaces.
Hocklicombi (5 mL/L) served as positive control. Larval mortality (35.5-97.5%),
pupation (0.0-35.0%) and adult emergence (0.0-20.0%) in surfaces treated with
Hocklicombi compared favourably with those treated with Novaluron (25.0-97.5%),
(2.5-60%) and (0.0-42.5%), respectively. A simulated field experiment was conducted
in which four batches of 5 kg of maize in miniature bags were pretreated with
0.4 mL/L Novaluron and 50 unsexed adults were introduced. This was left in a crib at
the University of Ghana farm for 60 days. The field experiment showed that after
60 days of storage there was a lower weight loss in the Hocklicombi (6.6%) and
Novaluron (6.8%) treatments compared to the negative control (11.3%).
759-767 | JRB | 2013 | Vol 3 | No 1

cited.
Research Journal
Authors:
Sackey I, Eziah VY and
Obeng-Ofori D.

Institution:
Department of Crop Science,
College of Agriculture and
Consumer Sciences, P. O.
Box LG 44, University of
Ghana, Legon.

Eziah VY.

Web Address:
http://jresearchbiology.com/
documents/RA0305.pdf.
Dates:
Article Citation:
Sackey I, Eziah VY and Obeng-Ofori D.
Bioefficacy of Novaluron, a chitin synthesis inhibitor against the tropical warehouse
moth, Ephestia cautella.
Original Research

INTRODUCTION
Maize (Zea mays) is one of the major staple food
crops in Ghana and it is susceptible to attack by several
insect pests including the tropical warehouse moth,
Ephestia cautella Walker (Lepidoptera: Pyralidae)
(CAB1, 2006). Ephestia cautella larva feeds on stored
products, damaging the product directly and form webs
on the surface. The webbing contains larval excreta and
exuviae which give unpleasant odour to the infested
commodity. Older larvae may leave the food to find
pupation sites in wall cracks.
In Ghana, E. cautella is controlled by the use of
residual insecticides usually, synthetic pyrethroids and
fumigants (CABI, 2006). The adverse effects of residual
pesticides such as poisoning, environmental and health
hazards and resistance development cannot be
overemphasized (Obeng-Ofori, 2007). Hence the use of
residual insecticides in stored product protection is
challenging. There is therefore, the need for new cost and
environment friendly alternatives with no adverse effect
on non-target organisms (Obeng-Ofori, 2007; Arthur and
Phillips, 2003). Some of these alternatives include
botanicals, insect growth regulators, microbial pathogens
among others (Arthur, 1996).
Novaluron (Rimon
10 EC) is a benzoylphenyl
urea group of insect growth regulators and a chitin
synthesis inhibitor. Novaluron has been registered as an
insecticide for food crops in several countries including
South Africa, Australia and Ghana (WHO, 2003; EPA,
2006). In Ghana, novaluron has been successfully used
in the laboratory against stored product pests
such as the rice moth, Corcyra cephalonica
Stainton (Lepidoptera: Pyralidae) (Sarbah, 2006), the
red flour beetle, Tribolium castaneum Herbst
(Coleoptera: Tenebrionidae) (Bakudie, 2006) and the
tropical warehouse moth, E. cautella (Ibrahim, 2008).
In Ghana, most of the work done on novaluron
focused on evaluating the effect of the chemical on
different developmental stages of insects in the
laboratory. There is no information on the use of
novaluron for stored product protection in warehouses or
cribs. Ephestia cautella is noted for feeding directly on
the grains and also, the mature larvae leave the
commodity in search of pupation sites in crevices, cracks
and storage containers. Therefore, treating these surfaces
to which the insect may be exposed will go a long way to
mitigate the losses caused by this pest. Hence, screening
Novaluron against E. cautella using the commodity and
storage surfaces as substrates is crucial in the
management of the pest. Such findings will contribute to
the efforts by farmers and warehouse managers to reduce
storage losses and contribute to the attainment of food
security in Ghana.
This study presents laboratory and field tests that
were carried out to determine the toxicity of novaluron to
the 5
th
instar larvae of E. cautella. Other tests were also
conducted to assess the efficacy of novaluron on
different surfaces against immature stages of E. cautella.

Insect cultures
The test insects were obtained from the
Entomology laboratory of the Crop Science Department.
Adult E. cautella were cultured on mixed substrate made
up of wheat powder, maize flour and glycerol (5:5:1).
Fifty adult E. cautella were introduced into each jar and
left under laboratory conditions of 272C and 55-60%
relative humidity for 30 days to allow for the
development of larval E. cautella. The set up was placed
on trays containing industrial oil to prevent the crawling
of other insects into the culture. The insects were reared
and handled using ethically acceptable standard
procedures in the laboratory.
Test chemicals
Novaluron (Rimon

10EC), 1-[3-chloro-4-(1, 1,
2-trifluoro-2-trifluoromethoxy-ethoxy) phenyl]-3-(2, 6
difluorobenzoyl) urea, produced by Makhteshim-Agan
Ltd (Israel) was used for the toxicity experiment and
Sackey et al., 2013
Hocklicombi (Hockley International Ltd. Poynton,
Stockport, U. K.) which contains 25% Fenitrothion and
5% Fenvalerate was used as a reference product.
Contact toxicity test
We adopted the method by (Eziah et al., 2011).
Concentrations of Novaluron (0.1, 0.2, 0.3, 0.4 and
0.5 mL/L) and 5 mL/L of Hocklicombi
were diluted in
distilled water and used for the assays. Distilled water
was used as negative control. Fifth instar larvae of both
sexes were transferred into clean Petri dishes and the
different dosages of the various concentrations was
topically (1 L) applied to the notal regions of the larvae
using a micro applicator. Each experimental unit
consisted of 10 larvae and was replicated for four times.
The treated insect larvae were then transferred into glass
petri dish containing food. The insect larvae were
examined for mortality 24, 48, 72, 96 h, 7 days and
14 days after treatment. Criterion for death was as
described by (Lloyd, 1969) in which insects were
presumed dead when they failed to move in a
coordinated manner after prodding with a blunt probe.
Data collected include larval mortality, percent pupation
and percent adult emergence were done after various
treatments and exposure periods.
Surface treatment
The surfaces chosen for the study were concrete,
plywood, glass and plastic which are among the
common surfaces encountered in structural insect pest
management. Individual concrete exposure arenas were
created in square bottoms of plastic containers (6x6 cm)
using a concrete patching material. Water-based slurry
was prepared by mixing 1 kg of Portland cement to 2 kg
of sand and 1 L of tap water and pouring 10 mL of the
slurry into the bottom of the plastic container to create a
treatment arena (Arthur, 1998b). Plywood arenas were
made by cutting rectangular disks from 1.25 cm thick
plywood to fit the plastic container then caulking the
margins to prevent the larvae from escaping the surface.
Plastic containers served as plastic surfaces and petri
dishes were used as glass surfaces for the surface
treatment.
Each of the four surfaces was treated with 4 mL
of water (negative control treatment) or an aqueous
solution of novaluron (0.5 mL/L) and Hocklicombi

(5 mL/L) (positive control). All treated arenas were
allowed to dry overnight and fifth instar larvae (N=10) of
E. cautella were exposed for 48 h. The larvae were then
transferred to new petri dishes containing food under
laboratory conditions of 272C and 55-60% relative
humidity. Post-treatment survival and mortality were
recorded daily. Number of surviving larvae that
successfully pupated and those that successfully emerged
as adults were recorded.
Field experiment
Maize grains were obtained from the Madina
(a suburb of Accra, Ghana) market and sieved to remove
all debris. Maize grains (5 kg) were sterilized in the oven
at 70C for 3 h after which they were left in desiccators
to cool. The grains were then treated with 0.4 mL/L
Novaluron or 5 mL/L Hocklicombi
. These dosages had

proven effective in laboratory experiments. Grains
treated with distilled water served as negative control.
Each treatment was replicated four times. Fifty unsexed
adults of E. cautella were put onto the treated grains in
each sack. The sacks were securely sealed by stitching
and stored in a grain crib at the University farm for
60 days. Prior to their treatment, subsamples were taken
from each sack for moisture and weight loss analyses
using the standard volume method was carried out
(Boxall, 1986). At the end of the storage period, the
contents of the sacks were sieved. The number of
both live and dead adult insects was recorded. Also,
subsamples of the maize grains were collected for
moisture and weight loss analyses as stated earlier.
Statistical analysis
Data involving percentages were arcsine
transformed and were analyzed using the Analysis
of Variance (ANOVA) with Genstat 9. 2
Sackey et al., 2013

(Lawes Agricultural Trust, 2007). Means were separated
using the Least Significant Difference (LSD) test at
5% probability level.

RESULTS
Contact toxicity test
The percent larval E. cautella mortality
following treatment with Novaluron and Hocklicombi

are presented in Table-1. Larval mortality varied with
insecticide concentration and exposure period. Lower
dosages of Novaluron (0.1-0.3 mL/L) caused less
than 50% larval mortality after 96 h of exposure
(Table 1). In contrast, novaluron concentrations of
0.4 mL/L and 0.5 mL/L caused between 50% to 80%
larval mortality after 72 to 96 h of exposure. After 96 h
exposure period, all dosages of Novaluron induced
significantly (p = 0.05) higher larval mortality compared
to the negative control. However, there was no
significant difference in larval mortality between 5 mL/L
Hocklicombi
and 0.5 mL/L Novaluron treatments. Also,

novaluron applied at 0.4 ml/L and 0.5 mL/L did not
differ significantly from each other after 96 h of
exposure.
Pupation and adult emergence of E. cautella
were observed in all insecticide treatments and the
negative control with the exception of Hocklicombi

treatment. The percentage pupation in larvae treated with
0.1 mL/L Novaluron (57.5-65.0%) was not significantly
different from the negative control (64.0-80.0%)
(Figure 1). However, all other concentrations of
Novaluron higher than 0.1 mL/L significantly (p = 0.05)
impaired pupation. There was no significant difference in
pupation 7 days after the exposure of E. cautella larvae
to 0.5 mL/L Novaluron and 5 mL/L Hocklicombi.
Also, after 14 days, percentage pupation recorded in
larvae treated with 0.4 mL/L and 0.5 ml/L was
comparable.
All levels of Novaluron concentrations
significantly reduced the development of F
1
of adult
E. cautella (Figure 2). The highest adult emergence
(77.5%) was recorded in the negative control and this
differed significantly (p = 0.05) from all other novaluron
concentrations applied. As concentration increased from
0.1 mL/L to 0.5 mL/L, adult emergence significantly
(p = 0.05) reduced from 50 to 2.5%. Also, the effect of
novaluron applied at 0.5 mL/L was comparable to
Hocklicombi
treatment in impairing the development of

adult E. cautella.
Surface treatment
Ephestia cautella larvae exposed on concrete
surfaces treated with Novaluron showed a lower
mortality than those exposed to concrete surfaces treated
with Hocklicombi
(Table 2). Mortality was also lower

on plastic and wood treated surfaces compared to
Hocklicombi treated surfaces. However, the percentage
mortality of E. cautella on glass surfaces treated with
Sackey et al., 2013
Treatments (ml/L) Mean(s.e) % larval mortality (h)
24 h 48 h 72 h 96 h
Control (Water) 0.00.0 0.00.0 0.00.0 0.00.0
5.0 mL/L (HC) 87.50.1 87.50.1 87.50.1 87.50.1
Novaluron
0.1 7.50.0 10.00.0 17.50.1 22.50.1
0.2 10.00.0 12.50.0 27.50.1 42.50.1
0.3 17.50.1 25.00.1 35.00.1 45.00.2
0.4 17.50.1 32.50.1 50.00.1 66.00.1
0.5 32.50.1 47.50.1 65.00.1 80.00.0
LSD (P < 0.05) 18.35 18.40 14.50 14.83
HC= Hocklicombi

s.e = standard error
Table 1 Mortality of larval E. cautella (%) after treatment
with novaluron and Hocklicombi
insecticides
novaluron was the same (97.5%) as those treated with
Hocklicombi. Surviving larvae were observed for
pupation and adult emergence. Fewer E. cautella larvae
pupated after exposure to concrete, plastic and wood
surfaces treated with Hocklicombi
but no pupation was

recorded on glass surfaces treated with Hocklicombi

(Table 3)
Fewer larvae pupated in glass surfaces-treated
with novaluron and this was not significantly different
from Hocklicombi
-treated glass surfaces. Generally,

percentage adult E. cautella that emerged was greater on
the untreated control for all the surfaces and differed
significantly (p = 0.05) from all insecticide treated
surfaces (Table 4). Mean percentage adult emergence of
E. cautela observed on glass and plastic surfaces treated
with novaluron and Hocklicombi
ranged from 0.0 to

25%. Thus, residual effects of novaluron and
Hocklicombi
significantly reduced the development of

E. cautella on glass and plastic surfaces.
Field experiment
Table 5 shows the dry weight loss of the treated
grains after 60 days of storage using the standard volume
method. Lower weight losses were observed in grains
treated with insecticides (6.6-6.8%) compared to grains
which were not treated (11.3%) using standard volume
methods.
DISCUSSION
The present study showed that Novaluron
concentrations of 0.4 mL/L and 0.5 mL/L significantly
affected the metamorphosis of E. cautella to the adult
stage. The effectiveness of novaluron at these dosages
compared favourably with Hocklicombi
. The insect
growth regulators ability to regulate metamorphosis in
the larvae through contact by topical application is
consistent with its mode of action. Tomlin (2005)
reported that novaluron was very effective on the larvae
of insects when absorbed by ingestion and contact
activity. The author also reported that the compound
causes abnormal endocuticular deposition and abortive
moulting.
Although pupation and adult emergence were
observed in all treatment levels, most of the larvae
treated with 0.4 mL/L and 0.5 mL/L Novaluron could not
emerge into adults 23 days after treatment. This may be
attributed to abnormal endocuticular deposition and
abortive moulting in the larvae (Tomlin, 2005). Also,
when cocoon covering the pupae were slightly removed,
pupae found were malformed compared to those in the
Sackey et al., 2013
Mean (%) s.e mortality
Insecticide Type of surface
Concrete Glass Plastic Wood Means
Control 0.0 0.0 0.00.0 2.5 0.0 0.0 0.0 0.70.0
Hocklicombi
43.0 0.1 97.50.0 45.0 0.1 35.0 0.1 55.00.0

Novaluron 25.00.1 97.50.0 17.5 0.1 25.0 0.1 41.10.0
Means 22.7 0.0 64.70.0 21.7 0.0 20.0 0.0 -
Table 2 Mortality of E. cautella larvae (%) after 7 days exposure on concrete, glass,
plastic and wood surfaces treated with Hocklicombi
and novaluron insecticides

LSD(P < 0.05): Main effects (insecticide = 1.21, surface= 1.39 Interaction (insecticide x surface)=2.4
Means (%) s.e for pupation
Control 92.50.0 95.00.0 95.00.0 97.50.0 95.00.0
Hocklicombi 35.00.1 0.00.0 35.00.1 25.00.0 23.80.0
Novaluron 55.00.1 2.50.0 60.00.1 47.50.1 43.10.0
Means 61.90.0 41.20.0 63.10.0 56.20.0 -
Table 3: Percentage pupation of E. cautella after 14 days exposure on concrete, glass,
plastic and wood surfaces treated with Hocklicombi

LSD(P < 0.05): Main effects (insecticide5.6, surface= 6.6) Interaction (insecticide x surface)=13.20

control. Adults that emerged were found not to be active
as those in the control. These findings are consistent with
reports by Amos and Williams (1974). According to
CABI (2006), pupal formation is completed in seven
days and development from egg to adult ranges from
29-31 days under optimum conditions of 32.5
o
C and
70% relative humidity. However, in the present study
under laboratory conditions of 272C and 55-60%
relative humidity, pupation extended up to 14 days and
adult emergence was also delayed up to 30 days in the
treated 5
th
instar larvae of E. cautella. Thus, novaluron
was found to prolong the development period of
E. cautella larvae to adults.
The ability of Novaluron to reduce the number of
new generations is consistent with the findings of
(Kostyukovsky et al., 2003) and Kostyukovsky and
Trostanetsky (2006). The authors found that novaluron
applied at 1 ppm reduced the number of new generations
of S. oryzae and R. dominica by 95% and also caused
total mortality of the 3
rd
instar larvae of T. castaneum.
The effectiveness of novaluron in preventing the
metamorphosis of E. cautella when applied at 0.4 mL/L
and 0.5 mL/L also confirms work done by Ibrahim
(2008). The author found that development of E. cautella
to adults was prevented when novaluron was applied at
0.4 mL/L and 0.6 mL/L.
These observations indicate that the effectiveness
of novaluron as a grain protectant depends on the species
of insect, dosage and exposure time. Wilson and Cryan
(1997) and Mulla et al., (2003) stated that the effects of
chitin synthesis inhibitors vary according to species,
development stage, time of application, kind of
compound and dose administered.
The residual effect of Hocklicombi
and
Novaluron were significantly greater on glass surfaces
than plastic, concrete or wood surfaces. Generally,
Hockicombi
significantly caused higher mortalities on

all the surfaces than novaluron. The high residual
efficacy of Hocklicombi
may be attributed to the

components of the compound. Hocklicombi
contains
fenitrothion and fenvalerate as its active ingredients.
These compounds have been reported by several
researchers to have high residual effects when used as
surface treatment against storage insects (Orui, 2004).
Both compounds are non-systemic insecticides with
contact and stomach activity (Tomlin, 2005).
In the present study, novaluron demonstrated
excellent residual effect on glass surfaces by preventing
the metamorphosis of E. cautella to the adult stage. The
residual effect on glass surfaces treated with novaluron
compared well with Hocklicombi
. However, on plastic,
concrete and wood surfaces, Novaluron was less
effective compared with Hocklicombi
but differed
significantly from the untreated control surfaces.
However, the residual effectiveness on plastic surfaces
showed better efficacy than on concrete and wood
surfaces.
The excellent effectiveness of Novaluron on
glass and plastic surfaces is consistent with work done by
(Atkinson et al., 1992). The authors found that when
hydropene, an insect growth regulator was sprayed on
non-absorbent surfaces such as glass and ceramic tile, the
Sackey et al., 2013
Means (%) s.e for adult emergence
Control 92.50.0 90.00.0 95.00.0 97.50.0 93.80.0
Hocklicombi 20.00.1 0.00.0 12.50.1 12.50.0 11.20.0
Novaluron 42.50.1 0.00.0 25.00.1 42.50.1 27.50.0
Means 50.60.0 32.50.0 45.00.0 48.10.0
LSD(P < 0.05): Main effects (insecticide5.9, surface= 6.34)= 6.34 Interaction Insecticide x surface=12.67
Table 4 Percentage adult emergence of E. cautella after 30 days exposure on concrete,
glass, plastic and wood surfaces treated with Hocklicombi

survival, number of oothecae and percentage of
cockroaches were more affected than on absorbent
surfaces of finished plywood and fibreboard. The low
mortality rates, pupation and adult E. cautella that
emerged after exposure to concrete and wood surfaces in
the current study can also be attributed to the
composition of these surfaces. Burkholder and Dicke
(1966) reported that new concrete surfaces contain high
levels of alkaline which hydrolyze residues and reduce
residual efficacy of insecticides hence, the low mortality
rates on concrete-treated surface in the present study was
not unexpected. Chadwick (1985) attributed low efficacy
of insecticides on plywood surfaces to vaporization,
chemical degradation, photodegradation and absorption
of insecticides into surfaces. Thus, the low mortalities
and higher survival rates observed in E. cautella exposed
to wood surfaces treated with the insecticides may be due
to the absorption of the insecticide into the wood
surfaces after treatment.
In the field experiment, all the insecticide
treatments significantly reduced dry weight loss in the
grains compared to the control. Novaluron was observed
to significantly reduce insect numbers in the treated
grains and also had a significantly lower dry weight loss.
Results from this study showed that novaluron
effectively protected maize grains from damage by
E. cautella. Grain weight losses calculated in the
Novaluron treatment compared well with those observed
in grains treated with Hocklicombi
.
Considering that
Novaluron selectively targets larval stages by inhibiting
chitin synthesis and therefore, minimizes its impact on
adults of non targeted insect species (Ishaaya et al.,
2001), Novaluron can be used in replacement of residual
insecticides like Hocklicombi
for treatment of maize

grains for storage.

CONCLUSION
The current study showed that Novaluron was
effective in controlling the tropical warehouse moth. The
application of Nuvaluron at 0.4 mL/L and 0.5 mL/L
treatments resulted in larval mortality ranging between
50-80% after 96 h of exposure. Also, the treatment of
concrete, wood, glass and plastic surfaces usually
encountered in structural insect pest management
systems with 0.5 mL/L Novaluron induced (25.0-97.5%)
larval mortality, (2.5-60%) pupation and ((0.0-42.5%)
adult emergence. These figures were comparable to
those obtained from surfaces treated with 5 mL/L
Sackey et al., 2013
Figure 1 Percentage pupation (meanss.e) of
E. cautella larvae after treatment with novaluron and
Hocklicombi
insecticides. d = days h= hours

Figure 2 Percentage adult emergence (meanss.e) of
E. cautella after treatment with novaluron and
Hocklicombi
insecticides. d = days
Dosage (mL/L) Mean dry weight loss (%)
Control 11.30.0
Hocklicombi 5 6.60.0
Novaluron 0.4 6.80.0
Table 5 Percent dry weight loss after 60 days of
storage using the standard volume method
LSD(P < 0.05) = 1.63
Hocklicombi
insecticide. In the field maize treated with

0.4 mL/L Novaluron
and infested with adult E. cautella

after 60 days of storage showed that there was a lower
weight loss in the Hocklicombi
(6.6%) and novaluron

(6.8%) treatments compared to the negative control
(11.3%). This work has proven that Novaluron
could
replace the synthetic insecticides that are used in the
management of this pest and should be included in the
management programmes for storage pests control.

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A Checklist of Butterflies of Meenachil River Basin, Kerala, India
Keywords:
Meenachil river, Endemic species, bio-indicators, anthropogenic pressure.
ABSTRACT:

Butterflies are highly sensitive to environmental change and are delicate
creatures that act as good bio-indicators of the health of an ecosystem. Meenachil
river basin has attracted considerable amount of public interest. A survey of the
butterflies conducted randomly revealed a total of 91 species belonging to five
families including three endemic species. Family Nymphalidae dominated in the study
area, followed by Hesperiidae and Lycaenidae. This area is currently under severe
anthropogenic pressure and minimizing these disturbances is important for the
long-term survival of specialist butterflies.
768-774 | JRB | 2013 | Vol 3 | No 1

cited.
Research Journal
Authors:
Vincy MV
1
, Brilliant R
2

and Pradeepkumar AP
3

Institution:
1. School of Environmental
Science, Mahatma Gandhi
University, Kottayam,
Kerala.

2. PG Department of
Environmental Sciences,
St. Johns College, Anchal,
Kerala.

3. Department of Geology,
University of Kerala,
Kariavattom, Kerala.

Vincy MV.

Email:
vincybrilliant@gmail.com

Web Address:
Dates:
Received: 21 Nov 2012 Accepted: 03 Dec 2012 Published: 04 Feb 2013
Article Citation:
Vincy MV, Brilliant R and Pradeepkumar AP.
A Checklist of Butterflies of Meenachil River Basin, Kerala, India.
Original Research

INTRODUCTION
Butterflies are the most beautiful and colourful
creatures on the earth and have a great aesthetic value.
India harbours about 1501 species of butterflies
(Haribal, 1992), 285 species are found in southern India
(Thomas, 1966), of which 45 species are endemic to
southern India. Butterflies, widely appreciated for the
aesthetic value are important as ecological indicators
(Chakravarthy et al., 1997) and flagship taxa in
biodiversity inventories (Lawton et al., 1998).
Meenachil river which is one of the important
river of Kottayam district in Kerala, emerges from
Western Ghats and confluences into Vembanad Lake.
This river has a total length of 78 km and has
a catchment area of 1272 km
2
. The entire Meenachil
watershed area geographically lies between 925 N
to 955 N latitude and 7630 E to 7700 E longitude.
The general elevation of the entire river basin ranges
from 77 m to 1156 m in the high lands and less than 2 m
in the low lands. The Meenachil river basin falls within
the realm of tropical climate. The temperature of the area
varies in between 24C and 32C throughout the year.
The annual rainfall varies from less than 100 cm to more
than 500 cm with an average of 300 cm. The occasional
rainfall is also received between the two seasons. Rubber
trees are extensively cultivated in vast areas in the entire
river basin. Besides rubber, other crops like spices,
paddies etc., are also cultivated in the river basin area
(Watershed Atlas, 1996).
Among insects, butterflies are the most
studied group. Larsen (1987a, b, c, 1988) made a detailed
survey of butterflies of Nilgiri Mountains and recorded
nearl y 300 speci es incl uding endemi cs.
In Kerala, documentation of butterflies on Silent Valley
National Park (Mathew and Rahamathulla, 1993)
an d Par a mbi kul a m Wi l dl i f e San ct uar y
(Sudeendrakumar et al., 2000) have been carried out.
The present paper presents a checklist and diversity of
butterfly populations in different altitude levels in
Meenachil river basin in Kerala, South India. However,
comprehensive long-term ecological studies to monitor
the butterfly population of the area remains as a serious
lacuna. Such studies are imperative to improve the
ecological utility of butterflies as indicator taxa.

The present study is an attempt to provide a
checklist of butterflies based on a four-year field study
from October 2008 to October 2012. Identification of
species was done using available literature (Evans, 1932;
Gunathilagaraj et al., 1998; Haribal, 1992; Palot et al.,
2003; Gay et al., 1992; Wynter-Blyth, 1957) and with
the help of experts. Species classification and scientific
names are as per Gunathilagaraj et al., (1998).

RESULT AND DISCUSSION
The study during the period indicate that the
habitats where butterflies were found and captured are
disturbed areas and are strongly influenced by
anthropogenic activities. These range from city lots to
pasture, abandoned fields, road sides, plantations,
riparian area, etc.
A total of 91 species belonging to 71 genera
distributed over five families were collected from the
monitoring sites, during the study period. The family
Nymphalidae dominated with 34 species followed
by Hesperiidae (20 spp.), Lycaenidae (18 spp.), Pieridae
(7 spp.), and Papilionidae (12 spp.). Even though, the
family Nymphalidae exhibited the maximum species
diversity, family Pieridae showed maximum species
density. Three butterfly species recorded from this region
have protected status under the Wildlife Protection Act,
1972 (Arora, 2003). They are Hypolimnas misippus and
Atrophaneura hector included under Schedule I Part IV
and one species Aeromachus pygmaeus in Schedule II
Part II. Further research with reference to ecology,
threats and conservation of butterflies in the area is in
progress.
Vincy et al., 2013

Vincy et al., 2013
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8
5
-
9
5

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2
6

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6
0
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5

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2
7

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6
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0

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2
8

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6
5
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7
5

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n

B
a
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a

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p
.

2
9

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m
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n

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m
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6
0
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8
0

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a

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a
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s

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a

3
0

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n

B
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3
8
-
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5

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O
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3
1

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4
5
-
5
5

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n

O
r
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3
2

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3
2
-
4
8

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m

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m
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n

3
3

C
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3
0
-
4
0

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m
m
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n

G
r
a
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3
4

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a
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A
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a

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a
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5
0
-
6
5

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m
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n

A
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a

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a

3
5

T
a
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l

Y
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n

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c
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a

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a
b
r
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c
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)

6
0
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7
5

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o
m
m
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n

H
y
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3
6

R
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c

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r
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m
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s

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D
r
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r
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5
0
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6
0

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m

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m
m
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n

3
7

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o
m
m
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n

L
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d

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r
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y
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5
0
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6
0

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m

C
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m
m
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n

3
8

C
o
m
m
a
n
d
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r

M
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d
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z
a

p
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s

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r
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m
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6
0
-
7
5

m
m

C
o
m
m
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n

O
c
h
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n
a
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,

M
u
s
s
a
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n
d
a

f
r
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n
d
o
s
a

3
9

C
o
m
m
o
n

L
a
s
c
a
r

P
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p
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r
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a

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o
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d
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a

(
S
t
o
l
l
)

4
5
-
5
0

m
m

C
o
m
m
o
n

A
c
a
c
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a

p
e
n
n
a
t
a

4
0

C
o
m
m
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n

S
a
i
l
o
r

N
e
p
t
i
s

h
y
l
a
s

(
L
i
n
n
a
e
u
s
)

5
0
-
6
0

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m

C
o
m
m
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n

D
a
l
b
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r
g
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a

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p
p
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,

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s
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,

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s
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a

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p
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a
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r
e
w
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a

s
p
p
.
,

B
o
m
b
a
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a
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a
b
a
r
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c
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m

4
1

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l
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p
p
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r

P
a
r
t
h
e
n
o
s

s
y
l
v
i
a

(
C
r
a
m
e
r
)

9
5
-
1
3
0

m
m

C
o
m
m
o
n

t
i
n
o
s
p
o
r
a

c
o
r
d
i
f
o
l
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a

4
2

C
o
m
m
o
n

B
a
r
o
n

E
u
t
h
a
l
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a

a
c
o
n
t
h
e
a

(
H
e
w
i
t
s
o
n

)

5
5
-
8
0

m
m

C
o
m
m
o
n

A
n
a
c
a
r
d
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u
m

o
c
c
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d
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t
a
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s
,

M
a
n
g
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f
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r
a

i
n
d
i
c
a
,

S
t
r
e
b
l
u
s

a
s
p
e
r

4
3

G
r
e
y

C
o
u
n
t

T
a
n
a
e
c
i
a

l
e
p
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d
e
a

(
B
u
t
l
e
r
)

6
5
-
8
5

m
m

C
o
m
m
o
n

C
a
r
e
y
a

a
r
b
o
r
e
a

4
4

C
o
m
m
o
n

M
a
p

C
y
r
e
s
t
i
s

t
h
y
o
d
a
m
a
s

(
B
o
i
s
d
u
v
a
l
)

5
0
-
6
0

m
m

N
o
t

C
o
m
m
o
n

F
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c
u
s

s
p
p
.

4
5

P
a
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n
t
e
d

L
a
d
y

V
a
n
e
s
s
a

c
a
r
d
u
i

(
L
i
n
n
a
e
u
s
)

5
5
-
7
0

m
m

C
o
m
m
o
n

B
l
u
m
e
a

s
p
p
.

4
6

A
n
g
l
e
d

C
a
s
t
e
r

A
r
i
a
d
n
e

a
r
i
a
d
n
e

(
L
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n
n
a
e
u
s
)

4
5
-
6
0
m
m

U
n
c
o
m
m
o
n

R
i
c
i
n
u
s

c
o
m
m
u
n
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s

4
7

C
o
m
m
o
n

C
a
s
t
e
r

A
r
i
a
d
n
e

m
e
r
i
o
n
e

(
C
r
a
m
e
r
)

4
5
-
6
0
m
m

C
o
m
m
o
n

R
i
c
i
n
u
s

c
o
m
m
u
n
i
s

4
8

C
h
o
c
o
l
a
t
e

P
a
n
s
y

J
u
n
o
n
i
a

i
p
h
i
t
a

(
C
r
a
m
e
r
)

5
5
-
8
0

m
m

C
o
m
m
o
n

4
9

G
r
e
y

P
a
n
s
y

J
u
n
o
n
i
a

a
t
l
i
t
e
s

(
L
i
n
n
a
e
u
s
)

5
5
-
6
5

m
m

C
o
m
m
o
n

5
0

P
e
a
c
o
c
k

P
a
n
s
y

J
u
n
o
n
i
a

a
l
m
a
n
a

(
L
i
n
n
a
e
u
s
)

6
0
-
6
5

m
m

C
o
m
m
o
n

O
s
b
e
c
k
i
a

s
p
p
.

5
1

L
e
m
o
n

P
a
n
s
y

J
u
n
o
n
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a

l
e
m
o
n
i
a
s

(
L
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n
n
a
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s
)

4
0
-
6
0

m
m

C
o
m
m
o
n

S
i
d
a

r
h
o
m
b
i
f
o
l
i
a

5
2

G
r
e
a
t

E
g
g
f
l
y

H
y
p
o
l
i
m
n
a
s

b
o
l
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n
a

(
L
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n
n
a
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s
)

7
0
-
1
1
0

m
m

C
o
m
m
o
n

S
i
d
a

r
h
o
m
b
i
f
o
l
i
a
,

H
i
b
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s
c
u
s

s
p
p
.

5
3

D
a
n
a
i
d

E
g
g
f
l
y
*

H
y
p
o
l
i
m
n
a
s

m
i
s
i
p
p
u
s

(
L
i
n
n
a
e
u
s
)

7
0
-
8
5

m
m

C
o
m
m
o
n

H
i
b
i
s
c
u
s

s
p
p
.

F
A
M
I
L
Y

L
Y
C
A
E
N
I
D
A
E

5
4

A
p
e

F
l
y

S
p
a
l
g
i
s

e
p
i
u
s

(
W
e
s
t
w
o
o
d
)

2
0
-
3
0

m
m

N
o
t

c
o
m
m
o
n

C
a
r
n
i
v
o
r
o
u
s

c
a
t
e
r
p
i
l
l
a
r
s

f
e
e
d

o
n

m
e
a
l
y

b
u
g
s

5
5

I
n
d
i
a
n

S
u
n
b
e
a
m

C
u
r
e
t
i
s

t
h
e
t
i
s

(
H
b
n
e
r
)

4
0
-
4
8

m
m

N
o
t

r
a
r
e

A
b
r
u
s

p
r
e
c
a
t
o
r
i
u
s
,

P
o
n
g
a
m
i
a

p
i
n
n
a
t
a

5
6

R
e
d

S
p
o
t

Z
e
s
i
u
s

c
h
r
y
s
o
m
a
l
l
u
s

(
H
b
n
e
r
)

3
8
-
4
4

m
m

N
o
t

r
a
r
e

C
a
t
e
r
p
i
l
l
a
r
s

f
e
e
d

o
n

a
n
t

l
a
r
v
a
e

5
7

Y
a
m
f
l
y

L
o
x
u
r
a

a
t
y
m
n
u
s

(
C
r
a
m
e
r
)

3
6
-
4
0

m
m

C
o
m
m
o
n

S
m
i
l
a
x

s
p
p
.
,

D
i
o
s
c
o
r
e
a

p
e
n
t
a
p
h
y
l
l
a

5
8

M
o
n
k
e
y

P
u
z
z
l
e

R
a
t
h
i
n
d
a

a
m
o
r

(
F
a
b
r
i
c
i
u
s
)

2
6
-
2
8

m
m

N
o
t

r
a
r
e

I
x
o
r
a

s
p
p
.

Vincy et al., 2013
5
9

S
l
a
t
e

F
l
a
s
h

R
a
p
a
l
a

m
a
n
e
a

(
H
e
w
i
t
s
o
n
)

3
0
-
3
3

m
m

C
o
m
m
o
n

A
c
a
c
i
a

p
e
n
n
a
t
a

6
0

C
o
m
m
o
n

S
i
l
v
e
r
l
i
n
e

C
i
g
a
r
i
t
i
s

v
u
l
c
a
n
u
s

(
F
a
b
r
i
c
i
u
s
)

2
6
-
3
4

m
m

C
o
m
m
o
n

Z
i
z
y
p
h
u
s

r
u
g
o
s
a
,

C
a
n
t
h
i
u
m

o
r
o
m
a
n
d
e
l
i
c
u
m
,

C
l
e
r
o
d
e
n
d
r
u
m

i
n
e
r
m
e

6
1

A
n
g
l
e
d

P
i
e
r
r
o
t

C
a
l
e
t
a

c
a
l
e
t
a

(
H
e
w
i
t
s
o
n
)

2
6
-
3
2

m
m

N
o
t

r
a
r
e

Z
i
z
y
p
h
u
s

r
u
g
o
s
a

6
2

B
a
n
d
e
d

B
l
u
e

P
i
e
r
r
o
t

D
i
s
c
o
l
a
m
p
a

e
t
h
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The study shows that the sustained interference
and disturbance seem to affect the occurrence and
numerical strength of each butterfly species. If this
situation goes unabated, the abundant butterflies may
become rare and the less abundant ones could disappear
permanently. Further, the decline in the number of
butterflies largely allows inbreeding which becomes fatal
in course of time. Modified habitats with reduced plant
cover contribute to warm conditions and these conditions
might allow some butterflies to extend their distribution
to different habitats. The butterflies which control certain
plant pets, if decline in number or disappear from the
habitat, plants too get affected because of the unchecked
plant pets. Therefore, the very presence of butterflies in
species and number may be taken as an indication of the
health of the habitat.

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Microbial production of glutaminase enzyme
Keywords:
Actinomycetes, Anticancer properties, Enzymes, Glutamic acid and
L-Glutaminase.
ABSTRACT:

Enzymes are proteins highly specific in their actions on substrates and serve
as biocatalysts. They are produced by cells in order to accelerate both the rate and
specificity of metabolic reactions. Microbial enzymes are known for their unique
characteristics over other sources due to their easy production on a commercial scale
and stability. Different microorganisms are known to produce various enzymes such as
bacteria, fungi and actinomycetes which produce a variety of extra-cellular and endo-
cellular enzymes. Some of these actinomycetes enzymes have been isolated from the
culture filtrates or the mycelium, concentrated and purified. Others have only been
demonstrated in the mycelium of the organism. However, the ability to produce a
variety of enzymes may be an attractive phenomenon in these microorganisms since
they are nutritionally quite versatile. Microbial L-glutaminase has recently gained
more attention due to its anticancer properties, in addition to its use as a flavor
enhancer in food industry by increasing the amount of glutamic acid content in the
fermented food .
775-779 | JRB | 2013 | Vol 3 | No 1

cited.

Research Journal
Authors:
Mario Khalil Habeeb

Institution:
Microbiology Department,
Faculty of Science, Ain
Shams University, 15566
El-Khalifa El-Mamoun
street, Abbassia, Cairo,
Egypt, Postal code: 11566.


Email:
mario_khalil87@yahoo.com,
mario_khalil@sci.asu.edu.eg

Telephone:
+20 (02) 22409635

Mobile:
+20 (0128) 3941815

Web Address:
Dates:
Received: 16 Jan 2013 Accepted: 22 Jan 2013 Published: 06 Feb 2013
Article Citation:
Microbial production of glutaminase enzyme.
OVER VIEW

INTRODUCTION
Enzymes are highly selective catalytic proteins
produced by living cells which may or may not contain a
non-protein prosthetic group (Underkofler et al., 1958).
Actinomycetes are considered to be preferred
enzymes sources due to their production of extracellular
enzymes. They are highly diverse group with numerous
members representing important source of microbial
enzymes. Actinomycetes genera are differentiated from
each other based on morphological, biochemical, and
physiological criteria. They act as decomposers of
complex animal and plant materials resulting in release
of simple substances, especially carbon and nitrogen
which is easily utilized by other organisms, thus
performing a vital role in life cycle. Due to their
significant biochemical activities, Actinomycetes are
used in commercial production of various substances
such as antibiotics and enzymes (Waksman, 1950).
Because of its industrial and pharmaceutical
applications, intensive research was conducted on
L-glutaminase recently. L-glutaminase is produced by
various terrestrial microorganisms such as Pseudomonas
sp., Acinetobacter sp., Escherichia coli, Bacillus sp.,
Hansenula sp., Candida sp., Aspergillus oryzae and
Beauveria bassiana (Sabu, 2003). Also few
marine microorganisms such as Micrococcus luteus,
Vibrio cholera and Pseudomonas fluorescens were
reported to produce the enzyme (Chandrasekaran, 1997).
Definition
L-glutaminase is classified as an amidohydrolase
enzyme which acts upon amide bonds of L-glutamine
generating L-glutamic acid and ammonia. It is present
in both microorganisms and mammalian tissues
(Ohshima et al., 1976b). Microbial sources of
glutaminase showed a great role in various applications
such as its use in fermented foods precisely in soy sauce
and other related types, in addition to its use as
anticancer agent which act by inhibition of glutamine
utilization by the cancerous cells resulting in selective
starvation of cancerous cells and their possible death
(Santana et al., 1968).
Glutaminase Producing microorganisms
Different types of organisms were reported
to produce glutaminase enzyme. However, The selection
of the right organism is very critical to obtain high yield
of the required enzyme (Akujobi et al., 2012).
L-glutaminases from E. coli, Pseudomonas sp.,
Bacillus sp., and Clostridium welchii have been isolated
and well studied (Wade et al., 1971). In addition to these
bacterial sources, the fungus Aspergillus oryzae showed
a great ability to produce this enzyme. Among
microorganisms, actinomycetes are widely recognized
as preferable L-glutaminase sources because they
generally produce extracellular enzymes, which facilitate
the enzyme recovery from the fermentation broth
such as glutaminase from Streptomyces rimosus
(Sivakumar et al., 2006).
Microbial Glutaminase Characteristics
Temperature is considered to be an important
factor affecting the enzyme stability, The optimum
temperature recorded by many glutaminases ranged from
40-50C. However, the temperature stability of
glutaminase I (Micrococcus glutaminase) of M. luteus
could be increased by the addition of 10% NaCl
(Moriguchi et al., 1994). The optimum temperature for
A. oryzae glutaminase was around 37-45C and remained
stable at up to 45C and the enzyme was completely
inactive at 55C (Nakadai and Nasuno, 1989).
It is interesting that the exposure of E. coli
glutaminase B to cold resulted in a reversible
inactivation of enzymatic activity, while subsequent
warming to 24C restored the activity. There was no
difference in the molecular weight of the cold inactivated
enzyme and the warm activated enzyme. The
conformational changes which probably occur upon
exposure to cold resulted from a weakening of the
interaction among hydrophobic groups in the protein
(Chou et al., 1993).
Habeeb., 2013
The salt-tolerance of glutaminase is an important
parameter in industrial processes that include
high-salinity. It was reported that the high-salt
concentration (nearly 3 M NaCl) used in the process
of soy sauce fermentation resulted in remarkable
inhibition of the koji mold (A. oryzae) Glutaminase
(Koibuchi et al., 2000).
Methods Used for Microbial Glutaminase Production
Two methods are known for the production of
microbial glutaminase.
Submerged (Liquid) Production Method
In this method, the sterile media together with
the enzyme producing organism were introduced into
large fermentors (Tanks) followed by constant mixing
and supply of sterile air (Schuegerl et al., 1991).
Actinomycetes glutaminases showed a high salt
tolerance in this production method. Reports showed that
Streptomyces rimosus isolated from estuarine fish
recorded high salt tolerance and the highest enzyme
production obtained at temperature 27C, pH 9.0 and
both glucose and malt extract proved to be the best
carbon and nitrogen sources for maximum enzyme
production (Imada et al., 1973).
Surface Production Method
This method includes the use of solid support on
which microorganisms are grown. Surface production
method (solid state fermentation) showed 25 to 30 fold
increase in enzyme production when compared with
submerged production (Sabu et al., 2000b).
Wheat bran was found to be a favorable support
for microorganisms in the process of glutaminase
production (Kashyap et al., 2002). In addition to wheat
bran many other solid supports showed high efficiency in
the enzyme production such as ground nut cake powder,
copra cake powder and sesamum oil cake (Prabhu and
Chandrasekaran, 1995). Polystyrene beads, supported by
mineral salts and glutamine are another form of solid
supports used for the enzyme production.

By using this method it was found that L-glutaminase
producing marine alkalophilic Streptomyces sp. SBU1
which was isolated from Cape Comorin coast,
India gave highest enzyme production after 4 days
of incubation and at 14% Corn steep liquor
(Krishnakumar et al., 2011).
Applications
L-glutaminase has received great attention due to
its valuable applications in several fields especially in
medicine and its use as an anticancer agent either alone
or together with any other agents is known as enzyme
therapy, In addition to its role as flavor enhancer by
increasing the glutamic acid content of food. Also
glutaminase applications extend to the enzyme utilization
as biosensor in analytical purposes by measuring the
levels of L-glutamine and finally in the manufacture of
fine chemicals such as theanine when used with bakers
yeast.
Glutaminase as Enzyme Therapy
Glutaminase can be used as alternative for cancer
treatment as enzyme therapy. The mechanism for
glutaminase therapy includes that L-Glutaminase act on
its substrate (L-glutamine) and breaks it down leading to
the selective destruction of the tumor cells accompanied
by inhibition of both protein and nucleic acid
biosynthesis due to glutamine starvation and this is
attributed to the inability of cancerous cells to synthesis
glutamine (Tanaka et al., 1988). This is due to the fact
that some types of cancerous cells utilize glutamine
greatly (Lazarus and Panasci, 1986). Concerning this
finding various enzymatic therapies developed to deprive
L-glutamine to cancerous cells (Roberts et al., 1970).
Glutaminase as Flavor Enhancer
Glutamate is a famous amino acid and
considered as a natural constituent of many fermented or
aged foods, such as soy sauce, fermented bean paste and
cheese (O`Mahony and Ishi, 1987). It gives these types
of food their desired taste (Chou and Hwan, 1994).
Glutamate (Glutamic acid) accumulated in these food
Habeeb., 2013

types as a result of protein hydrolysis by proteolytic
enzymes such as glutaminase and protease have a vital
role in food industry (Tambekar and Tambekar, 2011).
Glutaminase as biosensor
L-glutaminase is used as biosensor to monitor the
L-glutamine levels in body fluids. This technique is more
applicable than previously used methods and
characterized by its high specificity compared with cell
based sensors in addition to its fast response. This has led
to intensive use of glutaminase in clinical purposes
especially that is derived from mammalian tissues.
Glutaminase and Manufacture of Various Chemicals
Theanine (-l-glutamyl ethylamide) is
synthesized by theanine synthetase (EC 6.3.1.6) in plants
and known for its capability to inhibit stimulation by
caffeine, in order to enhance the effects of the anticancer
agents. Bacterial glutaminases together with bakers
yeast are used to produce theanine (Tachiki et al., 1998).
Also L-glutaminase is used in the manufacture of
glutamyl alkamides by the transfer of -glutamyl from
a donor molecule such as glutamine or glutathione to a
glutamyl acceptor like ethylamine or glycyl glycine by
catalysis.
Conclusion
Due to their important applications, Microbial
glutaminases gained much attention among the
commercially important enzymes. Their role in the
biotechnological industries, in addition to their medical
applications as anticancer agents created the need for
searching of high potential microorganisms strains. The
advantages of the microbial glutaminases - such as their
stability and large scale production - over other sources
made microorganisms represent a desirable source for
the enzyme production. This brief review revealed the
microbial sources of the enzyme and its characteristics,
in addition to the production methods and extended to its
various applications.

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Chandrasekaran M. 1997. Industrial enzymes from
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Chou CC, Yu RC, Tsai CT. 1993.
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Imada A, Igarasi S, Nakahama K and Isono M. 1973.
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Kashyap P, Sabu A, Pandey A, Szakacs G and Soccol
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Koibuchi K, Nagasaki H, Yuasa A, Kataoka J
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Krishnakumar S, Alexis R, Rajan and Ravikumar S.
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and Fukami H. 1998. -Glutamyl transfer reactions
by glutaminase from Pseudomonas nitroreducens
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Tambekar DH and Tambekar SD. 2011.
Partial characterization and optimization of protease
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Ammon HL, Roberts J, Weber IT and Wlodawer A.
1988. Structures of amidohydrolases. Amino acid
sequence of a glutaminase-asparaginase from
Acinetobacter glutaminasifrcans and preliminary
crystallographic data for an asparaginase from
Erwinia chrysanthemi. J Biol Chem., 263:8583-8591.

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Wade HE, Robinson HK and Phillips BW. 1971.
Asparaginase and glutaminase activities of bacteria.
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A review on the role of nutrients in development and
organization of periphyton
Keywords:
Aquatic ecosystem, Biofilm, carbon, primary productivity, phytoplankton.
ABSTRACT:

Periphyton communities have not received wider attention and often
misunderstood with biofilm for their nature of development and role in aquatic
ecosystem. To clarify its functional objective in aquatic ecosystem, present review
proposes a functional definition for periphyton in terms of ecological interactions
and also outlines its ecological role in nutrient sharing with other aquatic components.
The development and succession of periphyton is a function of nutrient and carbon (C)
sharing with its constituent parts and ambient environment. Through mechanisms like
entrapment, de novo synthesis, nutrient leakage, trophic upgrading etc., ambient
nutrients are routed to periphyton and transferred to upper trophic levels. Periphyton
communities stand next to phytoplankton for their contribution to primary
productivity, in nutrient rich aquatic environment. Unlike phytoplankton, nutrient
poor aquatic environment has no effect on periphytic primary productivity.
As periphyton communities are attached to substratum, their ability to assimilate
organic nutrient through substratum is an additional advantage over phytoplankton.
780-788 | JRB | 2013 | Vol 3 | No 1

cited.

Research Journal
Authors:
Saikia SK
1
, Nandi S
2
,
Majumder S
3
.

Institution:
1. Assistant Professor,
Aquatic Ecology Laboratory,
Department of Zoology,
Visva Bharati University,
Santiniketan, West Bengal,
India, Pin-731235.

2.

Research Fellows,
Santiniketan, West Bengal,
India.

3. Research Fellows,
Santiniketan, West Bengal.

Saikia SK.

Email:
surjyasurjya@gmail.com

Web Address:
Dates:
Received: 20 Nov 2012 Accepted: 10 Dec 2012 Published: 11 Feb 2013
Article Citation:
Saikia SK, Nandi S, Majumder S.
A review on the role of nutrients in development and organization of periphyton.
Original Research
INTRODUCTION
The term periphyton (peri round; phyton plant)
was proposed by (Behning, 1924) and popularized
by several authors (Cooke, 1956; Sladeckova, 1962;
Pieczynska, 1970). There exist a series of definitions
proposed for periphyton (Young, 1945; Neel, 1953;
Wetzel, 1963). (Wetzel, 1983) defined it as the micro
floral community living attached to any substrate
under water. (Stevenson, 1996) used it for describing
microorganisms such as algae and bacteria growing in
association with substrata. These communities play an
important role in water bodies, not only as important
primary producers and energy source for higher trophic
levels, but also by affecting the nutrient turnover and the
transfer of nutrients between the benthic and the pelagic
zone (review Saikia, 2011). The substrates of periphyton
commonly include submerged plants or plant parts,
rocks and sediments. Such substrate selection designs
periphyton as a medium in transferring and trophic
upgrading of nutrients. This property recognizes
periphyton as a tool for biofiltering excess nutrients from
polluted waters and for efficient nutrient transfer in
aquatic food chain.
Aquatic ecosystems mainly comprise of
freshwater and marine water bodies and their ecology
discusses the relationship of aquatic organisms and
its interaction with the immediate environment. The
principle biotic components primarily explained in the
recent past for their contributions to different interactions
in aquatic ecosystem are macrophytes, plankton
(Zooplankton and phytoplankton) and invertebrates
(benthos, nekton and neuston). Till the mid of
19
th
century, periphyton or associated organisms were
not given any biological credit for their role in aquatic
ecosystem. Probably, (Wetzel, 1963) in his evolutionary
review paper Primary productivity of periphyton in
Nature, for the first time made convincing remark on the
role of periphyton in aquatic ecosystem. Even today,
periphytic communities are ignored as a major
contributor of most of the nutrient inputs to aquatic
ecological cycles. The present review is an attempt to
outline periphyton as an integral and essential component
of aquatic ecosystem highlighting few areas recently
addressed on the role of periphyton in nutrient sharing in
aquatic processes.
Periphyton: A Nutrient Dependent Organization
(Whal, 1989) discussed settling pattern
of biofilm (Figure 1), in four phases: (i) surface
conditioning or adsorption of dissolved organic
compounds where macromolecules attach to submerged
surfaces following a spontaneous physical-chemical
process; (ii) primary colonization or bacterial settling
following surface conditioning and after their
colonization, bacteria start to produce EPS,
(iii) secondary colonization to bacterial layer and EPS
pool by eukaryotic unicellular microorganisms, mainly
protozoan, microalgae and cyanobacteria and (iv) settling
of eukaryotic multicellular organisms as a function of
nutrient sharing, grazing and predation. According to
(Wetzel, 1983), associated organisation from secondary
colonization onwards can be designated as periphyton.
In that way, it could be defined as an advanced
successional stage of biofilm. However, there could be
a fifth (v) phase; the tertiary colonization where
bacterioplankton colonized on the surfaces of unicellular
and filamentous secondary colonizer (e.g. diatom,
Oedogonium etc.). Several bacteria different from early
colonizer settle on algal surfaces at this stage
(Alldredge et al., 1993; Armstrong et al., 2000).
Periphyton are rich Carbon source
TEP with rich Carbon sources of
Glucopolysaccharides in aquatic environments
initiates early colonization of bacteria through
surface conditioning (Stoderegger and Herndl, 1999).
The bacterial EPS from early biofilm exists as a part of
dissolved organic matter (Lignell, 1990) as well as
particulate matter (Decho, 2000). It acts both as rich
organic Carbon storage (Freeman and Lock, 1995) and
Saikia et al., 2013
chief supplier of Carbon demand for organisms that feed
on periphytic aggregates (Decho and Moriarty, 1990;
Hoskins et al., 2003). Being polyanionic in nature
(Costerton et al., 1978), EPS further permits inorganic
nutrient entrapment through ion exchange processes
(Freeman et al., 1995) leading to storage of organic
Carbon in the biofilm. In addition, among the bacterial
fractions, cyanobacteria are important primary producers
and many of their species can x atmospheric Nitrogen
(Whitton and Potts, 1982). Chemical screening of
laboratory grown, commercially viable cyanobacteria
have revealed that they have a high nutritional value, in
terms of protein (Choi and Markakis, 1981).
During tertiary phase of periphyton development,
algal communities play indirect role in nutrient addition
to periphytic complex through their surfaces. A study on
algae-bacteria interactions on biotic surfaces revealed
that bacterial abundance is significantly higher in areas
of diatom colonization on substrates (Donnelly and
Herbert, 1999). These bacteria contribute to the
management of community metabolism of periphytic
matrix and can trap the metabolic products released
by bacterta on algal surface (Makk et al., 2003). Such
algae-bacteria interactions enrich periphytic organic
matrix with components of polysaccharides, proteins,
nucleic acid and other polymers (Davey and Otoole,
2000).
Periphytic pathway of nutrient transfer
The periphytic nutrient transfer pathway (PNP)
mainly involves ambient nutrient entrapment, storage
and transferring it to immediate higher trophic level. The
fate of PNP gets its initiation from the surface
conditioning phase of periphyton formation. As soon as
TEP prepares the substrate surface for colonization,
bacteria as initial colonizer develops micro-colonies
(Costerton, 1984) and through EPS, it supplies a
significant source of Carbon to periphytic complex
(Hobbie and Lee, 1980) (Figure 2). A PNP establishes
between dissolved organic in periphytic complex and
inorganic substances in the water column and the higher
trophic levels of the ecosystem (Hynes, 1970). In
general, the Carbon reserve of periphyton generates
through three mechanisms. The first mechanism supplies
energy through bacterial EPS. Bacterial EPS is rich in
carbohydrate, and some time vitamins and other
nutrients. During first-cryptic growth, the dying bacteria
leak metabolizable energy to immediate environment
(i.e. EPS) acting as nutrient source to neighbouring
periphyton strata. This property not only protects the
neighbours from starvation but also permits their
multiplication (Postgate, 1976). In a growing periphytic
assembly, cynobacteria and other early algal colonizers
share this Carbon source. In aged periphytic assembly,
the old mostly filamentous periphytic layer receives such
Carbon from overlying bacterial composition resulted
from tertiary phase of colonization. The second
mechanism consists of endogenous energy reserves.
These reserves consist of Carbon that is accumulated and
assimilated inside the microbial cell and can be
Saikia et al., 2013
Figure 1. Formation of periphytic complex on
natural substrate showing tertiary phase of coloni-
zation (Modified from Whale, 1989). TEP, Trans-
parent Exopolymer Prticles; EPS, Extracellular
Polymeric Substances

mobilized to ensure survival during starvation (Dawes
and Senior, 1973) and thereby recovery of periphytic
aggregates due to senescence. The third mechanism of
organic Carbon storage is the polysaccharide exudates
(Freeman and Lock, 1995) released by algae at tertiary
phase under nutrient (especially Phosphorus) limited
condition. The algal components release polysaccharide
exudates to EPS under Phosphorus limitation on which
tertiary phase bacteria flourish. In return, these bacteria
remineralize Phosphorus for algae. In addition, the ECM
with polyanionic by nature (Costerton et al., 1978) is
believed to permit nutrient entrapment through ion
exchange processes (Freeman et al., 1995). (Freeman
and Lock, 1995) proposed that the entrapment
mechanism may also permit the storage of organic
Carbon in the biofilm.
In transferring nutrient through PNP, the
bacterial Carbon enters to organisms in the next trophic
level as complex Carbon rich compounds. The Fatty acid
(FA) component of algae is under extensive research
now a day as Carbon rich compounds. Periphytic matrix
is dominated by algae and hence FA contributes to the
food quality of matured periphytic organization. In algae,
FA increases as a result of exposure to stressful
environmental conditions, such as high temperature,
nutrient extremes and harsh light conditions.
Polyunsaturated fatty acids (PUFAs) also affect many
physiological processes in living organisms and are
major nutrient constituents of polar lipids, and are
present in cell and chloroplast membranes. The
dominance of algae in periphytic canopy acts as rich
source of FA to animals grazing on periphyton.
Primary productivity of periphyton
The energetic relation of an ecosystem is
principally regulated by primary production. In aquatic
ecosystem, algae are dominant primary producers, and
responsible for both Carbon fixation and sequestration.
Periphyton with majority of algae might have significant
contribution to primary production of aquatic ecosystem.
However, very few investigations have been performed
on measurements of photosynthetic rates of algal
periphyton under natural conditions. (Wetzel, 1963)
pointed out technical/methodological difficulty in
assessing such parameters of periphyton under natural
condition. From an analysis on nutrient limiting and
nutrient rich lakes, it is obvious that periphyton
productivity contributes more than 30% of primary
productivity to the aquatic ecosystem (Figure 2a). On
comparison, it seems evident that the nutrient limited
aquatic ecosystems have more or less equal primary
productivity levels to nutrient rich aquatic ecosystems.
The same is not true in case of phytoplankton
(Figure 2b).

Saikia et al., 2013
Figure 2. (a) Primary productivity of Phytoplankton, Periphyton and Macrophytes from
aquatic ecosystems. (b) Primary productivity of Phytoplankton, Periphyton and Macrophyte
in nutrient limited (NL, n=17) and nutrient rich (NR, n=10) aquatic ecosystems. Data from
Vadeboncoeur and Steinman (2002).
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Phytoplankton Periphyton Macrophyte
Nutrient Regulated Biotic Interactions of Periphyton
Biotic interactions in aquatic ecosystems are
more complex than any other ecosystems for its variable
nature. Interactions between periphyton and biotic
components in aquatic ecosystem are primarily regulated
by nutrients and can be discussed under following
subheadings-
Plankton-periphyton interaction
Periphyton-macrophyte interaction
Grazer-periphyton interaction
Plankton-Periphyton interaction
The plankton-periphyton interaction is
principally regulated by light and nutrient availability in
the environment. Both the communities are composed of
common members of bacterial, algal and zooplanktonic
origin. However, on spatial ground, habitats of both
plankton and periphyton have differences in receiving
light and nutrients. Conceptual models revealed that
nutrient limited environments are dominated by
periphyton than plankton (Wetzel, 2001; Hansson, 1992).
Nutrient limitation results thin planktonic cover that
allows maximum light to pass through water column to
reach the bottom of the ecosystem facilitating
multiplication of periphytic population. Conversely,
plankton rich aquatic ecosystems limit growth of
periphyton due to limited light availability. Epiphytic
communities can better adsorb nutrients from sediments
or bottom of the system through macrophytes
(Burkholder and Wetzel, 1990).
Periphyton-substrate interaction
Substrate type plays a driving role in growth and
succession of periphyton. Being a substrate based
organization, periphyton have access to both organic
nutrients from substrate and inorganic nutrients from
water column. In nutrient rich environments, it receives
nutrients from water column (Eminson and Moss, 1980;
Burkholder, 1996). Here, similar to planktonic cells,
periphytic cells can use inorganic nutrients efficiently,
specifically dissolved organic Phosphorus and in nutrient
limited environments, it relies mainly on organic
nutrients from natural substrate. All artificial substrates
cannot serve as organic nutrient supplier to periphyton.
Substrates like sediments or seed grains acts as nutrient
diffusing substrate releasing nutrients to overlying
periphytic layer. (Hansson, 1989) showed that epipelion
can significantly lower nutrient availability in the water
column due to uptake of diffusing nutrients. (Hagerthey
and Kerfoot, 1998) demonstrated that inflowing ground
water is a significant source of nutrients for episammon
in nutrient limiting environment. These sediments act as
better nutrient source for periphyton (Burkholder, 1996).
Substrate based nutrient uptake by periphyton is further
related to depth, light availability, physical disturbances
etc.
Grazer-Periphyton interaction
Studies reported that several herbivore types
(e.g. gastropods, trichopteran larvae and fish) can
dramatically reduce periphytic biomass to only a few
percent of total biomass (Hillebrand et al., 2000).
Although grazing results reduction in periphytic biomass,
the total productivity of the periphytic complex increases
due to reduced competition among algal members
(Carpenter, 1986; Mc Cormick and Stevenson, 1989).
(Norberg 1999), using transparent incubation chambers,
measured a 4-fold increase in periphyton specific
productivity in grazed periphyton compared to ungrazed
controls. Moreover, the grazer presence increased the
Chlorophyll: biovolume ratio, especially reported from
streams (Hill and Knight, 1987). In addition to increase
in productivity, grazing and competition can modify the
species composition of periphytic algal assemblages
(Duffy and Hay, 2000; Nielsen, 2001), generating
heterogeneity through temporal or spatial scale on the
substrate. A top down effect of consumers on their prey
can be further accelerated by grazer and grazer excretion
of nutrients, removal of senescent cells, or increased
uptake of nutrients by the remaining cells (Lamberti
et al., 1987; Kahlert and Baunsgaard, 1999). Grazers
Saikia et al., 2013

may have strongest effects on Carbon:Phosphorus
and Nitrogen:Phosphorus, but Carbon:Nitrogen and
Carbon:Chlorophyll may remain unaffected (Hillebrand
and Kahlert, 2001). Hillebrand et al., (2008) described
three pathways for grazer mediated periphytic
interactions affecting nutrient stoichiometry. First, the
non algal component, which could be a dominant part
of the organic material of periphyton assemblage
(Frost et al., 2002) is reduced by unselective grazing.
Benthic invertebrates graze upon both detritus and algal
component of periphyton but only algae regenerate.
Therefore, grazing not only reduces non algal component
of periphyton, but also facilitates the growth of live
component within it. (Jones et al., 1999) suggested that
epiphytes can influence the nutritional quality of the
periphyton which grows on their surfaces, making it
more nutritious for grazing by invertebrates, particularly
snails. In return, these grazers might preferentially feed
on the periphyton and clear the plants of a potential
competitor for nutrients, with the plants and grazers both
gaining from this relationship. Secondly, in streams,
nutrient uptake of intact periphyton mats is often slower
than cell specific uptake rates as boundary effects reduce
the uptake ability of the benthic algae (Riber and Wetzel,
1987; Bothwell, 1989; Burkholder et al., 1990). Grazer
presence alters periphyton architecture, increases
periphytic heterogeneity and relative availability of
nutrients (through reducing Carbon: nutrient ratio) per
unit biomass enhancing periphytic nutrient uptake.
Thirdly, the excretion or egestion of nutrients or both by
grazers also increase the supply of nutrients to the
periphytic assemblages. Grazers may spatially recycle
nutrients that increase the availability and uptake of
nutrients by the periphyton. However, in streams,
grazers may increase the export of nutrients
(Mulholland et al., 1991).

CONCLUSION
Disrupted nutrient cycling is a major problem
both in freshwater and marine ecosystems and
periphyton could be a non-point manager of nutrient
cycle disruption and hence can overplay on plankton for
nutrient cycling in aquatic ecosystem. During renovative
practices, strategies of aquatic ecosystem health
managers greatly ignore the role of these substrate based
microorganisms. At the same time, it can play as an
efficient supplier of nutrient to its grazer under
controlled and well managed productive practices. It is
observed that at traditional level, farmers from different
parts of the world have been practicing periphyton to
feed aquacrops to convert periphytic energy biomass to
crop biomass (Saikia and Das, 2009). Such conversion of
biomass is an outcome of increased assimilation of
micro- and macro nutrients from periphytic complex in
the fish body through trophic upgrading (Saikia and
Nandi, 2010). Further researches on the mode of energy
transfer through periphytic food chain, enhanced nutrient
uptake under manipulative nutrient input, modelling on
applied periphytic ecology, ecotoxicology, Carbon
entrapment and delivery, directing nutrient and Carbon
sequestration both in marine and freshwater are needed
for better understanding of its role in aquatic ecosystem.

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Assessing heavy metal contamination of road side soil in urban area
Keywords:
Pollution, combustion, heavy metal enrichment, road side soils, enrichment
factor, Normalized scatter coefficient value, environmental pollution.
ABSTRACT:

Environmental pollution of heavy metals from automobiles has attained
much attention in the recent past. The pollution of soil by heavy metals is a serious
environmental issue. Heavy metals are released during different operations of the
road transport such as combustion, component wear, fluid leakage and corrosion of
metals lead, cadmium, copper and zinc which are the major metal pollutants of the
road side environment. The present research is conducted to study heavy metal
contamination in road side and industrial soil of Madurai city. The soil samples are
collected from three sites and analyzed for six heavy metals (Pb, Cu, Cr, Zn, Ni and Cd).
Their concentration and distribution in different depths (0 cm, 5 cm and 10 cm) were
determined. Heavy metal contents were analyzed by Atomic Absorption Spectroscopy
(AAS). The studies with Enrichment Factor (EF) indicate that lead has been enriched to
quite great extent while the Normalized Scatter Coefficient values (NSC) indicate
faster enrichment of cadmium. The level of heavy metals in road side soils were higher
as compared to their natural background levels. The results revealed that the heavy
metals are harmful to the road side vegetation, wild life and the neighbouring human
settlements.
789-796 | JRB | 2013 | Vol 3 | No 1

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
An International Scientific
Research Journal
Authors:
Sarala Thambavani D
1
,
Vidya Vathana M
2
.

Institution:
1. Associate Professor
Department of Chemistry,
Sri Meenakshi Govt. Arts
College (W), Madurai.

2. Assistant Professor,
Department of Chemistry,
Sacs M.A.V.M.M Engg.
College, Madurai.

Sarala Thambavani D.

Web Address:
Dates:
Received: 16 Jan 2012 Accepted: 27 Jan 2012 Published: 16 Feb 2013
Article Citation:
Sarala Thambavani D and Vidya Vathana M.
Assessing heavy metal contamination of road side soil in urban area.
An International Scientific Research Journal
Original Research

INTRODUCTION
Pollution in recent years has increased
considerably as a result of increasing human activities
such as burning of fossil fuels, industrial and automobile
exhaust emissions. The pollution of soils by
heavy metals from automobile sources is a serious
environmental issue. The majority of the heavy metals
are toxic to the living organisms and even those
considered as essential can be toxic if present in excess.
The heavy metals can impair important biochemical
processes posing a threat to human health, plant growth
and animal life (Jarup 2003; Michalke 2003; Silva et al.,
2005).
The waste products from vehicles that ply
highways contain some heavy metals inform of smokes.
Emissions from exhaust pipes of automobile engine and
contacts between different metallic objects in machines
contain such heavy metals as Lead (Pb), Zinc(Zn), Iron
(Fe), Copper (Cu), Chromium (Cr) and Cadmium (Cd)
and are major sources of pollution among soils (Turer
and Maynard, 2003).
Soils are usually regarded as an ultimate sink.
For heavy metals discharged into the environment (Banat
et al., 2005) and sediments can be sensitive indicators for
monitoring contaminants in aquatic environment (Pekey
et al., 2004). Therefore the environmental problem of
soil and sediment pollution by heavy metals has received
increasing attention in the last few decades in both
developing and developed countries throughout the
world (Zhang et al., 2007). Hence, inorder to monitor
heavy metal pollution in an area, due to the
anthropogenic activity (Sarala Thambavani and Vathana,
2011), the soil samples represent an excellent media
because heavy metals are usually deposited in the top
soil (Govil et al., 2001; Romic and Romic, 2003) and
help in knowing the sources of heavy metals and also
controlling and optimizing their effects on the human
health.

It is needless to say that the industrial activities
in the metropolitan cities of the world are responsible for
the addition of pollutants through chemical factories,
residential activities (Point sources) and vehicular traffic
(non-point sources) which are the primary sources of soil
pollution. The objective of this study, is to investigate the
effect of heavy metal pollution of soil along road sides.
The present study reports the role of industrial and
urban activities in the heavy metal contamination of the
soils in the Madurai industrial area with the objectives:
To assess the extent of heavy metal pollution
influenced by urban and industrial activities.
To predict the rate of heavy metals pollution in the
future if the activities are allowed with the same
pace.
To understand the variations in the behavior of
different heavy metal.

METHODS
Field Methodolgy
To understand the state of environment of the
Madurai area a detailed field survey was carried out and
after having identified possible sources of pollution a
part of Madurai area was selected. This area is under
intense human interference in terms of growing
urbanization (municipal sewage sludge, traffic pollution
in particular) and industrialization.
Selection of sampling site
In the present study stratified regular sampling
method was adopted for soil sample collection as in
geo-assessment of the variables estimated, the stratified
regular sampling is more suitable because this kind of
sampling draws homogenous error (Burgess et al., 1981).
Different sampling stations were selected and samples
are collected from the top layer of the soil using plastic
spatula after removing the debris, rock pieces and
physical contaminants. In order to have the background
concentration values of the heavy metal elements, three
soil samples were collected, each from 100 cm below
Thambavani and Vathana, 2013
ground level, which are least affected by anthropogenic
activities (Table1). The samples were placed in the clean
polythene bags, which were brought to the laboratory.
Laboratory Methodology
The samples were brought to the laboratory
where they are dried and mixed thoroughly to obtain the
representative samples. Soon after drying the debris and
other objects were hand picked up and the sample were
grounds in a mortar to break up the aggregates or lumps,
taking care not to break actual soil particles. Soil samples
were then passed through a 2 mm sieve in order to
collect granulometric fraction. Since trace metals are
often found mainly in clay and silt fractions of soil and
hence the size fraction <63 m sieve (wet sieving) and
was used to measure the concentration of the heavy
metals Lead, Copper, Chromium, Zinc, Nickel and
Cadmium from all the samples collected.
For this purpose the clay and silt fraction were
digested by acids to get the solution by taking 5 g of
sample into a 300 ml polypropylene wide-mouthed jar
and distilled water was added to make a total 200 ml.
Then it was acidified with 10 ml HF, 5 ml HClO
4
,
2.5 ml HCl and 2.5 ml HNO
3
in order to completely
digest the soil. This jar was shaken on an orbital shaker
for 16 h at 200-220 rpm before being filtered through
whatman filter paper (No.42) into acid washed bottles.
The solution was stored and heavy metal contents were
analyzed by Atomic Absorption Spectrophotometer as
per the method recommended by committee of soil
standard methods for analyses and measurement (1986).
The raw data obtained during the course of laboratory
analyses were stored in Microsoft Excel software and
further processed to obtain various parameters required
for interpretation.
RESULTS
The concentration of heavy metals Lead,
Copper, Chromium, Nickel and Cadmium in the soils
of Madurai industrial, traffic and residential area
were analyzed, collected at six sampling stations during
May 2011- Oct 2011. The range of the concentrations
found in different sampling stations are (i) Pb industrial
(24.81-42.37 mg/kg), traffic (26.80-5.32 mg/kg)
and residential (20.42-2.66 mg/kg) (ii) Cu industrial
and residential (10.5 -18.16 mg/kg) (iii) Cr industrial
and residential (25.12 mg/kg) (13.60-18.52 mg/kg)
( i v) Zn i ndust r i al ( 22. 5- 45. 6 mg/ kg) ,
traffic (22.32-25.46 mg/kg) and residential (22.24- 25.12
mg/kg) (v) Ni industrial (11.85-14.0 mg/kg), traffic
( 11.52 -14.80 mg/kg) and residential (11.70-13.9 mg/kg)
(vi) Cd industrial (1.24-4.32 mg/kg), traffic
(1.60-3.62 mg/kg) and residential (1.70-2.25 mg/kg).
The mean concentration for these heavy metals
from the surface soil have been calculated to be (i) Pb
industrial (33.23), traffic (41.50) and residential (24.31).
(ii) Cu industrial (12.97), traffic (15.03) and residential
(14.98). (iii) Cr industrial (24.33), traffic (17.53) and
residential (15.51). (iv) Zn industrial (29.78), traffic
(24.23) and residential (23.74). (v) Ni industrial (12.77),
traffic (13.72) and residential (12.99). (vi) Cd industrial
(2.94), traffic (2.59) and residential (1.92) respectively at
the confidence limits of 95%.
The concentration of heavy metals in all the
sampling stations exhibit an increasing trend over a very
short period of monitoring from May 2011-Oct 2011
(Figure 1). It was observed that the mean concentration
of Lead has been increased in all the three sampling
Table 1 Natural Local background concentration values (mg/kg) of the heavy elements of soils
Sampling stations Pb Cu Cr Zn Ni Cd
Industrial Area 5.14 9.44 9.89 11.32 11.28 0.32
Traffic Area 5.22 9.58 10.09 11.76 11.29 0.30
Residential Area 5.26 9.63 11.10 11.87 11.31 0.35

stations followed by Zinc, Chromium, Copper, Nickel
and Cadmium.
Accumulative Signature of Heavy Metals
An increasing trend has been found for the heavy
metal elements Lead, Copper, Chromium, Zinc Nickel
and Cadmium wherein the Lead and Cadmium are
getting accumulated with very rapid rate mainly due to
anthropogenic activities (Sayadi, 2009). In order to
assess the variations in the heavy metal accumulations in
the soils, the calculated measures that is Enrichment
Factor and Normalized Scatter Coefficient were used.
The Enrichment Factor (EF) is a ratio of the
concentrations of the heavy metals in the soil samples to
the corresponding concentration of natural background
concentration. EF is calculated with the help of the
formula given by Subramanian and Datta dilip (1998)
and presented in Table 2.

Normalized Scatter Coefficient (NSC) has been
calculated to asses the temporal variability of the heavy
metals in the soils. It helps to understand the increasing
or decreasing concentration of heavy metals in the soils
with the passage of time which is independent of the past
focusing only at the period of study. The NSC for any
element is calculated (Table 3) with the following
formula (Sayadi and Sayyed, 2010).
The NSC values + 100% indicates absolute
increase while-100% means absolute decrease. The value
of 0% can be regarded for no change in the parameters
under consideration.

DISCUSSION
In order to evaluate the rate of accumulation of
heavy metals in the soils the mean values for all heavy
metals studied were considered along with Enrichment
factor values of all six metals (Table 2), which clearly
indicate the highest enrichment of Cadmium followed by
Lead, Zinc, Chromium, Copper and Nickel in all the
three sampling stations of industrial, traffic and
residential area. The values of NSC for all six heavy
metal showed that Cadmium is increasing in soil
environment of industrial area followed by Zinc,
Chromium, Lead, Copper and Nickel. In traffic area
Lead is increasing in soil environment followed by
Cadmium, Chromium, Copper, Nickel and Zinc and in
residential area Copper is increasing in soil environment
followed by Chromium, Cadmium, Lead, Nickel and
Zinc.
It is observed that in all the sampling sites, Lead
shows highest concentration in soil and also have high
Pb Cu Cr Zn Ni Cd
4.6 1.1 1.7 1.9 1.1 3.8
5.3 1.1 1.8 2.1 1.1 4.1
5.7 1.4 2.2 2.3 1.0 8.8
6.9 1.5 2.7 2.5 1.1 11.8
7.7 1.5 2.9 2.9 1.2 13.1
8.3 1.7 3.5 4.0 1.2 13.5
Industrial Area
Pb Cu Cr Zn Ni Cd
5.1 1.1 1.4 1.9 1.0 5.3
5.6 1.4 1.6 1.9 1.2 6.6
6.9 1.5 1.6 2.1 1.2 8.3
8.7 1.7 1.8 2.1 1.3 9.5
10.1 1.8 1.8 2.1 1.3 9.9
11.2 1.9 2.1 2.2 1.3 12.1
Traffic Area
Pb Cu Cr Zn Ni Cd
3.9 1.1 1.2 1.9 1.0 4.9
4.2 1.3 1.3 1.9 1.1 4.9
4.7 1.3 1.3 2.0 1.2 5.3
4.8 1.8 1.4 2.0 1.2 5.7
5.0 1.9 1.5 2.1 1.2 5.7
5.1 1.9 1.7 2.1 1.2 6.4
Residential Area
Table 2 Enrichment Factor for heavy metals in the
soils
concentration in the last sampling
concentration in first sampling
NSC = x 100
concentration in the last sampling +
concentration in first sampling
EF = Value of a given metal concentration found on soil (mg/kg)
Natural local background concentration of the metal (mg/kg)
enrichment factor. Cadmium shows lowest concentration
in soil but is has quite high enrichment factor, while
Copper, Chromium, Zinc and Nickel shows higher metal
concentration but rather low EF when compared to lead.
The scatter plot of the mean concentration of
heavy metals was plotted against the EF for all the three
sampling sites (Figures 5,6,7). Per usual of the result
showed that Zinc is having high mean concentration but
it is not getting enriched in proportion to its mean
concentration. On the other hand Cadmium though
having lowest mean concentration has higher rate of
enrichment. Lead shows the highest mean concentration
and also corresponding highest enrichment factor.
The behavior of Zinc may be attributed to its
source mainly from weathering of the parent rock while
that of Cadmium and Lead mainly due to anthropogenic
activities. EF normally reveals the addition and or
removal of metal under consideration which is a result of
cumulative activity in the region. Hence the Enrichment
factor should denote the total enrichment and or
depletion of an element and cannot evaluate the trend for
the short term accumulation.
When the mean values of EF and NSC for all the
six heavy metals are studied at all the sampling stations
(Figures 8, 9,10) it can be stated that Cadmium has been
enriched to a quite greater extent followed by Lead, Zinc,
Chromium, Copper and Nickel at all the sampling sites.
On the other hand the Normalized Scatter Coefficient
value indicates that Cadmium has got enriched in faster
rate at industrial area followed by Zinc, Chromium,
Lead, Copper and Nickel. In traffic area Lead is getting
enriched in the faster rate followed by Cadmium,
Chromium, Copper, Zinc and Nickel. But in residential
area, the NSC value indicate that Cu is quite enriched
with the faster rate followed by Chromium, Cadmium,
Lead and Zinc.
Figure 2. Industrial Area
Figure 3. Traffic Area
Figure 4. Residential Area
Figure 1. Mean Concentrations of the heavy metals
on different sampling stations

CONCLUSION
The variation assessment of heavy metal
pollution by using Enrichment Factor and Normalized
Scatter Coefficient in the soil sample collected from the
study area between May 2011-Oct 2011 has revealed
significant increase in the six heavy metals (viz Pb, Cu,
Cr, Zn, Ni and Cd). Enrichment Factor values shows
that Cadmium has enriched to a greater extent followed
by Lead, Zinc, Chromium, Copper and Nickel.
Normalized Scatter Coefficient value indicate that Lead
is getting accumulated in a faster rate followed by
Cadmium, Chromium, Copper, Zinc and Nickel. In
summary the soils in the Madurai industrial, traffic and
residential area are significantly contaminated by heavy
metals and hence more attention to be paid to heavy
metal pollution particularly for Lead and Cadmium. In
Figure 7. Residential Area
N
o
r
m
a
l
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z
e
d

S
c
a
t
t
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r

C
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f
f
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c
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t

%

E
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r
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c
h
m
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n
t

F
a
c
t
o
r

Figure 8 INDUSTRIAL AREA
N
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m
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d

S
c
a
t
t
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r

C
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e
f
f
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%

Figure 9 TRAFFIC AREA
E
n
r
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c
h
m
e
n
t

F
a
c
t
o
r

E
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r
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c
h
m
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n
t

F
a
c
t
o
r

N
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m
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l
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S
c
a
t
t
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C
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f
f
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%

Figure 10. RESIDENTIAL AREA
Figure 5. Industrial Area
Figure 6. Traffic Area
order to prevent heavy metal contamination in the soils
from the Madurai city and to maintain the ecological
balance some immediate measures as per environmental
quality criteria, a need to be taken.

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Pb Cu Cr Zn Ni Cd
26.1 21.9 33.8 33.8 8.6 55.4
21.1 21.1 30.9 31.7 8.1 53.2
17.8 12.0 23.6 26.7 6.6 21.3
8.9 7.8 13.0 24.3 4.7 6.7
3.4 7.6 9.8 16.8 1.9 1.4
0 0 0 0 0 0
Industrial Area
Pb Cu Cr Zn Ni Cd
13.2 26.4 15.3 6.1 8.9 13.9
8.8 17.9 13.1 4.7 7.2 13.4
3.9 16.8 11.9 3.4 3.2 9.8
2.5 1.6 8.7 2.6 2.5 6.1
0.8 0.6 5.4 0.4 0.6 5.6
0 0 0 0 0 0
Residential Area
Pb Cu Cr Zn Ni Cd
37.0 25.9 19.5 6.6 12.5 38.7
32.9 14.6 15.8 4.9 5.7 29.1
23.0 10.9 14.0 1.9 2.7 18.3
12.5 5.5 8.0 0.9 2.0 12.1
5.2 4.1 7.3 7.3 0.8 9.5
0 0 0 0 0 0
Traffic Area
Table 3 Normalized Scatter Coefficient (%)of the
heavy metals in the soils of the study area

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Aim and Scope

Total amount of ingredients used for preparing the

, a chitin synthesis inhibitor against the tropical

. These dosages had

and 0.5 mL/L Novaluron treatments. Also,

treatment in impairing the development of

(Table 2). Mortality was also lower

but no pupation was

-treated glass surfaces. Generally,

ranged from 0.0 to

significantly reduced the development of

43.0 0.1 97.50.0 45.0 0.1 35.0 0.1 55.00.0

and novaluron insecticides

and novaluron insecticides

significantly caused higher mortalities on

may be attributed to the

for treatment of maize

insecticides. d = days h= hours

insecticide. In the field maize treated with

and infested with adult E. cautella

(6.6%) and novaluron

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