Journal of Research in Biology Volume 3 Issue 1 by Journal of Research in Biology

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CHANDRAMOHAN [Biochemist] College of Applied Medical Sciences, King Saud University.

Dr. Pankaj Sah [Environmental Science, Plant Ecology] Higher College of Technology (HCT), Al-Khuwair.

B.C. Behera [Natural product and their Bioprospecting] Agharkar Research Institute, Pune, INDIA.

Dr. Erkan Kalipci [Environmental Engineering] Selcuk University, Turkey.

Kuvalekar Aniket Arun [Biotechnology] Lecturer, Pune.

Dr Gajendra Pandurang Jagtap [Plant Pathology] College of Agriculture, India.

Mohd. Kamil Usmani [Entomology, Insect taxonomy] Aligarh Muslim university, Aligarh, india.

Dr. Arun M. Chilke [Biochemistry, Enzymology, Histochemistry] Shree Shivaji Arts, Commerce & Science College, India.

Dr. Lachhman Das Singla [Veterinary Parasitology] Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India.

Dr. AC. Tangavelou [Biodiversity, Plant Taxonomy] Bio-Science Research Foundation, India.

Vaclav Vetvicka [Immunomodulators and Breast Cancer] University of Louisville, Kentucky.

Nasroallah Moradi Kor [Animal Science] Razi University of Agricultural Sciences and Natural Resources, Iran

José F. González-Maya [Conservation Biology] Laboratorio de ecología y conservación de fauna Silvestre, Instituto de Ecología, UNAM, México.

T. Badal Singh [plant tissue culture] Panjab University, India

Dr. Kalyan Chakraborti [Agriculture, Pomology, horticulture] AICRP on Sub-Tropical Fruits, Bidhan Chandra Krishi Viswavidyalaya, Kalyani, Nadia, West Bengal, India. Dr. Monanjali Bandyopadhyay [Farmlore, Traditional and indigenous practices, Ethno botany] V. C., Vidyasagar University, Midnapore. M.Sugumaran [Phytochemistry] Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram District. Prashanth N S [Public health, Medicine] Institute of Public Health, Bangalore. Tariq Aftab Department of Botany, Aligarh Muslim University, Aligarh, India. Manzoor Ahmad Shah Department of Botany, University of Kashmir, Srinagar, India. Syampungani Stephen School of Natural Resources, Copperbelt University, Kitwe, Zambia. Iheanyi Omezuruike OKONKO Department of Biochemistry & Microbiology, Lead City University, Ibadan, Nigeria. Sharangouda Patil Toxicology Laboratory, Bioenergetics & Environmental Sciences Division, National Institue of Animal Nutrition and Physiology (NIANP, ICAR), Adugodi, Bangalore. Jayapal Nandyal, Kurnool, Andrapradesh, India. T.S. Pathan [Aquatic toxicology and Fish biology] Department of Zoology, Kalikadevi Senior College, Shirur, India. Aparna Sarkar [Physiology and biochemistry] Amity Institute of Physiotherapy, Amity campus, Noida, INDIA. Dr. Amit Bandyopadhyay [Sports & Exercise Physiology] Department of Physiology, University of Calcutta, Kolkata, INDIA . Maruthi [Plant Biotechnology] Dept of Biotechnology, SDM College (Autonomous), Ujire Dakshina Kannada, India. Veeranna [Biotechnology] Dept of Biotechnology, SDM College (Autonomous), Ujire Dakshina Kannada, India. RAVI [Biotechnology & Bioinformatics] Department of Botany, Government Arts College, Coimbatore, India. Sadanand Mallappa Yamakanamardi [Zoology] Department of Zoology, University of Mysore, Mysore, India. Anoop Das [Ornithologist] Research Department of Zoology, MES Mampad College, Kerala, India.

Dr. Satish Ambadas Bhalerao [Environmental Botany] Wilson College, Mumbai Rafael Gomez Kosky [Plant Biotechnology] Instituto de Biotecnología de las Plantas, Universidad Central de Las Villas Eudriano Costa [Aquatic Bioecology] IOUSP - Instituto Oceanográfico da Universidade de São Paulo, Brasil M. Bubesh Guptha [Wildlife Biologist] Wildlife Management Circle (WLMC), India Rajib Roychowdhury [Plant science] Centre for biotechnology visva-bharati, India. Dr. S.M.Gopinath [Environmental Biotechnology] Acharya Institute of Technology, Bangalore. Dr. U.S. Mahadeva Rao [Bio Chemistry] Universiti Sultan Zainal Abidin, Malaysia. Hérida Regina Nunes Salgado [Pharmacist] Unesp - Universidade Estadual Paulista, Brazil Mandava Venkata Basaveswara Rao [Chemistry] Krishna University, India. Dr. Mostafa Mohamed Rady [Agricultural Sciences] Fayoum University, Egypt. Dr. Hazim Jabbar Shah Ali [Poultry Science] College of Agriculture, University of Baghdad , Iraq. Danial Kahrizi [Plant Biotechnology, Plant Breeding,Genetics] Agronomy and Plant Breeding Dept., Razi University, Iran Dr. Houhun LI [Systematics of Microlepidoptera, Zoogeography, Coevolution, Forest protection] College of Life Sciences, Nankai University, China. María de la Concepción García Aguilar [Biology] Center for Scientific Research and Higher Education of Ensenada, B. C., Mexico Fernando Reboredo [Archaeobotany, Forestry, Ecophysiology] New University of Lisbon, Caparica, Portugal Dr. Pritam Chattopadhyay [Agricultural Biotech, Food Biotech, Plant Biotech] Visva-Bharati (a Central University), India

Table of Contents (Volume 3 - Issue 1) Serial No

Accession No

RA0304

Title of the article

Anatomical variation in the olfactory apparatus of marine teleosts.

Page No

742-746

Biswas S, Datta NC, Sarkar SK and De SK.

RA0306

The use of purple yam (Dioscorea trifida) as a health-promoting

747-758

ingredient in bread making. Teixeira AP, Oliveira IMA, Lima ES and Matsuura T.

RA0305

Bioefficacy of Novaluron速, a chitin synthesis inhibitor against the

759-767

tropical warehouse moth, Ephestia cautella. Sackey I, Eziah VY and Obeng-Ofori D.

RA0308

A Checklist of Butterflies of Meenachil River Basin, Kerala, India.

768-774

Vincy MV, Brilliant R and Pradeepkumar AP. 5

RA0325

Microbial production of glutaminase enzyme.

775-779

Mario Khalil Habeeb. 6

RA0307

A review on the role of nutrients in development and organization of

780-788

periphyton. Saikia SK, Nandi S, Majumder S.

RA0187

Assessing heavy metal contamination of road side soil in urban area.

Sarala Thambavani D and Vidya Vathana M.

789-796

Journal of Research in Biology

An International Open Access Research Journal

Original Research

Journal of Research in Biology

Anatomical variation in the olfactory apparatus of marine teleosts Authors: Biswas S1, Datta NC2, Sarkar SK1 and De SK1. Institution: 1. Department of Zoology, Vidyasagar University, Midnapore (West) - 721102, West Bengal, India. 2. 110/20 B. T. Road, Kolkata - 700108, West Bengal, India.

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.

Corresponding author: Subrata Kumar De. Email: skdvu@yahoo.co.in

Keywords: Olfactory, Rosette, Scombridae, Carangidae, Platycephalidae, etc.

Phone No: +91 03222 275329

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

Web Address: http://www.jresearchbiology.com/ documents/RA0304pdf.

Dates: Received: 08 Nov 2012

Accepted: 20 Nov 2012

Published: 09 Jan 2013

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, noncommercial, distribution and reproduction in all medium, provided the original work is properly cited.

Journal of Research in Biology An International Open Access Research Journal

742-748 | JRB | 2013 | Vol 3 | No 1

www.jresearchbiology.com

Biswas et al., 2013 and Platycephalus scaber (Linnaeus, 1758), an estuarine

INTRODUCTION Olfaction is an important type of chemoreception which is mediated through well developed, complex and

amphidromous fish to unfold their structural components and functional significance in olfaction.

organized olfactory system in vertebrates (Freitag et al., 1999). This system generally develops from the specialized tissue, called olfactory placode (von Kupffer,

MATERIALS AND METHODS Adult,

sex-independent

specimens

1894). In the early stage of development, placodes are

R. kanagurta, S. commersonianus and P. scaber were

formed from the preplacodal ectoderm of the anterior

collected from the coastal regions of Bengal, India.

region of the embryo, medial to epidermis and lateral to

Specimens were preserved in 10% formaldehyde

both neural crest and neural plate (Knouff, 1935).

solution for 24 h and brought to the laboratory. The

The developmental process leading to the formation of

olfactory apparatus of these species were dissected out

fish olfactory organ, is little diverse (Hansen and

and separately mount on grease free slide by using

Zielinski, 2005). The peripheral olfactory organ is the

glycerine. The olfactory apparatus of respective species

first chemosensory organ to develop in fish preceding the

was examined under light microscope (LM).

systems of solitary chemosensory cells (Kotrschal et al., 1997) and taste (Hansen et al., 2003). Fish generally

RESULTS

possesses a paired olfactory organ located at the anterior

R. kanagurta (Figure 1A) possesses two pairs of

part of the head (Derivot, 1984; Hansen and Zeiske,

nostril viz., anterior nostril and posterior nostril

1998; DĂ¸ving, 2003; De and Sarkar, 2009). The olfactory

(Figure 1B). The anterior nostril is an oval shaped

chambers, olfactory rosette, accessory nasal sacs,

structure, encircled by a thick lip like ridge of skin and

olfactory bulbs, olfactory nerve tracts and brain are the

located at the dorsal region of the snout where as the

major

apparatus

posterior nostril is situated in front of the eye.

(Hamdani and DĂ¸ving, 2007). The anatomy of the

R. kanagurta shows a slit like structure i.e., posterior

olfactory apparatus shows wide range of structural

nostril which is located at a moderate distance from the

diversity regarding the shape and size of the olfactory

anterior one (Figure 1B). The olfactory apparatus of the

rosette, number of the olfactory lamellae, occurrence of

said species is present at the antero-dorsal side of the

accessory nasal sacs, etc. among the teleostean groups

snout in between the anterior and posterior nostril

(Kleerekoper, 1969). The anatomical details of the

(Figure 1C). The multilamellar olfactory rosette is oval

olfactory apparatus in several species belonging to the

in shape and is situated at the floor of the olfactory

diverse teleostean taxa with respect to their habitat are

chamber (Figures 1D and 1E). The triangular olfactory

still an obscure part in sensory biology. The sensory

lamellae vary from 60 to 70 in number per rosette and

systems of fishes show notable adaptations according to

are arranged pinnately on the raphe (Figure 1E). The

habitat and mode of life in comparison with the higher

accessory nasal sacs are clearly marked in the olfactory

vertebrates (Bone and Moore, 2008). The present study

apparatus of R. kanagurta. The lacrimal sac is conical in

focused on the true anatomy of the olfactory apparatus in

shape and located at the antero-medial region of

three species of different ecological habitat viz.,

lachrymal bone in association with olfactory rosette

Rastrelliger

a marine

(Figures 1C and 1D). The ethmoidal sac is connected at

oceanodromous fish; Scomberoides commersonianus

the posterior part of the olfactory rosette and situated

(Lacepede, 1801), a brackish water amphidromous fish

within a groove at the antero-lateral extension of the

743

components

kanagurta

fish

(Cuvier,

olfactory

1816),

Journal of Research in Biology (2013) 3(1): 742-748

Biswas et al., 2013 Plate - 1

OLR LS ETS

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).

frontal bone (Figures 1C and 1D). The olfactory nerve

(Figure 2B). The olfactory rosette is circular in shape and

tracts are arised from the base of olfactory rosette and the

consisting

length varies from 16 mm to 18 mm respectively. The

(Figures 2C and 2D). The number of the olfactory

distal part of the olfactory nerve tracts are connected

lamellae ranges from 100 to 140 per rosette (Figure 2D).

with olfactory lobes of the brain. The size of the

The accessory nasal sacs are not distinct in the olfactory

olfactory lobes is comparatively small in R. kanagurta

apparatus. The olfactory nerve tracts are arised from the

(Figure 1C).

base of the olfactory rosette and the length is ranges from

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 Journal of Research in Biology (2013) 3(1): 742-748

two

pairs

olfactory

lamellae

10 mm to 12 mm. The olfactory lobe is comparatively large in size (Figure 2C). Interestingly,

the

nostrils

scaber

(Figure 3A) are situated at the antero-dorsal region to the 744

Biswas et al., 2013 Plate - 2

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). eye and lying far apart from each other. The anterior

DISCUSSION

nostril is oval in shape and has a tongue like nasal flap

Olfactory systems of fish are among the most

where as the posterior nostril is valvular (Figure 3B).

highly developed olfactory senses of vertebrates

The olfactory rosette is relatively large in size and oval

(Kleerekoper, 1969). The sense of olfaction is mediated

in shape (Figures 3C and 3D). The number of the

through olfactory apparatus associated with nostrils. The

olfactory lamellae varies from 50 to 76 in number per

nostrils are responsible for incurrent and excurrent of

rosette (Figure 3D). The absence of accessory nasal sacs

water during water ventilation (Nevitt, 1991). The

is noted in the olfactory apparatus of P. scaber. The

structure of the anterior and posterior nostril varies

length of the olfactory nerve tracts ranges from

among the teleostean fishes (Kapoor and Ojha, 1972).

22 mm - 24 mm. and it is well connected with the

The presence of nasal flaps in between the both nostrils

olfactory lobe of the brain (Figure 3C).

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

745

Journal of Research in Biology (2013) 3(1): 742-748

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). olfactory rosette perhaps arrangement

pattern

several type of

possesses well developed accessory nasal sacs but

arrangement may help in the particular sensitivity to

S. commersonianus and P. scaber has no accessory nasal

certain

steroids,

sacs. The movement of jaws and its associated muscles

prostaglandins, etc. (Theisen et al., 1991). Anatomically

are also very significant for the water ventilation

the lamellar surface in S. commersonianus is much closer

(Nevitt, 1991). Accessory nasal sacs may be found in the

due to the short distance between anterior and posterior

olfactory organs of fishes with widely variable life styles

nostril than R. kanagurta and P. scaber. Therefore, the

and habitats, both marine and fresh water which are not

water soluble odorants may travel short distance to

confined to one particular situation (Cox, 2008).

interact with comparatively greater olfactory lamellar

The olfactory nerve may convey the chemical cues

surface of S. commersonianus. The water ventilation is

during water ventilation to the brain (Hamdani and

assisted by the pumping mechanism of accessory nasal

DĂ¸ving, 2007). The olfactory information plays an

sacs (Theisen et al., 1991) and may provide the ability

important role in different behaviour of fish such as

1965). amino

This

to sniff (Nevitt, 1991; Cox, 2008). R. kanagurta

lamellar

components

(Holl,

adopt

acids,

Journal of Research in Biology (2013) 3(1): 742-748

746

Biswas et al., 2013 searching

foods,

discrimination

avoidance

predators,

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

the

pollutants.

The

Cox JPL. 2008. Hydrodynamic aspects of fish olfaction. Journal of the Royal Society Interface, 5(23):575-593. Derivot JH. 1984. Functional anatomy of the peripheral olfactory system of the African lungfish Protopterus annectens

Owen:

macroscopic,

microscopic,

and

morphometric analysis. American Journal of Anatomy, 169(2):177-192.

xenotoxification of ocean especially the acidification

De SK and Sarkar SK. 2009. Morphoanatomy of

may also impair the ability of olfactory discrimination of

olfactory apparatus of Pseudapocryptes lanceolatus

coastal and marine species (Munday et al., 2009). Thus,

(Bloch and Schneider)

it is necessary to examine the effect of specific toxic

Ecology,

agent at a subcellular level of olfactory structures in marine teleolsts.

Journal Environment

and

27(4):1646-1648.

Døving KB. 2003. The fish olfactory system: It’s role in normal

biology

and

toxicological

research.

Proceedings of the Seventh International Symposium,

CONCLUSION This comparative anatomical study on the

Tallinn, Estonia. 149-158.

olfactory apparatus along with brain in three different

Freitag J, Beck A, Ludwig, G, von Buchholtz L, Breer

marine teleost belonging to the diverse ecological habitat

H. 1999. On the origin of the olfactory receptor family:

shows much structural variation according to the

receptor genes of the jawless fish (Lampetra fluviatilis).

changing environment which may be significant for

Gene, 226(2):165-174.

ecomorphology

and

evolutionary

aspects

neurobiology (Kotrschal et al., 1998). However, the olfactory system of marine, estuarine and coastal or migratory species may experience rapid fluctuations of

Hamdani EH, Døving KB. 2007. The functional organization

the

fish

olfactory

system.

Prog Neurobiol, 82(2):80-86.

environmental inorganic ions (Hubbard et al., 2000), so

Hansen A and Zeiske E. 1998. The peripheral olfactory

it could be an interesting part to identify the cellular

organ of the zebrafish, Danio rerio: an ultrastructural

components that are involved in the ion regulation of the

study. Chem Senses, 23:39-48.

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.

Hansen A, Rolen SH, Anderson KT, Morita Y, Caprio J, Finger, TE. 2003. Correlation between olfactory receptor cell type and function in the channel catfish. J Neurosci., 23:9328-9339. Hansen A and Zielinski, BS. 2005. Diversity in the olfactory epithelium of bony fishes: development,

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Teichmann H, 1954. Vergle chende ntersuchungen an

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der Nase der Fische. Zeitschrift fur Morphologie und Ă&#x2013;kologie der Tiere, 43(2):171-212. Theisen B, Zeiske E, Silver WL, Marui T, Caprio J. 1991. Morphological and physiological studies on the olfactory organ of the striped eel catfish, Plotosus lineatus. Marine Biology, 110(1):127-135. Journal of Research in Biology (2013) 3(1): 742-748

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An International Open Access Research Journal

Original Research

Journal of Research in Biology

The use of purple yam (Dioscorea trifida) as a health-promoting ingredient in bread making Authors: Teixeira AP1, Oliveira IMA1, Lima ES1 and Matsuura T2.

ABSTRACT:

Corresponding author: Antonia Paiva Teixeira.

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.

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 Institution: acceptance test and microbiological analysis of the formulations 10%, 15% and 20% 1. Faculdade de Ciências were also performed. Except for lipids, the centesimal composition and caloric values Farmacêuticas (FCF), revealed no statistically significant differences. An addition of purple yam in natura up Universidade Federal do to 20%, instead of wheat flour in ordinary bread (0%), can be made with no effect on Amazonas (UFAM), Rua the diet’s energy. The free radical scavenging, 2.2-diphenyl-1-picryl-hydrazyl (DPPH) Alexandre Amorim, 330, Aparecida, CEP: 69010-330, 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. Manaus, AM, Brasil. The acceptance test applied to compare the three formulations of purple yam breads 2. Instituto de Ciências revealed a significant difference only in the attribute colour. Purple yam breads Biológicas (ICB), showed no preferable differences. Results highlight the feasibility of purple yam bread Universidade Federal do as a health-promoting food in the Amazon region. Amazonas (UFAM), Av. General Rodrigo Octávio Jordão Ramos, 3000, Keywords: Campus Universitário, Purple yam (Dioscorea trifida); antioxidant activity; health-promoting food; Coroado I CEP: 69077-000, Amazon region. Manaus, AM, Brasil.

Email: nietapt@yahoo.com.br Web Address: http://www.jresearchbiology.com document/ RA0306.pdf.

Dates: Received: 15 Nov 2012

Accepted: 27 Nov 2012

Published: 09 Jan 2013

Journal of Research in Biology An International Open Access Research Journal

747-758 | JRB | 2013 | Vol 3 | No 1

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Teixeira et al., 2013 INTRODUCTION

pies and porridges. Nevertheless, this species has

Yams belong to the family Dioscoreaceae, genus

undergone little scientific investigation, so little is known

Dioscorea (Pedralli, 1988; 1997; Pedralli et al., 2002;

about its management techniques, genetic improvement,

Pedralli, 2004). This family is made up by 6 to 9 genera

nutritional potential, industrial use, storage procedures,

comprising over 600 species distributed throughout the

characterization, uses as natural dye, as well as its use as

Worldâ&#x20AC;&#x2122;s tropical, subtropical and temperate regions

a health-promoting ingredient, among others.

(Barroso et al., 1974; Pedralli, 1988; 1997; Melo

By and large, the bread consumed throughout the

Filho et al., 2000; Pedralli et al., 2002; Pedralli, 2004).

world is made mostly of wheat flour, salt and yeast.

The yams (Dioscorea spp.) yield tubers, which are very

Many other ingredients, have been incorporated into

important as staple, nutritional and healthy food, and are

bread formulation, so as to increase its diversity and

still used as an ingredient in traditional Chinese herbal

product appeals (Hsu et al., 2004).

medicine. They show a worldwide distribution, and are

A few studies have highlighted the great

found in many tropical countries, in South-Eastern Asia

potential of purple yam in bread making. In this case,

and Western Africa, where the species were introduced

yam flour may replace part of the wheat flour, improving

by cultivators (Rasper and Coursey, 1967; Akanbi et al.,

bread quality, as well as adding economical advantages

1996; Omonigho and Ikenebomeh, 2000; Lin et al.,

to it (Abramo, 1990; Hurtado et al., 1997; Litvin et al.,

2005). They can also be found in some American

1998; Omonigho and Ikenebomeh, 2000; Ratti, 2001).

countries, particularly in Brazil, where one can find them

Hsu et al., (2004) demonstrated the presence

in all regions, from the Amazon down to the Southern

antioxidants

the

flour

part of the country (Chu and Figueiredo-Ribeiro, 1991;

(Dioscorea purpurea), in five formulations of breads

Pedralli, 1997; 2004).

prepared with this tuberâ&#x20AC;&#x2122;s

flour,

purple

yam

with excellent

Purple yam (Dioscorea trifida) is an American

acceptance in Taiwan supermarkets. Contado et al.,

native species, which was domesticated by Amerindians,

(2009) showed yam (Dioscorea spp.) mucilage-based

with the cultivar distribution possibly pointing out its

loaf to present good public acceptance as to flavor,

domestication in Brazilian and Guyana border areas,

aroma and texture with sensory attributes, demonstrating

followed by dissemination throughout the Caribbean

the use of this tuber to be feasible as improvers in bread

islands (Pedralli, 1988; Pedralli et al., 2002; Pedralli,

making.

2004). D. trifida shows a wide distribution in Central and

The following aspects motivated the use of

South America, from the Caribbean to Peru. In Brazil it

purple yam (Dioscorea trifida) in natura as a bread

is found all the way from the Amazon right down to the

manufacturing health-promoting

Southern region. The species is associated to forest

present work: 1) its significant world consumption,

environments-Amazonian highland tropical rainforests,

presenting a considerable, expanding tillage alternative

Coastal Atlantic Forest in Southeastern Brazil and,

(Rasper and Coursey, 1967; Abramo, 1990; IITA, 2007);

mesophytic (seasonable) and gallery forests (Pedralli,

2) although, as yet incipient, an increase on the

1997).

production of this tuber in the State of Amazonas, Brazil, Here in the Amazonian region, purple yam

especially

Caapiranga

and

ingredient,

Careiro

in the

Castanho

(D. trifida) may be consumed in the following ways:

municipalities is being observed. According to the

baked, boiled, mashed, as ingredients for soups and meat

Instituto de Desenvolvimento AgropecuĂĄrio do Estado

stews, and in the formulation of flour for making cakes,

do Amazonas (IDAM) in 2008, 110 families of the

748

Journal of Research in Biology (2013) 3(1): 747-758

Teixeira et al., 2013 Caapiranga municipality yielded 2,475 tonnes in an area

(edible portion), in Amazonas

of 165 ha; and 3) the presence of antioxidants in purple

types most easily identified are: roxinho (light purple

yam, which increases the nutritional capacity in breads

flesh); roxo (mid purple flesh); roxão (dark purple flesh);

made from this tuber (Hsu et al., 2004).

branco (white flesh); and misto (white-purple flesh)

The main aim of the present study was to evaluate the potential of purple yam yield in the State of

State Townships. The

(Figure 1). Purple yam samples were collected at two

Amazonas, Brazil as a health-promoting ingredient in

Amazonas

bread making. On this context, it determined the

Castanho. Due to the seasonality and availability of these

centesimal composition, caloric value, and antioxidant

tubers in the region, the centesimal composition analyses

properties of purple yam as well as of breads made from

of yams and breads were performed with Caapiranga

this tuber in natura. Then, it undertook an organoleptic

samples. Yam and bread antioxidant and bread sensory

characteristic assessment of the breads, following tasters’

and microbiological analyses were carried out with

panel acceptance criteria. This purple yam species is, for

Careiro Castanho samples.

the very first time, being used in the Amazonian region,

Purple yam bread elaboration

as a feasible alternative for bread making.

Townships:

Caapiranga

and

Careiro

On account of the probability of getting breads with higher antioxidant concentration (Hsu et al., 2004), roxão (dark purple flesh) type samples were used in the

MATERIALS AND METHODS Species

identification

and

purple

yam

tuber

present study (Figure 1C). Yams in natura, for replacing wheat flour, were washed, peeled, weighed, ground

(Dioscorea trifida) collection Identification of the species Dioscorea trifida

in the liquidizer together with yeast, oil and water.

was accomplished by comparisons with a voucher

Then, this mixture was added to the previously mixed

herbarium specimen (Exsicata number 1353) deposited

dry ingredients (wheat flour, powdered milk, sugar and

at the National Research Institute of Amazonia (INPA)

salt). Bread manufacturing formulations can be seen at

Herbarium. It is very common to find the purple yam

(Table 1). Homogenization (30 min.), dough underwent

(D. trifida) exhibiting several color hues of its flesh

initial fermentation (60 min.), intermediate time for

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) roxão (dark purple flesh); D) branco (white flesh); and E) misto (white-purple flesh). Journal of Research in Biology (2013) 3(1): 747-758

749

Purple Yam (D.trifida)*** 76.43 ± 0.50 1.13 ± 0.69 1.83 ± 0.13 1.80 ± 0.05

0.90 24.30

0.90 23.00

0.78 ± 0.02 18.04 ± 0.66

100.00

96.00

89.64 ± 4.52

(Montaldo, 1977), **(TACO 2006), ***Present study

750

0.0862 0.2587 0.0683 0.1438

269.17 ± 21.82a 53.43 ± 3.13a 1.20 ± 0.05a

280.64 ± 3.95a 49.45 ± 2.26a 1.38 ± 0.07a 1.84 ± 0.07a

294.45 ± 15.63 a

Caloric value (kcal/100 g)

290.73 ± 7.56a 50.95 ± 2.79a

53.41 ± 4.07a 1.41 ± 0.03a 1.91 ± 0,10a

2.34 ± 0.37a 10.06 ± 2,12a

0.2699 0.0370

Yam (D. alata)** 73.70 0.10 2.30 7.30

Values exhibiting different letters in the same column indicate statistically significant differences (P <0.05).

Moisture (%) Lipid (%) Protein (%) Crude Fiber (%) Ash (%) Carbohydrate (%) Caloric value (Kcal/100g)

Yam (D. spp.)* 72.60 0.20 2.00 0.60

0.0752

Parameters

Table 2. Yam centesimal composition.

4.74 ± 0.10b

centesimal composition analyses were subjected to

35.09 ±5.62a

Findings obtained on the bread formulation

20%

according to the method of (AOAC, 2005).

9.82 ± 1,24a

Carbohydrate and caloric values were determined

4.87 ± 0.37ab

described by the Instituto Adolfo Lutz-IAL (2008).

32.65 ± 1.30a

fiber contents were determined according to procedures

15%

in triplicate. Moisture, ashes, lipid, proteins and crude

10.67 ± 0,41a

in four formulations: 0%, 10%, 15% and 20%, were done

4.24 ± 0,04a

(Dioscorea trifida), and purple yam incorporated breads

31.03 ± 0.83a

Centesimal composition analyses of purple yam

10%

its incorporated breads

1.18 ± 0.28a

Centesimal composition analyses of purple yam and

1.95 ± 0.08a

displaying the product’s labeling.

11.62 ± 0.81a

room temperature and packed in polyethylene bags

4.49 ± 0.12ab

bread. After being prepared the breads were cooled to

29.79 ± 2.04a

time for baking (30 min.) are followed for making yam

bread shaping (25 min.), final fermentation (60 min.),

Carbohydrate (%)

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

Ash (%)

Crude Fiber (%)

20% 400 100 10 5 10 10 10 250 545

Protein (%)

0% 500 0 10 5 10 10 10 250 545

Lipid (%)

Wheat flour (g) Purple yam (g) Sugar (g) Salt (g) Yeast (g) Milk powder (g) Oil (g) Water (mL) Total (g) †

Type of bread* 10% 15% 450 425 50 75 10 10 5 5 10 10 10 10 10 10 250 250 545 545

Moisture (%)

Ingredients

Bread

Table 1. Purple yam (D. trifida) incorporated bread formulations.

Table 3. Centesimal composition and caloric value mean and standard deviation on account of the dry material (except for the moisture content) of the four analyzed purple yam incorporated bread formulations (0%, 10%, 15% and 20%). Probability (P) values calculated from Kruskal-Wallis ANOVA followed by post hoc tests are shown.

Teixeira et al., 2013

Journal of Research in Biology (2013) 3(1): 747-758

Teixeira et al., 2013 statistical analysis through the statistical software

(2004), with some modifications, where 2 mg of DPPH

package (Statsoft STATISTICA 8.0 2007). Given to the

were dissolved into 15 mL of methanol, and applied so

number of sampled observations (n=3), Kruskal-Wallis

as to determine the antioxidant activity of samples of

ANOVA and post hoc tests were applied as a

purple yam and its incorporated breads in the four

non-parametric alternative to Fisher ANOVA, for

aforementioned formulations. A micro plate bearing

independent data, in the comparison among the bread

96 well was used. Thirty microliters (30 µL) of the

formulations.

methanolic extract, plus 170 µL of methanol (used as the

Findings showing significance level of (P<0.05)

blank) were placed in the wells. The reading was

were considered as statistically significant.

performed on an Elisa reader (DXL 800-BECKMAN

Preparation of purple yam and its incorporated

COULTER) at a wavelength of 492 nm, using triplicate

breads methanolic extract

samples. Then, 100 µL of the DPPH solution were

Samples of purple yam (Dioscorea trifida),

added, and the material was stored in a dark place for

were peeled and ground with the aid of a knife. They

30 min, and the reading was repeated as soon as this time

were then dehydrated in a laboratory oven at 60°C for

was over. Two hundred microliters (200 µL) of methanol

24 h. Purple yam incorporated breads of four

added to 100 µl of the DPPH solution were used as

formulations: 0%, 10%, 15%, and 20%, were cut into

the control. Thirty microliters (30 µL) of quercetin

1 cm thick slices, and dehydrated in a laboratory oven at

(10 µg/mL), 170 µL of methanol and 100 µl of the

40°C for 24 h (Hsu et al., 2004). Dehydrated yams and

DPPH solution, were used as the standard. The following

breads were ground with pestle and mortar, weighed at

formula was used so as to calculate the antioxidant

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

A sample - A blank % AA = 100 -

x 100 A control

added with 5 mL of methanol and left in a rotary shaker for 24 h. The material was centrifuged at 2,500 RPM

activity percentage

for 10 min so as to obtain the supernatant (methanolic

Antioxidant activity determination through the lipid

extract). The antioxidant activity of the samples was

peroxidation (LPO) method in purple yam and its

determined by the free radical scavenging, 2.2-diphenyl-

incorporated breads

1-picryl-hydrazyl (DPPH) and lipid peroxidation (LPO)

The determination of the antioxidant activity of

methods. The latter method evaluates the inhibition of

the samples through the LPO method was carried out

free radicals generated during the

acid

according to the method reported by Duarte-Almeida

peroxidation, and is based on spectrophotometric

et al., (2006), based on the methodology originally

measurements of discoloration (oxidation) of ß-carotene,

described by Marco (1968), and later modified by Miller

induced by linoleic acid oxidative degradation products

(1971).

(Marco, 1968; Miller, 1971; Duarte-Almeida et al.,

an Erlenmeyer flask, containing 50 µL of linoleic acid,

2006).

200 µL of tween 80 (emulsifying agent), 150 µL of

Antioxidant activity determination through free

ß-carotene solution at 2 mg/mL in chloroform, and

radicals scavenging methods (DPPH) in purple yam

500 µL of chloroform. The mixture was then subjected to

and its incorporated breads

evaporation in nitrogen till there was no more

linoleic

The

reactive

mixture

was

prepared

DPPH method, following the methodologies

chloroform left. Later, the mixture of 25 mL of

described by Shimada et al., (1992) and Hsu et al.,

previously oxygen saturated water was added, and during

Journal of Research in Biology (2013) 3(1): 747-758

751

Teixeira et al., 2013 a period of 30 min it was homogenized through vigorous

software package (Statsoft STATISTICA 8.0 2007). The

shaking.

Shapiro-Wilk test rejected the frequency distribution

The reactive mixture showed to be clear with

normality of the three tested bread formulations, in all

absorbency ranging from 0.6 to 0.7 at a wavelength of

their sensory attributes. However, the Levene test

492 nm. A 96 well bearing micro plate was used. Two

accepted the homocedasticity (homogeneity of variances)

hundred forty microliters (240 µL) of the reactive

among the formulations for all sensory attributes. As

mixture and 10 µL of the methanolic extract samples

frequency

were placed in the wells. Ten microliters (10 µL) of

homogeneity are basic assumptions made for the

methanol and an equal volume of butylhydroxytoluene

application of parametric tests, such as Fisher’s

(BHT) at a concentration of 40 µg/mL were used as

ANOVA, and as these assumptions were not attended to,

control and standard, respectively. The micro plate was

the Friedman ANOVA followed by post hoc tests were

incubated at 50ºC to speed up the oxidation reactions and

applied as a non-parametric alternative for paired data in

start

slope

bread comparisons. Findings presenting significance

readings of samples, control and BHT (in triplicate) were

level of (P<0.05) were considered as statistically

performed readily, in an Elisa reader at a wavelength of

significant.

492 nm every 15 min for 135 min. The following

Microbiological analysis of purple yam breads

β-carotene

discoloration.

Discoloration

formula was used so as to calculate the oxidation

Following Brazilian

A2 sample - A1 sample % I = 100 -

x 100 A2 control - A1 control

distribution

(in

the

National

Portuguese,

normality

and

recommendation

Health

Agência

Surveillance

Nacional

variance

the

Agency Vigilância

Sanitária, ANVISA), based on Ruling Number 12 (RDC,

inhibition percentage:

2001), we carried out the microbiological analysis so as

Sensory analysis of purple yam incorporated breads

to verify Coliforms and Salmonella in samples of the

The acceptance test of purple yam in natura incorporated breads counted with the participation of 78

three purple yam bread formulation samples through the membrane filtration method (APHA, 2001).

non-trained volunteer judges. Each one of them was provided with an answering card bearing a 9 point

RESULTS AND DISCUSSION

hedonic scale (9-like extremely to 1-dislike extremely),

Centesimal composition and caloric value of purple

adapted from Stone et al., (1993) and Silva et al., (2005).

yam

The judges were provided with three purple yam

Moisture (76.43±0.50), protein (1.83±0.13) and

incorporated bread samples, produced from three

ash (0.78±0.02) contents, as well as the caloric value

formulations (10%, 15% and 20%) (Table 1). Samples

(89.64±4.52) of purple yam (D. trifida) samples analyzed

were served in white, disposable plastic plates; encoded

in the present study (Table 2) show to be near

with three randomly chosen numbers. Samples were

those

evaluated according to their sensory qualities: global

(Dioscorea spp.) and those found in the Brazilian

feel, aroma, flavor, color and texture. Judges were

Food Composition Table TACO (2006), for the yam

advised to always rinse their mouth with water before

(D. alata). Lipid content (1.13±0.69) stayed well above

testing the next sample.

that presented by Montaldo (1991) and TACO (2006).

presented

Montaldo

(1991)

for

yam

The findings obtained on the acceptance test

Crude fiber content (1.80±0.05) is above the value

were submitted to statistical analysis through statistical

observed by Montaldo (1991), and well below that

752

Journal of Research in Biology (2013) 3(1): 747-758

Teixeira et al., 2013 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). Moisture Lipid Protein Crude Fiber Ash Carbohydrate Caloric value Bread (%) (%) (%) (%) (%) (%) (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 presented in TACO (2006). The high fiber content

ordinary bread loaf (OBL) (Anton et al., 2006) and

presented by TACO (2006) might be due to the

whole bread loaf (WBL) (TACO, 2006) (Table 4). One

enzymatic gravimetric method employed in the analyses.

notices, a high fiber content (6.90%) in the whole bread

That method warrants a higher precision for determining

loaf (WBL) (TACO, 2006), relative to the remaining

the dietary fiber as compared to the acid digestion

breads. It can be highlighted that in whole bread

methodology used in the present study as well as

composition, we have the presence of grain-composed

by Montaldo

(1991). Total carbohydrate content

whole flour, almost wholly made up of bran, germ and

(18.04Âą0.66) is well below Montaldo (1991) and TACO

endosperm (FDA, 2006). By and large, all other values

(2006) values. The remaining differences in centesimal

show to be approximate. All differences found may be

composition values presented by Montaldo (1991) and in

related to formulations employed in the preparation of

the present study might be related to the different soil

those breads.

types being employed on planting the tubers and/or to the

Antioxidant activity determination through the free

different species being utilized. Nevertheless, the

radical scavenging method (DPPH) in purple yam

different values presented in TACO (2006) may be

and its incorporated breads

related to the different yam species being analyzed. Centesimal composition and caloric value of purple

Antioxidant activity (% AA) of the methanolic extract pertaining to purple yam (Dioscorea trifida)

yam incorporated breads 100

Based on data from Kruskal-Wallis (ANOVA) 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

90 Antioxidant activity (%)

followed by post hoc tests (Table 3), it may be asserted

80 70

Purple yam 0% Bread

10% Bread

15% Bread

20% Bread Quercetin

30 20 10 0 40

160

330

Concentration (mg/mL)

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 Journal of Research in Biology (2013) 3(1): 747-758

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. 753

Teixeira et al., 2013 samples in the concentrations of 330, 160, 80 and

LPO method confirmed the antioxidant activity (% I) in

40 mg/mL, as determined by the DPPH method, were

purple yam (55.80±4.85) and its breads from the three

higher than 70%, reaching a maximum of 88.13±0.12.

formulations (10%, 15% and 20%), with the values of

This plainly shows this species to exert DPPH radical

46.16±4.90; 48.20±3.72 and 49.13±2.79, respectively.

scavenging activity (Figure 2). This same figure reveals purple yam incorporated breads prepared in 10%, 15%

0.9 0.8

and 20% formulations, to also present a certain and

53.71±1.01

maximum

percentile

values,

respectively. Those findings are above the values

Abs 492

antioxidant activity, reaching 43.32±1.18; 48.13±1.17

0.7

Blank

0.6

Purple yam 0% Bread

0.5

10% Bread

0.4

15% Bread 20% Bread

0.3

presented by Hsu et al., (2004) (20-40% approximately),

BHT

0.2 0.1

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,

105

120

135

Time (min)

Figure 3. (Dioscorea extracts in blank and method.

Discoloration slope of purple yam trifida) and its incorporated bread four formulations: 0%, 10%, 15%, 20%, BHT, as determined through the LPO

present free radical scavenging activity (Kim et al., 90

2007; Jing and Kitts, 2000; Hsu et al., 2004; Michalska

80 70

it was confirmed that the antioxidant activity rose as the

percentage of purple yam substituting wheat flour increased. The high free radical scavenging activity

Inhibition (%)

et al., 2008). Corroborating data from Hsu et al., (2004),

50 40 30

observed by Hsu et al., (2004) in flour of Taiwan purple

yam (D. purpurea), was also detected in the Amazonian

10 0

region’s purple yam (D. trifida).

Purple yam

Antioxidant activity determination through the lipid per oxidation (LPO) method in purple yam and its incorporated breads Discoloration slope (Figure 3) and free radical

0% Bread

10% Bread

15% Bread

20% Bread

BHT

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.

inhibition activity (Figure 4) determined through the 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. Tests Shapiro-Wilk P Levene P

Bread% 10 15 20

Color 0.0009 < 0.0001 < 0.0001 0.0519

Aroma < 0.0001 0.0009 0.0001 0.5580

Flavor 0.0023 0.0008 0.0002 0.2306

Texture 0.0044 0.0019 0.0090 0.8415

Overall impression 0.0001 < 0.0001 0.0005 0.5184

Values were considered statistically significant at (P< 0.05). 754 Journal of Research in Biology (2013) 3(1): 747-758

Teixeira et al., 2013

(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 the purple yam amount being added to the bread the

0.0608

7 6.88 ± 1.33ª

0.5264

6 6.22 ± 1.61ª

0.9641

6 6.41 ± 1.43ª

0.6285

6,5

6 6.59 ± 1.14ª 6 5.94 ± 1.67ª 6 6.44 ± 1.22ª 6

6 6.54 ± 1.23ª 6 6.03 ± 1.50ª 6

Mean Median

attractive kind of color. It was observed that the larger

Choosing a determined food should depend Journal of Research in Biology (2013) 3(1): 747-758

6.15 to 6.97).

Bread

higher the mean score obtained (values ranging from

Values exhibiting different letters in the same column point out statistically significant differences (P< 0.05).

bread formulations revealed a significant difference

0.0001

applied for comparing the three purple yam incorporated

6.68 ± 1.20ª

Friedman ANOVA followed by post hoc tests

(P <0.05).

6.97 ± 1.55b

acceptance test (i.e. statistically significant values at

20%

Levene test on all sensory attributes evaluated in the

6.51 ± 1.34ª

homocedasticity among the formulations through the

through the Shapiro-Wilk test, and the acceptance of the

6.29 ± 1.30ab

incorporated bread formulations (10%, 15% e 20%)

15%

distribution normality of the three tested purple yam

6.28 ± 1.44ª

Table 5 shows the rejection of the frequency

Sensory analysis of purple yam incorporated breads

6.71 ± 1.22ª

2004).

antioxidant activity in yams (Hou et al., 2001; Hsu et al.,

6.15 ± 1.46ª

in plants, might be the active components for this

10%

In fact, polyphenols and anthocyanins, usually detected

Mean

(Carreno-Diaz and Grau, 1977; Escudero et al., 2010).

Median

(Rasper and Coursey, 1967) and D. trifida L.

Mean

these pigments were detected in purple yams, D. alata

Median

incorporated breads analyzed in the present study, since

Mean

detected in the purple yam (D. trifida) and its

Median

be partly responsible for the antioxidant activities

Mean

et al., 2004; Michalska et al., 2008). Anthocyanins might

Overall impression

products (Kim et al., 2007; Jing and Kitts, 2000; Hsu

Table 6. Sensory evaluation results of the three purple yam incorporated bread formulations. Probability (P) value was obtained through Friedman ANOVA followed by post hoc test.

resulted from the development of Maillard reaction

Texture

presented some antioxidant ability which might have

Flavor

Moreover, bread with no addition of purple yam (0%)

Aroma

substituting wheat flour in the breads increased.

Color

antioxidant activity rose as the percentage of purple yam

Median

As it was observed by the DPPH method, the

755

Teixeira et al., 2013 Table 7. Purple yam incorporated breads microbiological analysis. Microorganism

10% Absent Absent

Coliforms (at 45°C/g) Salmonella sp/25 g

15% Absent Absent

Type of bread 20% Absent Absent

Based on RDC (2001)* 102 Absent

*Ruling Number 12 (RDC 2001) recommended by the Brazilian National Health Surveillance Agency (in Portuguese, Agência Nacional de Vigilância Sanitária - ANVISA). mainly on its nutritional value. Nevertheless, color,

ACKNOWLEDGEMENTS

aroma and texture are the factors usually guiding the

The authors are indebted to the Fundação de

consumer’s preference rate. Of these three factors, color

Amparo à Pesquisa do Estado do Amazonas (FAPEAM)

interferes the most on the product’s preference (Bobbio

for the master scholarship granted to Antonia Paiva

and Bobbio, 2001).

Teixeira. To Dr. Antonio José Inhamuns da Silva and

Given that there were no preferential differences

MSc. Cynthia Tereza Corrêa da Silva of Universidade

among the other sensory attributes, the three breads

Federal do Amazonas (UFAM) for kindly having

evaluated can be considered approved.

allowed to carry out the centesimal composition analyses

Microbiological analysis

in their laboratories. To MSc. Antonio Fábio Lopes de

Considering that the microbiological analysis

Sousa, Arleilson de Sousa Lima and Ana Cláudia dos

was negative for Coliforms and Salmonella (Table 7), the

Santos for their invaluable aid in undertaking of the

purple yam incorporated breads may be considered

laboratory analyses. To Misters Claudio Adriano

proper for human consumption, as long as they have

Cardoso Amanajás and Francisco de Oliveira Batista for

been properly handled.

their logistical support in the collection of purple yam samples.

CONCLUSIONS Through such findings, one concludes that any

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Pedralli, G. 1997. Revisão taxonômica das espécies de Dioscoreaceae (R.Br.) Lindley da Cadeia do Espinhaço, Minas Gerais e Bahia. Tese doutorado PG - Botânica, Universidade de São Paulo (USP), São Paulo, Brazil.

Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing Open Access and Quick spreading You retain your copyright

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2002. Uso de nomes populares para as espécies de

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Journal of Research in Biology

An International Open Access Research Journal

Original Research

Journal of Research in Biology

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

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 Institution: Department of Crop Science, to treat four surfaces (concrete, wood, glass and plastic) usually encountered in structural insect pest management systems and the larvae exposed to these surfaces. College of Agriculture and Hocklicombi® (5 mL/L) served as positive control. Larval mortality (35.5-97.5%), Consumer Sciences, P. O. pupation (0.0-35.0%) and adult emergence (0.0-20.0%) in surfaces treated with Box LG 44, University of Hocklicombi® compared favourably with those treated with Novaluron (25.0-97.5%), Ghana, Legon. (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%). Corresponding author: Eziah VY.

Keywords: Novaluron, Hocklicombi®, Ephestia cautella, warehouse moth, chitin, loss assessment.

Web Address: http://jresearchbiology.com/ documents/RA0305.pdf.

Article Citation: Sackey I, Eziah VY and Obeng-Ofori D. Bioefficacy of Novaluron®, a chitin synthesis inhibitor against the tropical warehouse moth, Ephestia cautella. Journal of Research in Biology (2013) 3(1): 759-767 Dates: Received: 08 Nov 2012

Accepted: 27 Nov 2012

Published: 17 Jan 2013

Journal of Research in Biology An International Open Access Research Journal

759-767 | JRB | 2013 | Vol 3 | No 1

www.jresearchbiology.com

Sackey et al., 2013 laboratory. There is no information on the use of

INTRODUCTION Maize (Zea mays) is one of the major staple food

novaluron for stored product protection in warehouses or

crops in Ghana and it is susceptible to attack by several

cribs. Ephestia cautella is noted for feeding directly on

insect pests including the tropical warehouse moth,

the grains and also, the mature larvae leave the

Ephestia cautella Walker (Lepidoptera: Pyralidae)

commodity in search of pupation sites in crevices, cracks

(CAB1, 2006). Ephestia cautella larva feeds on stored

and storage containers. Therefore, treating these surfaces

products, damaging the product directly and form webs

to which the insect may be exposed will go a long way to

on the surface. The webbing contains larval excreta and

mitigate the losses caused by this pest. Hence, screening

exuviae which give unpleasant odour to the infested

Novaluron against E. cautella using the commodity and

commodity. Older larvae may leave the food to find

storage surfaces as substrates is crucial in the

pupation sites in wall cracks.

management of the pest. Such findings will contribute to

In Ghana, E. cautella is controlled by the use of

the efforts by farmers and warehouse managers to reduce

residual insecticides usually, synthetic pyrethroids and

storage losses and contribute to the attainment of food

fumigants (CABI, 2006). The adverse effects of residual

security in Ghana.

pesticides such as poisoning, environmental and health hazards

and

resistance

development

cannot

This study presents laboratory and field tests that

were carried out to determine the toxicity of novaluron to

overemphasized (Obeng-Ofori, 2007). Hence the use of

the 5th instar larvae of E. cautella. Other tests were also

residual insecticides in stored product protection is

conducted to assess the efficacy of novaluron on

challenging. There is therefore, the need for new cost and

different surfaces against immature stages of E. cautella.

environment friendly alternatives with no adverse effect on non-target organisms (Obeng-Ofori, 2007; Arthur and

MATERIALS AND METHODS

Phillips, 2003). Some of these alternatives include

Insect cultures

botanicals, insect growth regulators, microbial pathogens among others (Arthur, 1996).

The test insects were obtained from the Entomology laboratory of the Crop Science Department.

Novaluron (Rimon 10 EC) is a benzoylphenyl

Adult E. cautella were cultured on mixed substrate made

urea group of insect growth regulators and a chitin

up of wheat powder, maize flour and glycerol (5:5:1).

synthesis inhibitor. Novaluron has been registered as an

Fifty adult E. cautella were introduced into each jar and

insecticide for food crops in several countries including

left under laboratory conditions of 27±2°C and 55-60%

South Africa, Australia and Ghana (WHO, 2003; EPA,

relative humidity for 30 days to allow for the

2006). In Ghana, novaluron has been successfully used

development of larval E. cautella. The set up was placed

pests

on trays containing industrial oil to prevent the crawling

cephalonica

of other insects into the culture. The insects were reared

such

the

laboratory

the

rice

against

stored

moth,

product

Corcyra

Stainton (Lepidoptera: Pyralidae) (Sarbah, 2006), the

and handled

red

procedures in the laboratory.

flour

beetle,

Tribolium

castaneum

Herbst

(Coleoptera: Tenebrionidae) (Bakudie, 2006) and the tropical warehouse moth, E. cautella (Ibrahim, 2008).

using

ethically acceptable

standard

Test chemicals Novaluron (Rimon ® 10EC), 1-[3-chloro-4-(1, 1,

In Ghana, most of the work done on novaluron

2-trifluoro-2-trifluoromethoxy-ethoxy) phenyl]-3-(2, 6

focused on evaluating the effect of the chemical on

difluorobenzoyl) urea, produced by Makhteshim-Agan

different developmental stages of insects in the

Ltd (Israel) was used for the toxicity experiment and

760

Journal of Research in Biology (2013) 3(1): 759-767

Sackey et al., 2013 Hocklicombi (Hockley International Ltd. Poynton,

dishes were used as glass surfaces for the surface

Stockport, U. K.) which contains 25% Fenitrothion and

treatment.

5% Fenvalerate was used as a reference product.

Each of the four surfaces was treated with 4 mL of water (negative control treatment) or an aqueous

Contact toxicity test We adopted the method by (Eziah et al., 2011).

solution of novaluron (0.5 mL/L) and Hocklicombi ®

Concentrations of Novaluron (0.1, 0.2, 0.3, 0.4 and

(5 mL/L) (positive control). All treated arenas were

0.5 mL/L) and 5 mL/L of Hocklicombi were diluted in

allowed to dry overnight and fifth instar larvae (N=10) of

distilled water and used for the assays. Distilled water

E. cautella were exposed for 48 h. The larvae were then

was used as negative control. Fifth instar larvae of both

transferred to new petri dishes containing food under

sexes were transferred into clean Petri dishes and the

laboratory conditions of 27±2°C and 55-60% relative

different dosages of the various concentrations was

humidity. Post-treatment survival and mortality were

topically (1 µL) applied to the notal regions of the larvae

recorded daily. Number of surviving larvae that

using a micro applicator. Each experimental unit

successfully pupated and those that successfully emerged

consisted of 10 larvae and was replicated for four times.

as adults were recorded.

The treated insect larvae were then transferred into glass

Field experiment

petri dish containing food. The insect larvae were

Maize grains were obtained from the Madina

examined for mortality 24, 48, 72, 96 h, 7 days and

(a suburb of Accra, Ghana) market and sieved to remove

14 days after treatment. Criterion for death was as

all debris. Maize grains (5 kg) were sterilized in the oven

described by (Lloyd, 1969) in which insects were

at 70°C for 3 h after which they were left in desiccators

presumed dead when they failed to move in a

to cool. The grains were then treated with 0.4 mL/L

coordinated manner after prodding with a blunt probe.

Novaluron or 5 mL/L Hocklicombi®. These dosages had

Data collected include larval mortality, percent pupation

proven effective in laboratory experiments. Grains

and percent adult emergence were done after various

treated with distilled water served as negative control.

treatments and exposure periods.

Each treatment was replicated four times. Fifty unsexed

Surface treatment

adults of E. cautella were put onto the treated grains in

The surfaces chosen for the study were concrete,

each sack. The sacks were securely sealed by stitching

plywood, glass and plastic which are among the

and stored in a grain crib at the University farm for

common surfaces encountered in structural insect pest

60 days. Prior to their treatment, subsamples were taken

management. Individual concrete exposure arenas were

from each sack for moisture and weight loss analyses

created in square bottoms of plastic containers (6x6 cm)

using the standard volume method was carried out

using a concrete patching material. Water-based slurry

(Boxall, 1986). At the end of the storage period, the

was prepared by mixing 1 kg of Portland cement to 2 kg

contents of the sacks were sieved. The number of

of sand and 1 L of tap water and pouring 10 mL of the

both live and dead adult insects was recorded. Also,

slurry into the bottom of the plastic container to create a

subsamples of the maize grains were collected for

treatment arena (Arthur, 1998b). Plywood arenas were

moisture and weight loss analyses as stated earlier.

made by cutting rectangular disks from 1.25 cm thick

Statistical analysis

plywood to fit the plastic container then caulking the

Data

involving

percentages

were

arcsine

margins to prevent the larvae from escaping the surface.

transformed and were analyzed using the Analysis

Plastic containers served as plastic surfaces and petri

Journal of Research in Biology (2013) 3(1): 759-767

Variance

(ANOVA)

with

Genstat

9.2 761

Sackey et al., 2013 (Lawes Agricultural Trust, 2007). Means were separated

different from the negative control (64.0-80.0%)

using the Least Significant Difference (LSD) test at

(Figure 1). However, all other concentrations of

5% probability level.

Novaluron higher than 0.1 mL/L significantly (p = 0.05) impaired pupation. There was no significant difference in

RESULTS

pupation 7 days after the exposure of E. cautella larvae

Contact toxicity test

to 0.5 mL/L Novaluron and 5 mL/L Hocklicombi®.

The

percent

larval

cautella

mortality

Also, after 14 days, percentage pupation recorded in

larvae treated with 0.4 mL/L and 0.5 ml/L was

following treatment with Novaluron and Hocklicombi

are presented in Table-1. Larval mortality varied with

comparable.

insecticide concentration and exposure period. Lower

All

levels

Novaluron

concentrations

dosages of Novaluron (0.1-0.3 mL/L) caused less

significantly reduced the development of F1 of adult

than 50% larval mortality after 96 h of exposure

E. cautella (Figure 2). The highest adult emergence

(Table 1). In contrast, novaluron concentrations of

(77.5%) was recorded in the negative control and this

0.4 mL/L and 0.5 mL/L caused between 50% to 80%

differed significantly (p = 0.05) from all other novaluron

larval mortality after 72 to 96 h of exposure. After 96 h

concentrations applied. As concentration increased from

exposure period, all dosages of Novaluron induced

0.1 mL/L to 0.5 mL/L, adult emergence significantly

significantly (p = 0.05) higher larval mortality compared

(p = 0.05) reduced from 50 to 2.5%. Also, the effect of

to the negative control. However, there was no

novaluron applied at 0.5 mL/L was comparable to

significant difference in larval mortality between 5 mL/L

Hocklicombi® treatment in impairing the development of

Hocklicombi® and 0.5 mL/L Novaluron treatments. Also,

adult E. cautella.

novaluron applied at 0.4 ml/L and 0.5 mL/L did not

Surface treatment

differ significantly from each other after 96 h of exposure.

Ephestia cautella larvae exposed on concrete surfaces treated with Novaluron showed a lower

Pupation and adult emergence of E. cautella

mortality than those exposed to concrete surfaces treated

were observed in all insecticide treatments and the

with Hocklicombi® (Table 2). Mortality was also lower

negative control with the exception of Hocklicombi ®

on plastic and wood treated surfaces compared to

treatment. The percentage pupation in larvae treated with

Hocklicombi® treated surfaces. However, the percentage

0.1 mL/L Novaluron (57.5-65.0%) was not significantly

mortality of E. cautella on glass surfaces treated with

Table 1 Mortality of larval E. cautella (%) after treatment with novaluron and Hocklicombi® insecticides Treatments (ml/L) Control (Water) 5.0 mL/L (HC) Novaluron 0.1 0.2 0.3 0.4 0.5 LSD (P < 0.05) HC= Hocklicombi® s.e = standard error 762

24 h 0.0±0.0 87.5±0.1 7.5±0.0 10.0±0.0 17.5±0.1 17.5±0.1 32.5±0.1 18.35

Mean±(s.e) % larval mortality (h) 48 h 72 h 0.0±0.0 0.0±0.0 87.5±0.1 87.5±0.1 10.0±0.0 12.5±0.0 25.0±0.1 32.5±0.1 47.5±0.1 18.40

17.5±0.1 27.5±0.1 35.0±0.1 50.0±0.1 65.0±0.1 14.50

96 h 0.0±0.0 87.5±0.1 22.5±0.1 42.5±0.1 45.0±0.2 66.0±0.1 80.0±0.0 14.83

Journal of Research in Biology (2013) 3(1): 759-767

Sackey et al., 2013 Table 2 Mortality of E. cautella larvae (%) after 7 days exposure on concrete, glass, plastic and wood surfaces treated with Hocklicombi ® and novaluron insecticides Mean (%) ± s.e mortality Insecticide Type of surface Concrete Glass Plastic Wood Means Control 0.0 ± 0.0 0.0±0.0 2.5 ± 0.0 0.0 ± 0.0 0.7±0.0 Hocklicombi® 43.0 ± 0.1 97.5±0.0 45.0 ± 0.1 35.0 ± 0.1 55.0±0.0 Novaluron 25.0±0.1 97.5±0.0 17.5 ± 0.1 25.0 ± 0.1 41.1±0.0 Means 22.7± 0.0 64.7±0.0 21.7 ± 0.0 20.0 ± 0.0 LSD(P < 0.05): Main effects (insecticide = 1.21, surface= 1.39 Interaction (insecticide x surface)=2.4 novaluron was the same (97.5%) as those treated with

which were not treated (11.3%) using standard volume

Hocklicombi®. Surviving larvae were observed for

methods.

pupation and adult emergence. Fewer E. cautella larvae

DISCUSSION

pupated after exposure to concrete, plastic and wood ®

The present study showed that Novaluron

surfaces treated with Hocklicombi but no pupation was

concentrations of 0.4 mL/L and 0.5 mL/L significantly

affected the metamorphosis of E. cautella to the adult

recorded on glass surfaces treated with Hocklicombi (Table 3)

stage. The effectiveness of novaluron at these dosages

Fewer larvae pupated in glass surfaces-treated

compared favourably with Hocklicombi ®. The insect

with novaluron and this was not significantly different

growth regulator’s ability to regulate metamorphosis in

from Hocklicombi®-treated glass surfaces. Generally,

the larvae through contact by topical application is

percentage adult E. cautella that emerged was greater on

consistent with its mode of action. Tomlin (2005)

the untreated control for all the surfaces and differed

reported that novaluron was very effective on the larvae

significantly (p = 0.05) from all insecticide treated

of insects when absorbed by ingestion and contact

surfaces (Table 4). Mean percentage adult emergence of

activity. The author also reported that the compound

E. cautela observed on glass and plastic surfaces treated

causes abnormal endocuticular deposition and abortive

with novaluron and Hocklicombi 25%.

Thus,

residual

effects

ranged from 0.0 to of

novaluron

moulting.

and

Although pupation and adult emergence were

Hocklicombi significantly reduced the development of

observed in all treatment levels, most of the larvae

E. cautella on glass and plastic surfaces.

treated with 0.4 mL/L and 0.5 mL/L Novaluron could not

Field experiment

emerge into adults 23 days after treatment. This may be

Table 5 shows the dry weight loss of the treated

attributed to abnormal endocuticular deposition and

grains after 60 days of storage using the standard volume

abortive moulting in the larvae (Tomlin, 2005). Also,

method. Lower weight losses were observed in grains

when cocoon covering the pupae were slightly removed,

treated with insecticides (6.6-6.8%) compared to grains pupae found were malformed compared to those in the Table 3: Percentage pupation of E. cautella after 14 days exposure on concrete, glass, plastic and wood surfaces treated with Hocklicombi ® and novaluron insecticides Means (%) ± s.e for pupation Insecticide Type of surface Concrete Glass Plastic Wood Means Control 92.5±0.0 95.0±0.0 95.0±0.0 97.5±0.0 95.0±0.0 Hocklicombi 35.0±0.1 0.0±0.0 35.0±0.1 25.0±0.0 23.8±0.0 Novaluron 55.0±0.1 2.5±0.0 60.0±0.1 47.5±0.1 43.1±0.0 Means 61.9±0.0 41.2±0.0 63.1±0.0 56.2±0.0 LSD(P < 0.05): Main effects (insecticide5.6, surface= 6.6) Interaction (insecticide x surface)=13.20 Journal of Research in Biology (2013) 3(1): 759-767

763

Sackey et al., 2013 control. Adults that emerged were found not to be active

development stage, time of application, kind of

as those in the control. These findings are consistent with

compound and dose administered.

reports by Amos and Williams (1974). According to

The residual effect

of Hocklicombi® and

CABI (2006), pupal formation is completed in seven

Novaluron were significantly greater on glass surfaces

days and development from egg to adult ranges from

than plastic, concrete or wood surfaces. Generally,

29-31 days under optimum conditions of 32.5 C and

Hockicombi® significantly caused higher mortalities on

70% relative humidity. However, in the present study

all the surfaces than novaluron. The high residual

under laboratory conditions of 27±2°C and 55-60%

efficacy of Hocklicombi® may be attributed to the

relative humidity, pupation extended up to 14 days and

components of the compound. Hocklicombi® contains

adult emergence was also delayed up to 30 days in the

fenitrothion and fenvalerate as its active ingredients.

treated 5 instar larvae of E. cautella. Thus, novaluron

These compounds have been reported by several

was found to prolong the development period of

researchers to have high residual effects when used as

E. cautella larvae to adults.

surface treatment against storage insects (Orui, 2004).

The ability of Novaluron to reduce the number of new generations is consistent with the findings of

Both compounds are non-systemic insecticides with contact and stomach activity (Tomlin, 2005).

(Kostyukovsky et al., 2003) and Kostyukovsky and

In the present study, novaluron demonstrated

Trostanetsky (2006). The authors found that novaluron

excellent residual effect on glass surfaces by preventing

applied at 1 ppm reduced the number of new generations

the metamorphosis of E. cautella to the adult stage. The

of S. oryzae and R. dominica by 95% and also caused

residual effect on glass surfaces treated with novaluron

total mortality of the 3 instar larvae of T. castaneum.

compared well with Hocklicombi®. However, on plastic,

The effectiveness of novaluron in preventing the

concrete and wood surfaces, Novaluron was less

metamorphosis of E. cautella when applied at 0.4 mL/L

effective compared with Hocklicombi® but differed

and 0.5 mL/L also confirms work done by Ibrahim

significantly from the untreated control surfaces.

(2008). The author found that development of E. cautella

However, the residual effectiveness on plastic surfaces

to adults was prevented when novaluron was applied at

showed better efficacy than on concrete and wood

0.4 mL/L and 0.6 mL/L.

surfaces.

These observations indicate that the effectiveness

The excellent effectiveness of Novaluron on

of novaluron as a grain protectant depends on the species

glass and plastic surfaces is consistent with work done by

of insect, dosage and exposure time. Wilson and Cryan

(Atkinson et al., 1992). The authors found that when

(1997) and Mulla et al., (2003) stated that the effects of

hydropene, an insect growth regulator was sprayed on

chitin synthesis inhibitors vary according to species,

non-absorbent surfaces such as glass and ceramic tile, the

Table 4 Percentage adult emergence of E. cautella after 30 days exposure on concrete, glass, plastic and wood surfaces treated with Hocklicombi ® and novaluron insecticides Insecticide Control Hocklicombi Novaluron Means

Concrete 92.5±0.0 20.0±0.1 42.5±0.1 50.6±0.0

Means (%) ± s.e for adult emergence Type of surface Glass Plastic 90.0±0.0 95.0±0.0 0.0±0.0 12.5±0.1 0.0±0.0 25.0±0.1 32.5±0.0 45.0±0.0

Wood 97.5±0.0 12.5±0.0 42.5±0.1 48.1±0.0

Means 93.8±0.0 11.2±0.0 27.5±0.0

LSD(P < 0.05): Main effects (insecticide5.9, surface= 6.34)= 6.34 Interaction Insecticide x surface=12.67 764

Journal of Research in Biology (2013) 3(1): 759-767

Sackey et al., 2013

Figure 1 Percentage pupation (means±s.e) of E. cautella larvae after treatment with novaluron and Hocklicombi® insecticides. d = days h= hours

Figure 2 Percentage adult emergence (means±s.e) of E. cautella after treatment with novaluron and Hocklicombi® insecticides. d = days

survival, number of oothecae and percentage of

grains compared to the control. Novaluron was observed

cockroaches were more affected than on absorbent

to significantly reduce insect numbers in the treated

surfaces of finished plywood and fibreboard. The low

grains and also had a significantly lower dry weight loss.

mortality rates, pupation and adult E. cautella that

Results from this study showed that novaluron

emerged after exposure to concrete and wood surfaces in

effectively protected maize grains from damage by

the current study can also be attributed to the

E. cautella. Grain weight losses calculated in the

composition of these surfaces. Burkholder and Dicke

Novaluron treatment compared well with those observed

(1966) reported that new concrete surfaces contain high

in grains treated with Hocklicombi®. Considering that

levels of alkaline which hydrolyze residues and reduce

Novaluron selectively targets larval stages by inhibiting

residual efficacy of insecticides hence, the low mortality

chitin synthesis and therefore, minimizes its impact on

rates on concrete-treated surface in the present study was

adults of non targeted insect species (Ishaaya et al.,

not unexpected. Chadwick (1985) attributed low efficacy

2001), Novaluron can be used in replacement of residual

of insecticides on plywood surfaces to vaporization,

insecticides like Hocklicombi® for treatment of maize

chemical degradation, photodegradation and absorption

grains for storage.

of insecticides into surfaces. Thus, the low mortalities and higher survival rates observed in E. cautella exposed

CONCLUSION

to wood surfaces treated with the insecticides may be due

The current study showed that Novaluron was

to the absorption of the insecticide into the wood

effective in controlling the tropical warehouse moth. The

surfaces after treatment.

application of Nuvaluron at 0.4 mL/L and 0.5 mL/L

In the field experiment, all the insecticide

treatments resulted in larval mortality ranging between

treatments significantly reduced dry weight loss in the

50-80% after 96 h of exposure. Also, the treatment of

Table 5 Percent dry weight loss after 60 days of storage using the standard volume method

concrete, wood, glass and plastic surfaces usually

Dosage (mL/L) Control Hocklicombi 5 Novaluron 0.4

systems with 0.5 mL/L Novaluron induced (25.0-97.5%)

Mean dry weight loss (%) 11.3±0.0 6.6±0.0 6.8±0.0

LSD(P < 0.05) = 1.63 Journal of Research in Biology (2013) 3(1): 759-767

encountered in structural insect pest management 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 765

Sackey et al., 2013 Hocklicombi® insecticide. In the field maize treated with

Bakudie E. 2006. Susceptibility of Tribolium castaneum

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

to novaluron on maize and rice. Bachelor of Science

after 60 days of storage showed that there was a lower

Dissertation. Department of Crop Science, University of

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.

Ghana, Legon, 36. Boxall RA. 1986. A critical review of the methodology for assessing farm grain losses after harvest. Tropical Development and Research Institute Report G191, Viii 139. Burkholder WE and Dicke RJ. 1966. The toxicity of

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Journal of Research in Biology

An International Open Access Research Journal

Original Research

Journal of Research in Biology

A Checklist of Butterflies of Meenachil River Basin, Kerala, India Authors: Vincy MV1, Brilliant R2 and Pradeepkumar AP3

ABSTRACT:

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

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.

2. PG Department of Environmental Sciences, St. Johnâ&#x20AC;&#x2122;s College, Anchal, Kerala. 3. Department of Geology, University of Kerala, Kariavattom, Kerala. Corresponding author: Vincy MV.

Keywords: Meenachil river, Endemic species, bio-indicators, anthropogenic pressure.

Email: vincybrilliant@gmail.com

Article Citation: Vincy MV, Brilliant R and Pradeepkumar AP. A Checklist of Butterflies of Meenachil River Basin, Kerala, India. Journal of Research in Biology (2013) 3(1): 768-774

Web Address:

http://jresearchbiology.com/ documents/RA0308.pdf.

Dates: Received: 21 Nov 2012

Accepted: 03 Dec 2012

Published: 04 Feb 2013

Journal of Research in Biology An International Open Access Research Journal

768-774 | JRB | 2013 | Vol 3 | No 1

www.jresearchbiology.com

Vincy et al., 2013 Meenachil river basin in Kerala, South India. However,

INTRODUCTION Butterflies are the most beautiful and colourful

comprehensive long-term ecological studies to monitor

creatures on the earth and have a great aesthetic value.

the butterfly population of the area remains as a serious

India harbours about 1501 species of butterflies

lacuna. Such studies are imperative to improve the

(Haribal, 1992), 285 species are found in southern India

ecological utility of butterflies as indicator taxa.

(Thomas, 1966), of which 45 species are endemic to southern India. Butterflies, widely appreciated for the

MATERIALS AND METHODS

aesthetic value are important as ecological indicators

The present study is an attempt to provide a

(Chakravarthy et al., 1997) and ‘flagship taxa” in

checklist of butterflies based on a four-year field study

biodiversity inventories (Lawton et al., 1998).

from October 2008 to October 2012. Identification of

Meenachil river which is one of the important

species was done using available literature (Evans, 1932;

river of Kottayam district in Kerala, emerges from

Gunathilagaraj et al., 1998; Haribal, 1992; Palot et al.,

Western Ghats and confluences into Vembanad Lake.

2003; Gay et al., 1992; Wynter-Blyth, 1957) and with

This river has a total length of 78 km and has

the help of experts. Species classification and scientific

a catchment area of 1272 km2. The entire Meenachil

names are as per Gunathilagaraj et al., (1998).

watershed area geographically lies between 9°25’ N to 9°55’ N latitude and 76°30’ E to 77°00’ E longitude.

RESULT AND DISCUSSION

The general elevation of the entire river basin ranges

The study during the period indicate that the

from 77 m to 1156 m in the high lands and less than 2 m

habitats where butterflies were found and captured are

in the low lands. The Meenachil river basin falls within

disturbed

the realm of tropical climate. The temperature of the area

anthropogenic activities. These range from city lots to

varies in between 24°C and 32°C throughout the year.

pasture, abandoned fields, road sides, plantations,

The annual rainfall varies from less than 100 cm to more

riparian area, etc.

areas and are

strongly influenced

than 500 cm with an average of 300 cm. The occasional

A total of 91 species belonging to 71 genera

rainfall is also received between the two seasons. Rubber

distributed over five families were collected from the

trees are extensively cultivated in vast areas in the entire

monitoring sites, during the study period. The family

river basin. Besides rubber, other crops like spices,

Nymphalidae dominated with 34 species followed

paddies etc., are also cultivated in the river basin area

by Hesperiidae (20 spp.), Lycaenidae (18 spp.), Pieridae

(Watershed Atlas, 1996).

(7 spp.), and Papilionidae (12 spp.). Even though, the

Among

most

family Nymphalidae exhibited the maximum species

studied group. Larsen (1987a, b, c, 1988) made a detailed

diversity, family Pieridae showed maximum species

survey of butterflies of Nilgiri Mountains and recorded

density. Three butterfly species recorded from this region

n earl y

en demi cs.

have protected status under the Wildlife Protection Act,

In Kerala, documentation of butterflies on Silent Valley

1972 (Arora, 2003). They are Hypolimnas misippus and

National Park (Mathew and Rahamathulla, 1993)

Atrophaneura hector included under Schedule I Part IV

an d

San ctuar y

and one species Aeromachus pygmaeus in Schedule II

(Sudeendrakumar et al., 2000) have been carried out.

Part II. Further research with reference to ecology,

The present paper presents a checklist and diversity of

threats and conservation of butterflies in the area is in

butterfly populations in different altitude levels in

progress.

769

300

insects,

speci es

Par a m bi kula m

butterflies

are

in cl udin g

Wi ld li fe

the

Journal of Research in Biology (2013) 3(1): 768-774

Not rare Very Common Common Common Common Not rare Not rare

120-150 mm 80-100 mm 80-110 mm 100-130 mm 90-110 mm 140-170 mm 140-190 mm 40-50 mm 55-80 mm 50-70 mm 80-100 mm 55-70 mm 66-83 mm 35-50 mm 90-100 mm

Papilio polymnestor (Cramer) Papilio demoleus (Linnaeus) Papilio paris (Linnaeus) Atrophaneura aristolochiae (Fabricius) Atrophaneura pandiyana (Moore) Atrophaneura hector (Linnaeus) Troides helena (Linnaeus) Troides minos (Cramer) Eurema hecabe (Linnaeus)

Journal of Research in Biology (2013) 3(1): 768-774 Catopsilia pomona (Fabricius) Catopsilia pyranthe (Linnaeus) Hebomoia glaucippe (Linnaeus) Appias lyncida (Cramer) Delias eucharis (Drury) Leptosia nina (Fabricius) Tirumala limniace (Cramer)

Common Emigrant

70-80 mm

Danaus chrysippus (Linnaeus) Parantica aglea (Stoll)

Plain Tiger

Glassy Blue Tiger

70-85 mm

75-95 mm 72-100 mm

Tirumala septentrionis (Butler) Danaus genutia (Cramer)

Dark Blue Tiger Stripped Tiger

21 22

15 Mottled Emigrant 16 Great Orange Tip 17 Chocolate Albatross 18 Common Jezebel 19 Psyche FAMILY NYMPHALIDAE 20 Blue Tiger

5 Blue Mormon 6 Lime Butterfly 7 Paris Peacock 8 Common Rose 9 Malabar Rose 10 Crimson Rose* 11 Common Birdwing 12 Southern Birdwing FAMILY PIERIDAE 13 Common Grass Yellow

Common

Common Common

Common

Common Common Common Common Common

Common

Common Very Common

90-100 mm 90-100 mm

Papilio clytia (Linnaeus) Papilio polytes (Linnaeus)

Common Mime Common Mormon

Common

3 4

85-100 mm

Graphium agamemnon (Linnaeus)

Common

Status

Tailed Jay

80-90 mm

Wingspan

Graphium sarpedon (Linnaeus)

Scientific Name

Sl. No. Common Name FAMILY PAPILIONIDAE 1 Common Blue Bottle Plants visited

Lantana camara, Ageratum conyzoides, Crotalaria retusa Ageratum conyzoides Tridax procumbens, Lantana spp., Crotalaria retusa Calotropis spp., Ageratum conyzoides, Tridax procumbens,Crotalaria retusa, Calotropis spp., Ageratum conyzoides, Stachytarpheta spp., Crotalaria retusa

Caesalpinia spp., Cassia tora, C. fistula, Acacia spp. Bauhinia racemosa, C. fistula, C. tora, Butea monosperma C. fistula, C. tora Capparis spp. Capparis spp. Dendrophthoe falcata Cleome viscosa

Aristolochia indica, Thottea siliquosa

Litsea chinensis, Polyalthia longifolia, Cinnamomum malabatrum, Persea odoratissima, P. macrantha Polyalthia longifolia, Cinnamomum spp., Annona reticulata, A. squamosa Litsea chinensis Citrus spp., Glycosmis arborea, Murraya koenigii, curry leaf plant Citrus limona, Glycosmis arborea Glycosmis arborea, Murraya koenigii Citrus spp. Thottea siliquosa Thottea siliquosa Aristolochia indica, Thottea siliquosa

Table 1. List of butterfly species collected in the study areas and the plants visited by them

Vincy et al., 2013

770

771 65-75 mm 60-80 mm 38-55 mm 45-55 mm 32-48 mm 30-40 mm 50-65 mm 60-75 mm 50-60 mm 50-60 mm 60-75 mm 45-50 mm 50-60 mm 95-130 mm 55-80 mm 65-85 mm 50-60 mm 55-70 mm 45-60mm 45-60mm 55-80 mm 55-65 mm 60-65 mm 40-60 mm 70-110 mm 70-85 mm 20-30 mm 40-48 mm 38-44 mm 36-40 mm 26-28 mm

Lethe europa (Fabricius) Elymnias hypermnestra (Linnaeus) Mycalesis perseus (Fabricius) Orsotriaena medus (Fabricius) Ypthima baldus (Fabricius) Ypthima huebneri (Kirby) Acraea violae (Fabricius) Cirrochroa thais (Fabricius) Cupha erymanthis (Drury) Phalanta phalantha (Drury) Moduza procris (Cramer) Pantoporia hordonia (Stoll) Neptis hylas (Linnaeus) Parthenos sylvia (Cramer) Euthalia aconthea (Hewitson ) Tanaecia lepidea (Butler) Cyrestis thyodamas (Boisduval) Vanessa cardui (Linnaeus) Ariadne ariadne (Linnaeus) Ariadne merione (Cramer) Junonia iphita (Cramer) Junonia atlites (Linnaeus) Junonia almana (Linnaeus) Junonia lemonias (Linnaeus) Hypolimnas bolina (Linnaeus) Hypolimnas misippus (Linnaeus) Spalgis epius (Westwood) Curetis thetis (H端bner) Zesius chrysomallus (H端bner) Loxura atymnus (Cramer) Rathinda amor (Fabricius)

Common Lascar Common Sailor

Clipper Common Baron

39 40

41 42

43 Grey Count 44 Common Map 45 Painted Lady 46 Angled Caster 47 Common Caster 48 Chocolate Pansy 49 Grey Pansy 50 Peacock Pansy 51 Lemon Pansy 52 Great Eggfly 53 Danaid Eggfly* FAMILY LYCAENIDAE 54 Ape Fly 55 Indian Sunbeam 56 Red Spot 57 Yamfly 58 Monkey Puzzle

28 29 30 31 32 33 34 35 36 37 38

60-75 mm 60-80 mm

Polyura athamas (Drury) Melanitis leda (Linnaeus)

Common Nawab Common Evening Brown Bamboo Treebrown Common Palmfly Common Bushbrown smooth-eyed bushbrown Common Fivering Common Fourring Tawny Coster Tamil Yeoman Rustic Common Leopard Commander

26 27

85-95 mm

Euploea core (Cramer)

Common Crow

Not common Not rare Not rare Common Not rare

Common Not Common Common Uncommon Common Common Common Common Common Common Common

Common Common

Common Common Common Common Common Common Common Common Common Common Common

Common Common

Common

Carnivorous caterpillars feed on mealy bugs Abrus precatorius, Pongamia pinnata Caterpillars feed on ant larvae Smilax spp., Dioscorea pentaphylla Ixora spp.

Osbeckia spp. Sida rhombifolia Sida rhombifolia, Hibiscus spp. Hibiscus spp.

Ochreinauclea missionis, Mussaenda frondosa Acacia pennata Dalbergia spp., Zizyphus spp., Thespesia populnea, Grewia spp., Bombax alabaricum tinospora cordifolia Anacardium occidentalis, Mangifera indica, Streblus asper Careya arborea Ficus spp. Blumea spp. Ricinus communis Ricinus communis

Hydnocarpus spp.

Grasses Aporosa lindleyana, Passiflora foetida

Bambusa spp. Areca catechu, Cocos nucifera Oryza spp. Oryza sativa

Ichnocarpus frutescens, Hemidesmus indicus, Ficus spp., Streblus asper, Ageratum conyzoides, Crotalaria spp., Chromolaena odorata Acacia pennata, Adenanthera pavonina Oryza sativa, Panicum spp.

Vincy et al., 2013

Journal of Research in Biology (2013) 3(1): 768-774

25-35 mm 19-26 mm 30-36 mm 16-30 mm 40-50 mm 45-55 mm 20-27 mm 26-35 mm 40-46 mm 45-50 mm 37-44 mm 23-30 mm 22-28 mm 24-28 mm 33-36 mm 30-36 mm 36-42 mm

Catochrysops strabo (Fabricius) Zizina otis (Fabricius) Talicada nyseus (Guérin) Neopithecops zalmora (Butler) Abisara echerius (Stoll) Celaenorrhinus leucocera (Kollar) Spialia galba (Fabricius) Sarangesa dasahara (Moore) Coladenia indrani (Moore) Tagiades gana (Moore) Tagiades litigiosa (Möschler) Taractrocera ceramas Taractrocera maevius (Fabricius) Oriens goloides (Moore) Telicota ancilla (Herrrich-Schäffer) Borbo cinnara (Wallace)

Journal of Research in Biology (2013) 3(1): 768-774 Polytremis lubricans (HerrrichSchäffer) Suastus gremius (Fabricius) Gangara thyrsis Fabricius) Matapa aria (Moore) Iambrix salsala (Moore) Notocrypta curvifascia (Felder & Felder) Udaspus folus (Cramer) Aeromachus pygmaeus (Fabricius) Halpe homolea (Hewitson)

Indian Palm Bob Gaint Red eye Common Redeye Chestnut Bob Restricted Demon

Grass Demon Pygmy Scrub Hopper** Indian Ace

84 85 86 87 88

89 90 91

40-48 mm 20-22 mm 30-36 mm

32-45 mm 70-76 mm 40-55 mm 26-30 mm 38-50 mm

Common Common Common

Common Not rare Common Common Common

Not common

Common Common Common Common Not rare Not rare Common Common Common Common Common

Common Common Common Common Common

Common

Not rare Common Common Common Common

Common Common

Bamboo

Zingiber spp.

Grasses and bamboos Costus speciosus

Calamus spp., Caryota urens, Cocos nucifera Calamus rotang, Caryota urens, Cocos nucifera

Cocos nucifera, Oryza spp., Saccharum spp. Oryza sativa, Pennisetum spp., Ischaemum spp., Cymbopogon spp.

Smilax spp. Oryza sativa, grasses Grasses

Sida rhombifolia, Hibiscus spp. Asystasia spp. Mallotus philippinensis, Desmodium spp.

Glycosmis pentaphylla

Acacia pennata Zizyphus rugosa, Canthium oromandelicum, Clerodendrum inerme Zizyphus rugosa Ziziphus spp. Zizyphus rugosa, Z. jujuba Acacia catechu, Mimosa spp. Butea monosperma, Crotalaria spp., Pongamia pinnata Butea monosperma, Pongamia pinnata, Abrus precatorius Desmodium spp. Fabaceae spp.

* - indicates species coming under Schedule I Part IV and ** - Schedule II Part II of The Wildlife (Protection) Act, 1972

Contigous Swift

67 Forget me not 68 Lesser Grass Blue 69 Red Pierrot 70 Quaker 71 Plum Judy FAMILY HESPERIIDAE 72 Common Spotted Flat 73 Indian Skipper 74 Common Small Flat 75 Tricolour Pied Flat 76 Suffused Snow Flat 77 Water Snow Flat 78 Tamil Grass Dart 79 Common Grass Dart 80 Common Dartlet 81 Dark Palm Dart 82 Rice Swift

27-40 mm

Jamides celeno (Cramer)

Common Cerulean

26-32 mm 26-30 mm 24-34 mm 18-25 mm 25-34 mm

Caleta caleta (Hewitson) Discolampa ethion (Cramer) Castalius rosimon (Fabricius) Prosotas nora (C. Felder) Jamides bochus (Stoll)

Angled Pierrot Banded Blue Pierrot Common Pierrot Common Line Blue Dark Cerulean

61 62 63 64 65

30-33 mm 26-34 mm

Rapala manea (Hewitson) Cigaritis vulcanus (Fabricius)

Slate Flash Common Silverline

59 60

Vincy et al., 2013

772

Vincy et al., 2013 The study shows that the sustained interference

Mathew G and Rahamathulla VK. 1993. Studies on

and disturbance seem to affect the occurrence and

the butterflies of Silent Valley National Park. Entomon,

numerical strength of each butterfly species. If this

18:185-192.

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

Larsen TB. 1987a. The butterflies of the Nilgiri mountains of South India (Lepidoptera: Rhopalocera). Journal of the Bombay Natural History Society, 84: 2643.

cover contribute to warm conditions and these conditions

Larsen TB. 1987b. The butterflies of the Nilgiri

might allow some butterflies to extend their distribution

mountains of South India (Lepidoptera: Rhopalocera).

to different habitats. The butterflies which control certain

Journal of the Bombay Natural History Society, 84: 291-

plant pets, if decline in number or disappear from the

316.

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.

Larsen TB. 1987c. The butterflies of the Nilgiri mountains of South India (Lepidoptera: Rhopalocera). Journal of the Bombay Natural History Society, 84: 560584.

REFERENCES

Larsen TB. 1988. The butterflies of the Nilgiri

Arora K. 2003. Forest Laws. The Wildlife Protection

mountains of South India (Lepidoptera: Rhopalocera).

Act, 1972 as amended by the Wildlife (Protection)

Journal of the Bombay Natural History Society, 85: 26-

Amendment Act, 2002 (Act 16 of 2003). Published by

43.

Professional Book Publishers, New Delhi, 85.

Lawton, JH Bignell D E Bolton B Bloemers GF

Chakravarthy AK Rajagopal D and Jagannatha R.

Eggleton P Hammond PM Hodda M Holts RD

1997. Insects as bio indicators of conservation in the

Larsen TB Mawdsley NA Stork NE Srivastava DS

tropics. Zoosâ&#x20AC;&#x2122; Print, 12:21-25.

and Watt AD. 1998. Biodiversity inventories indicator

Evans WH. 1932. Identification of Indian Butterflies. Bombay Natural History Society, Bombay, 454. Gay T Kehimkar ID and Punetha JC. 1992. Common Butterflies of India. Oxford University Press, Bombay. Gunathilagaraj K. 1998. Some South Indian Butterflies. Tamil Nadu, India: Nilgiri Wildlife and Environments Association, Udhagamandalam, Nilgiris, 274.

taxa and effect of habitat modification in tropical forest. Nature, 391: 72-76. Palot J Balakrishnan VC and Kambrath B. 2003. Keralathile Chitrasalabhangal. Malabar Natural History Society, Calicut, Kerala, 195. Sudheendrakumar VV Binoy CF Suresh PV and Mathew G. 2000. Habitat associations of butterflies in the Parambikulam Wildlife Sanctuary, Kerala, India.

Haribal M. 1992. The butterflies of Sikkim, Himalaya

Journal of the Bombay Natural History Society, 97: 193-

and their natural history. Nataraj Publishers, Dehradun,

201.

217.

773

Journal of Research in Biology (2013) 3(1): 768-774

Vincy et al., 2013 Thomas S. 1966. Bulletin of the Madras Government Museum - Descriptive Catalog of the Butterflies, Natural History Section Vol. VII No. 1. Watershed Atlas. 1996. Kerala State Land Use board, Govt. of Kerala Publications, Kerala. Wynter-Blyth MA. 1957. Butterflies of Indian Region. Bombay Natural History Society, Bombay.

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An International Open Access Research Journal

OVER VIEW

Journal of Research in Biology

Microbial production of glutaminase enzyme 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.

Corresponding author: Mario Khalil Habeeb.

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 endocellular 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 .

Email: Keywords: mario_khalil87@yahoo.com, Actinomycetes, Anticancer properties, Enzymes, Glutamic mario_khalil@sci.asu.edu.eg L-Glutaminase.

Telephone: +20 (02) 22409635

Mobile: +20 (0128) 3941815

acid and

Article Citation: Mario Khalil Habeeb. Microbial production of glutaminase enzyme. Journal of Research in Biology (2013) 3(1): 775-779 Dates: Received: 16 Jan 2013

Accepted: 22 Jan 2013

Published: 06 Feb 2013

Web Address:

http://jresearchbiology.com/ documents/RA0325.pdf.

Journal of Research in Biology An International Open Access Research Journal

775-779 | JRB | 2013 | Vol 3 | No 1

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Habeeb., 2013 starvation of cancerous cells and their possible death

INTRODUCTION Enzymes are highly selective catalytic proteins produced by living cells which may or may not contain a

(Santana et al., 1968). Glutaminase Producing microorganisms

non-protein prosthetic group (Underkofler et al., 1958).

Different types of organisms were reported

Actinomycetes are considered to be preferred

to produce glutaminase enzyme. However, The selection

enzymes sources due to their production of extracellular

of the right organism is very critical to obtain high yield

enzymes. They are highly diverse group with numerous

of the required enzyme (Akujobi et al., 2012).

members representing important source of microbial

L-glutaminases

enzymes. Actinomycetes genera are differentiated from

Bacillus sp., and Clostridium welchii have been isolated

each other based on morphological, biochemical, and

and well studied (Wade et al., 1971). In addition to these

physiological criteria. They act as decomposers of

bacterial sources, the fungus Aspergillus oryzae showed

complex animal and plant materials resulting in release

a great ability to produce this enzyme. Among

of simple substances, especially carbon and nitrogen

microorganisms, actinomycetes are widely recognized

which is easily utilized by other organisms, thus

as preferable L-glutaminase sources because they

performing a vital role in life cycle. Due to their

generally produce extracellular enzymes, which facilitate

significant biochemical activities, Actinomycetes are

the enzyme recovery from the fermentation broth

used in commercial production of various substances

such

such as antibiotics and enzymes (Waksman, 1950).

(Sivakumar et al., 2006).

Because of its industrial and pharmaceutical

from

glutaminase

coli,

from

Pseudomonas

Streptomyces

sp.,

rimosus

Microbial Glutaminase Characteristics

applications, intensive research was conducted on

Temperature is considered to be an important

L-glutaminase recently. L-glutaminase is produced by

factor affecting the enzyme stability, The optimum

various terrestrial microorganisms such as Pseudomonas

temperature recorded by many glutaminases ranged from

sp., Acinetobacter sp., Escherichia coli, Bacillus sp.,

40-50ยบC.

Hansenula sp., Candida sp., Aspergillus oryzae and

glutaminase I (Micrococcus glutaminase) of M. luteus

Beauveria

few

could be increased by the addition of 10% NaCl

marine microorganisms such as Micrococcus luteus,

(Moriguchi et al., 1994). The optimum temperature for

Vibrio cholera and Pseudomonas fluorescens were

A. oryzae glutaminase was around 37-45ยบC and remained

reported to produce the enzyme (Chandrasekaran, 1997).

stable at up to 45ยบC and the enzyme was completely

Definition

inactive at 55ยบC (Nakadai and Nasuno, 1989).

bassiana

(Sabu,

2003).

Also

However,

the

temperature

stability

L-glutaminase is classified as an amidohydrolase

It is interesting that the exposure of E. coli

enzyme which acts upon amide bonds of L-glutamine

glutaminase B to cold resulted in a reversible

generating L-glutamic acid and ammonia. It is present

inactivation of enzymatic activity, while subsequent

warming to 24ยบC restored the activity. There was no

both

(Ohshima

microorganisms et

al.,

and

1976b).

mammalian

Microbial

tissues

sources

difference in the molecular weight of the cold inactivated

glutaminase showed a great role in various applications

enzyme

such as its use in fermented foods precisely in soy sauce

conformational changes which probably occur upon

and other related types, in addition to its use as

exposure to cold resulted from a weakening of the

anticancer agent which act by inhibition of glutamine

interaction among hydrophobic groups in the protein

utilization by the cancerous cells resulting in selective

(Chou et al., 1993).

776

and

the

warm

activated

enzyme.

The

Journal of Research in Biology (2013) 3(1): 775-779

Habeeb., 2013 The salt-tolerance of glutaminase is an important parameter

high-salinity.

industrial It

processes

was reported that

that

include

the high-salt

By using this method it was found that L-glutaminase producing marine alkalophilic Streptomyces sp. SBU1 which

was isolated from

Cape Comorin

coast,

concentration (nearly 3 M NaCl) used in the process

India gave highest enzyme production after 4 days

of soy sauce fermentation resulted in remarkable

inhibition of the koji mold (A. oryzae) Glutaminase

(Krishnakumar et al., 2011).

(Koibuchi et al., 2000).

Applications

incubation

and

14%

Corn

steep

liquor

Methods Used for Microbial Glutaminase Production

L-glutaminase has received great attention due to

Two methods are known for the production of

its valuable applications in several fields especially in

microbial glutaminase.

medicine and its use as an anticancer agent either alone

Submerged (Liquid) Production Method

or together with any other agents is known as enzyme

In this method, the sterile media together with

therapy, In addition to its role as flavor enhancer by

the enzyme producing organism were introduced into

increasing the glutamic acid content of food. Also

large fermentors (Tanks) followed by constant mixing

glutaminase applications extend to the enzyme utilization

and supply of sterile air (Schuegerl et al., 1991).

as biosensor in analytical purposes by measuring the

Actinomycetes

salt

levels of L-glutamine and finally in the manufacture of

tolerance in this production method. Reports showed that

fine chemicals such as theanine when used with bakerâ&#x20AC;&#x2122;s

Streptomyces rimosus isolated from estuarine fish

yeast.

recorded high salt tolerance and the highest enzyme

Glutaminase as Enzyme Therapy

glutaminases

showed

high

production obtained at temperature 27ÂşC, pH 9.0 and

Glutaminase can be used as alternative for cancer

both glucose and malt extract proved to be the best

treatment as enzyme therapy. The mechanism for

carbon and nitrogen sources for maximum enzyme

glutaminase therapy includes that L-Glutaminase act on

production (Imada et al., 1973).

its substrate (L-glutamine) and breaks it down leading to

Surface Production Method

the selective destruction of the tumor cells accompanied

This method includes the use of solid support on

by inhibition of both protein and nucleic acid

which microorganisms are grown. Surface production

biosynthesis due to glutamine starvation and this is

method (solid state fermentation) showed 25 to 30 fold

attributed to the inability of cancerous cells to synthesis

increase in enzyme production when compared with

glutamine (Tanaka et al., 1988). This is due to the fact

submerged production (Sabu et al., 2000b).

that some types of cancerous cells utilize glutamine

Wheat bran was found to be a favorable support

greatly (Lazarus and Panasci, 1986). Concerning this

for microorganisms in the process of glutaminase

finding various enzymatic therapies developed to deprive

production (Kashyap et al., 2002). In addition to wheat

L-glutamine to cancerous cells (Roberts et al., 1970).

bran many other solid supports showed high efficiency in

Glutaminase as Flavor Enhancer

the enzyme production such as ground nut cake powder,

Glutamate is a

famous amino acid and

copra cake powder and sesamum oil cake (Prabhu and

considered as a natural constituent of many fermented or

Chandrasekaran, 1995). Polystyrene beads, supported by

aged foods, such as soy sauce, fermented bean paste and

mineral salts and glutamine are another form of solid

cheese (O`Mahony and Ishi, 1987). It gives these types

supports used for the enzyme production.

of food their desired taste (Chou and Hwan, 1994). Glutamate (Glutamic acid) accumulated in these food

Journal of Research in Biology (2013) 3(1): 775-779

777

Habeeb., 2013 types as a result of protein hydrolysis by proteolytic

REFERENCES

enzymes such as glutaminase and protease have a vital

Akujobi CO, Odu NN, Okorondu SI and Ike GN. 2012. Production of protease by Pseudomonas aeruginosa and Staphylococcus aureus isolated from abattoir environment. Journal of Research in Biology. 2(2):077-082

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

(γ-l-glutamyl

ethylamide)

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 baker’s yeast are used to produce theanine (Tachiki et al., 1998). Also L-glutaminase is used in the manufacture of γ –glutamyl alkamides by the transfer of γ-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.

778

Chandrasekaran M. 1997. Industrial enzymes from marine microorganisms. J Mar Biotech., 5:86-89. Chou CC, Y u RC, Tsai CT. 1993. Production of glutaminase by Actinomucor elegans, Actinomucor taiwanensis and Aspergillus oryzae. J Chinese Agric Chem Soc., 31:78-86. Chou CC, Hwan CH. 1994. Effect of ethanol on the hydrolysis of protein and lipid during the ageing of a Chinese fermented soya bean curdsufu. J Sci Food Agric., 66(3):393-398. Imada A, Igarasi S, Nakahama K and Isono M. 1973. Asparaginase and Glutaminase activities of microorganisms. J Gen Microbiol., 76:85-99. Kashyap P, Sabu A, Pandey A, Szakacs G and Soccol CR. 2002. Extra-cellular L-glutaminase production by Zygosaccharomyces rouxii under solid-state fermentation. Process Biochem., 38(3):307-312. Koibuchi K, Nagasaki H, Yuasa A, Kataoka J and Kitamoto K. 2000. Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae. Appl Microbiol Biotechnol., 54 (1):59-68. Krishnakumar S, Alexis R, Rajan and Ravikumar S. 2011. Extracellular production of L-glutaminase by marine alkalophilic Streptomyces sp. SBU1 isolated from Cape Comorin coast. Ind J Geo-Marine Sci., 40(5):717721. Lazarus P, Panasci LC. 1986; Characterization of L-Threonine and L-glutamine transport in murine P388 leukaemia cells in vitro. Biochim Biophys Acta 856 (3):488-495. Moriguchi M, Sakai K, Tateyama R, Furuta Y and Wakayama M. 1994. Isolation and characterization of salt-t olerant glutaminase from marine Micrococcus luteus K-3. J Ferment Bioeng. 77(6):621625. Journal of Research in Biology (2013) 3(1): 775-779

Habeeb., 2013 Nakadai T, Nasuno S. 1989. Use of glutaminase for soy sauce made by Koji or a preparation of proteases from Aspergillus oryzae. J Ferment Bioeng., 67(3):158-162.

production from newly isolated Cohnella thermotolerans from Lonar Lake. Journal of Research in Biology. 1(4):292-298.

Ohshima M, Yamamoto T and Soda K. 1976b. Further characterization of glutaminase isozymes from Pseudomonas aeruginosa. Agri Biologi Chem. 40(11):2251-2256.

Tanaka S, Robinson EA, Appella E, Miller M, 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.

O`Mahony M and Ishi M. 1987. The umami taste concept: Implications for the dogma of four basic tastes in Umami. Marcel Dekker, New York. 75-93. Prabhu GN, Chandrasekaran M. 1995. Polystyrene an inert carrier for glutaminase production by marine Vibrio costicola under Solid state fermentation. World J Microbiol Biotechnol., 11(6):683-684. Roberts J, Holcenberg JS and Dolowy WC. 1970. Antineoplastic activity of highly purified bacterial glutaminase. Nature 227:1136-1137. Sabu A. 2003. Sources, properties and applications of microbial therapeutic enzymes. Ind J Biotechnol., 2 (3):334-341.

Underkofler LA, Barton RR and Rennert SS. 1958. Production of microbial enzymes and their applications. Appl Microbiol., 6(3):212-221. Wade HE, Robinson HK and Phillips BW. 1971. Asparaginase and glutaminase activities of bacteria. J Gene Microbiol. 69:299-312. Waksman SA. 1950. The actinomycetes: nature, occurance and activities. Waverly press, Baltimore, U.S.A.

Santana CF de, Pinto Kde V, Moreira LC and Lacerda AL. 1968. Action of swine kidney L-glutaminase on Ehrlich carcinoma. Rev Inst Antibiot. ; 8(1):105-107. Schuegerl K, Brandes L, Dullau T, Holzhauer-Rieger K, Hotop S and Huebner U. 1991. Fermentation monitoring and control by on-line flow injection and liquid chromatography. Anal Chim Acta. ; 249(1):87100. Sivakumar K, Sahu MK, Manivel PR and Kannan L. 2006. Optimum conditions for L-glutaminase production by actinomycete strain isolated from estuarine fish, Chanos chanos. Ind J Exp Biol., 44(3):256-258. Tachiki T, Yamada T, Mizuno K, Ueda M, Shiode J and Fukami H. 1998. Îł-Glutamyl transfer reactions by glutaminase from Pseudomonas nitroreducens IFO 12694 and their application for the syntheses of theanine and Îł-glutamylmethylamide. Biosci Biotechnol Biochem., 62:1279-1283. Tambekar DH and Tambekar SD. 2011. Partial characterization and optimization of protease Journal of Research in Biology (2013) 3(1): 775-779

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Journal of Research in Biology

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Original Research

Journal of Research in Biology

A review on the role of nutrients in development and organization of periphyton Authors: Saikia SK1 , Nandi S2 , Majumder S3 .

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 Institution: and also outlines its ecological role in nutrient sharing with other aquatic components. 1. Assistant Professor, Aquatic Ecology Laboratory, 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 Department of Zoology, entrapment, de novo synthesis, nutrient leakage, trophic upgrading etc., ambient Visva Bharati University, Santiniketan, West Bengal, nutrients are routed to periphyton and transferred to upper trophic levels. Periphyton communities stand next to phytoplankton for their contribution to primary India, Pin-731235. productivity, in nutrient rich aquatic environment. Unlike phytoplankton, nutrient 2. Research Fellows, poor aquatic environment has no effect on periphytic primary productivity. Aquatic Ecology Laboratory, As periphyton communities are attached to substratum, their ability to assimilate Department of Zoology, organic nutrient through substratum is an additional advantage over phytoplankton. Visva Bharati University, Santiniketan, West Bengal, India. 3. Research Fellows, Aquatic Ecology Laboratory, Department of Zoology, Keywords: Visva Bharati University, Aquatic ecosystem, Biofilm, carbon, primary productivity, phytoplankton. Santiniketan, West Bengal.

Corresponding author: Saikia SK.

Article Citation: Saikia SK, Nandi S, Majumder S. A review on the role of nutrients in development and organization of periphyton. Journal of Research in Biology (2013) 3(1): 780-788

Email: surjyasurjya@gmail.com

Dates: Received: 20 Nov 2012

Web Address: http://jresearchbiology.com/ documents/RA0307.pdf.

Journal of Research in Biology An International Open Access Research Journal

Accepted: 10 Dec 2012

Published: 11 Feb 2013

780-788 | JRB | 2013 | Vol 3 | No 1

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Saikia et al., 2013 contributor of most of the nutrient inputs to aquatic

INTRODUCTION The term „periphyton‟ (peri round; phyton plant)

ecological cycles. The present review is an attempt to

was proposed by (Behning, 1924) and popularized

outline periphyton as an integral and essential component

by several authors (Cooke, 1956; Sladeckova, 1962;

of aquatic ecosystem highlighting few areas recently

Pieczynska, 1970). There exist a series of definitions

addressed on the role of periphyton in nutrient sharing in

proposed for „periphyton‟ (Young, 1945; Neel, 1953;

aquatic processes.

Wetzel, 1963). (Wetzel, 1983) defined it as the micro

Periphyton: A Nutrient Dependent Organization

„floral‟ community living attached to any substrate

(Whal,

1989)

discussed

settling

pattern

under water. (Stevenson, 1996) used it for describing

of „biofilm‟ (Figure 1), in four phases: (i) surface

microorganisms such as algae and bacteria growing in

conditioning

association with substrata. These communities play an

compounds where macromolecules attach to submerged

important role in water bodies, not only as important

surfaces following a spontaneous physical-chemical

primary producers and energy source for higher trophic

process; (ii) primary colonization or bacterial settling

levels, but also by affecting the nutrient turnover and the

following

transfer of nutrients between the benthic and the pelagic

colonization,

zone (review Saikia, 2011). The substrates of periphyton

(iii) secondary colonization to bacterial layer and EPS

commonly include submerged plants or plant parts,

pool by eukaryotic unicellular microorganisms, mainly

rocks and sediments. Such substrate selection designs

protozoan, microalgae and cyanobacteria and (iv) settling

periphyton as a medium in transferring and „trophic

of eukaryotic multicellular organisms as a function of

upgrading‟ of nutrients. This property recognizes

nutrient sharing, grazing and predation. According to

periphyton as a tool for biofiltering excess nutrients from

(Wetzel, 1983), associated organisation from secondary

polluted waters and for efficient nutrient transfer in

colonization onwards can be designated as „periphyton‟.

aquatic food chain.

In that way, it could be defined as an advanced

Aquatic

dissolved

conditioning

bacteria

start

and to

organic

after

their

produce

EPS,

freshwater and marine water bodies and their ecology

a fifth (v) phase; the tertiary colonization where

discusses the relationship of aquatic organisms and

bacterioplankton colonized on the surfaces of unicellular

its interaction with the immediate environment. The

and filamentous secondary colonizer (e.g. diatom,

principle biotic components primarily explained in the

Oedogonium etc.). Several bacteria different from early

recent past for their contributions to different interactions

colonizer settle on algal surfaces at this stage

(Alldredge et al., 1993; Armstrong et al., 2000).

are

comprise

surface

successional stage of biofilm. However, there could be

ecosystem

mainly

adsorption

aquatic

ecosystems

macrophytes,

plankton

(Zooplankton and phytoplankton) and invertebrates (benthos, nekton and neuston). Till the mid of th

Periphyton are rich Carbon source TEP

with

rich

Carbon

sources

aquatic

environments

19 century, periphyton or „associated organisms‟ were

Glucopolysaccharides

not given any biological credit for their role in aquatic

initiates

ecosystem. Probably, (Wetzel, 1963) in his evolutionary

surface conditioning (Stoderegger and Herndl, 1999).

review paper „Primary productivity of periphyton‟ in

The bacterial EPS from early biofilm exists as a part of

Nature, for the first time made convincing remark on the

dissolved organic matter (Lignell, 1990) as well as

role of periphyton in aquatic ecosystem. Even today,

particulate matter (Decho, 2000). It acts both as rich

periphytic communities are ignored as a major

organic Carbon storage (Freeman and Lock, 1995) and

781

early

colonization

bacteria

through

Journal of Research in Biology (2013) 3(1): 780-788

Saikia et al., 2013 chief supplier of Carbon demand for organisms that feed

management of community metabolism of periphytic

on periphytic aggregates (Decho and Moriarty, 1990;

matrix and can trap the metabolic products released

Hoskins et al., 2003). Being polyanionic in nature

by bacterta on algal surface (Makk et al., 2003). Such

(Costerton et al., 1978), EPS further permits inorganic

algae-bacteria interactions enrich periphytic organic

nutrient entrapment through ion exchange processes

matrix with components of polysaccharides, proteins,

(Freeman et al., 1995) leading to storage of organic

nucleic acid and other polymers (Davey and O‟toole,

Carbon in the biofilm. In addition, among the bacterial

2000).

fractions, cyanobacteria are important primary producers

Periphytic pathway of nutrient transfer

and many of their species can ﬁx atmospheric Nitrogen

The periphytic nutrient transfer pathway (PNP)

(Whitton and Potts, 1982). Chemical screening of

mainly involves ambient nutrient entrapment, storage

laboratory grown, commercially viable cyanobacteria

and transferring it to immediate higher trophic level. The

have revealed that they have a high nutritional value, in

fate of PNP gets its initiation from the surface

terms of protein (Choi and Markakis, 1981).

conditioning phase of periphyton formation. As soon as

During tertiary phase of periphyton development,

TEP prepares the substrate surface for colonization,

algal communities play indirect role in nutrient addition

bacteria as initial colonizer develops micro-colonies

to periphytic complex through their surfaces. A study on

(Costerton, 1984) and through EPS, it supplies a

algae-bacteria interactions on biotic surfaces revealed

significant source of Carbon to periphytic complex

that bacterial abundance is significantly higher in areas

(Hobbie and Lee, 1980) (Figure 2). A PNP establishes

of diatom colonization on substrates (Donnelly and

between dissolved organic in periphytic complex and

Herbert, 1999). These bacteria contribute to the

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

Figure 1. Formation of periphytic complex on natural substrate showing tertiary phase of colonization (Modified from Whale, 1989). TEP, Transparent Exopolymer Prticles; EPS, Extracellular Polymeric Substances Journal of Research in Biology (2013) 3(1): 780-788

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 782

Saikia et al., 2013 mobilized to ensure survival during starvation (Dawes

physiological processes in living organisms and are

and Senior, 1973) and thereby recovery of periphytic

major nutrient constituents of polar lipids, and are

aggregates due to senescence. The third mechanism of

present in cell and chloroplast membranes. The

organic Carbon storage is the polysaccharide exudates

dominance of algae in periphytic canopy acts as rich

(Freeman and Lock, 1995) released by algae at tertiary

source of FA to animals grazing on periphyton.

phase under nutrient (especially Phosphorus) limited

Primary productivity of periphyton

condition. The algal components release polysaccharide

The energetic relation of an ecosystem is

exudates to EPS under Phosphorus limitation on which

principally regulated by primary production. In aquatic

tertiary phase bacteria flourish. In return, these bacteria

ecosystem, algae are dominant primary producers, and

remineralize Phosphorus for algae. In addition, the ECM

responsible for both Carbon fixation and sequestration.

with polyanionic by nature (Costerton et al., 1978) is

Periphyton with majority of algae might have significant

believed to permit nutrient entrapment through ion

contribution to primary production of aquatic ecosystem.

exchange processes (Freeman et al., 1995). (Freeman

However, very few investigations have been performed

and Lock,

the entrapment

on measurements of photosynthetic rates of algal

mechanism may also permit the storage of organic

periphyton under natural conditions. (Wetzel, 1963)

Carbon in the biofilm.

pointed

1995) proposed

that

out

technical/methodological

difficulty in

In transferring nutrient through PNP, the

assessing such parameters of periphyton under natural

bacterial Carbon enters to organisms in the next trophic

condition. From an analysis on nutrient limiting and

level as complex Carbon rich compounds. The Fatty acid

nutrient rich lakes, it is obvious that periphyton

(FA) component of algae is under extensive research

productivity contributes more than 30% of primary

now a day as Carbon rich compounds. Periphytic matrix

productivity to the aquatic ecosystem (Figure 2a). On

is dominated by algae and hence FA contributes to the

comparison, it seems evident that the nutrient limited

food quality of matured periphytic organization. In algae,

aquatic ecosystems have more or less equal primary

FA increases as a result of exposure to stressful

productivity levels to nutrient rich aquatic ecosystems.

environmental conditions, such as high temperature,

The same is not true in case of phytoplankton

nutrient

(Figure 2b).

extremes

and

harsh

light

conditions.

Annual Primary Production in percentage

Polyunsaturated fatty acids (PUFAs) also affect many

Phytoplankton

Periphyton

Macrophyte

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). 783

Journal of Research in Biology (2013) 3(1): 780-788

Saikia et al., 2013 Nutrient Regulated Biotic Interactions of Periphyton

limited environments, it relies mainly on organic

Biotic interactions in aquatic ecosystems are

nutrients from natural substrate. All artificial substrates

more complex than any other ecosystems for its variable

cannot serve as organic nutrient supplier to periphyton.

nature. Interactions between periphyton and biotic

Substrates like sediments or seed grains acts as nutrient

components in aquatic ecosystem are primarily regulated

diffusing substrate releasing nutrients to overlying

by nutrients and can be discussed under following

periphytic layer. (Hansson, 1989) showed that epipelion

subheadings-

can significantly lower nutrient availability in the water

Plankton-periphyton interaction

column due to uptake of diffusing nutrients. (Hagerthey

Periphyton-macrophyte interaction

and Kerfoot, 1998) demonstrated that inflowing ground

Grazer-periphyton interaction

water is a significant source of nutrients for episammon

Plankton-Periphyton interaction

in nutrient limiting environment. These sediments act as

The

plankton-periphyton

interaction

better nutrient source for periphyton (Burkholder, 1996).

principally regulated by light and nutrient availability in

Substrate based nutrient uptake by periphyton is further

the environment. Both the communities are composed of

related to depth, light availability, physical disturbances

common members of bacterial, algal and zooplanktonic

etc.

origin. However, on spatial ground, habitats of both

Grazer-Periphyton interaction

plankton and periphyton have differences in receiving

Studies reported that several herbivore types

light and nutrients. Conceptual models revealed that

(e.g. gastropods, trichopteran larvae and fish) can

nutrient

dramatically reduce periphytic biomass to only a few

periphyton than plankton (Wetzel, 2001; Hansson, 1992).

percent of total biomass (Hillebrand et al., 2000).

Nutrient limitation results thin planktonic cover that

Although grazing results reduction in periphytic biomass,

allows maximum light to pass through water column to

the total productivity of the periphytic complex increases

reach

facilitating

due to reduced competition among algal members

multiplication of periphytic population. Conversely,

(Carpenter, 1986; Mc Cormick and Stevenson, 1989).

plankton rich aquatic ecosystems limit growth of

(Norberg 1999), using transparent incubation chambers,

periphyton due to limited light availability. Epiphytic

measured a 4-fold increase in periphyton specific

communities can better adsorb nutrients from sediments

productivity in grazed periphyton compared to ungrazed

controls. Moreover, the grazer presence increased the

limited

the

bottom

environments

bottom

the

system

are

dominated

ecosystem

through

macrophytes

(Burkholder and Wetzel, 1990).

Chlorophyll: biovolume ratio, especially reported from

Periphyton-substrate interaction

streams (Hill and Knight, 1987). In addition to increase

Substrate type plays a driving role in growth and

in productivity, grazing and competition can modify the

succession of periphyton. Being a substrate based

species composition of periphytic algal assemblages

organization, periphyton have access to both organic

(Duffy and Hay, 2000; Nielsen, 2001), generating

nutrients from substrate and inorganic nutrients from

heterogeneity through temporal or spatial scale on the

water column. In nutrient rich environments, it receives

substrate. A top down effect of consumers on their prey

nutrients from water column (Eminson and Moss, 1980;

can be further accelerated by grazer and grazer excretion

Burkholder, 1996). Here, similar to planktonic cells,

of nutrients, removal of senescent cells, or increased

periphytic cells can use inorganic nutrients efficiently,

uptake of nutrients by the remaining cells (Lamberti

specifically dissolved organic Phosphorus and in nutrient

et al., 1987; Kahlert and Baunsgaard, 1999). Grazers

Journal of Research in Biology (2013) 3(1): 780-788

784

Saikia et al., 2013 may have strongest effects on Carbon:Phosphorus

CONCLUSION

and Nitrogen:Phosphorus, but Carbon:Nitrogen and

Disrupted nutrient cycling is a major problem

Carbon:Chlorophyll may remain unaffected (Hillebrand

both

and Kahlert, 2001). Hillebrand et al., (2008) described

periphyton could be a non-point manager of nutrient

three

periphytic

cycle disruption and hence can overplay on plankton for

interactions affecting nutrient stoichiometry. First, the

nutrient cycling in aquatic ecosystem. During renovative

non algal component, which could be a dominant part

practices,

of the organic material of periphyton assemblage

managers greatly ignore the role of these substrate based

(Frost et al., 2002) is reduced by unselective grazing.

microorganisms. At the same time, it can play as an

Benthic invertebrates graze upon both detritus and algal

efficient supplier of nutrient to its grazer under

component of periphyton but only algae regenerate.

controlled and well managed productive practices. It is

Therefore, grazing not only reduces non algal component

observed that at traditional level, farmers from different

of periphyton, but also facilitates the growth of live

parts of the world have been practicing periphyton to

component within it. (Jones et al., 1999) suggested that

feed aquacrops to convert periphytic energy biomass to

epiphytes can influence the nutritional quality of the

crop biomass (Saikia and Das, 2009). Such conversion of

periphyton which grows on their surfaces, making it

biomass is an outcome of increased assimilation of

more nutritious for grazing by invertebrates, particularly

micro- and macro nutrients from periphytic complex in

snails. In return, these grazers might preferentially feed

the fish body through trophic upgrading (Saikia and

on the periphyton and clear the plants of a potential

Nandi, 2010). Further researches on the mode of energy

competitor for nutrients, with the plants and grazers both

transfer through periphytic food chain, enhanced nutrient

gaining from this relationship. Secondly, in streams,

uptake under manipulative nutrient input, modelling on

nutrient uptake of intact periphyton mats is often slower

applied periphytic ecology, ecotoxicology, Carbon

than cell specific uptake rates as boundary effects reduce

entrapment and delivery, directing nutrient and Carbon

the uptake ability of the benthic algae (Riber and Wetzel,

sequestration both in marine and freshwater are needed

1987; Bothwell, 1989; Burkholder et al., 1990). Grazer

for better understanding of its role in aquatic ecosystem.

pathways

presence

alters

for

grazer

periphyton

mediated

architecture,

in freshwater and marine ecosystems and

strategies

of aquatic

ecosystem

health

increases

periphytic heterogeneity and relative availability of

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788

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Assessing heavy metal contamination of road side soil in urban area Authors: Sarala Thambavani D1, Vidya Vathana M2.

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.

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.

Corresponding author: Sarala Thambavani D.

Keywords: Pollution, combustion, heavy metal enrichment, road side soils, enrichment factor, Normalized scatter coefficient value, environmental pollution.

Web Address:

Article Citation: Sarala Thambavani D and Vidya Vathana M. Assessing heavy metal contamination of road side soil in urban area. Journal of Research in Biology (2013) 3(1): 789-796

http://jresearchbiology.com/ documents/RA0187.pdf.

Dates: Received: 16 Jan 2012

Accepted: 27 Jan 2012

Published: 16 Feb 2013

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.

Journal of Research in Biology An International Scientific Research Journal

789-796 | JRB | 2013 | Vol 3 | No 1

www.jresearchbiology.com

Thambavani and Vathana, 2013 It is needless to say that the industrial activities

INTRODUCTION Pollution

increased

in the metropolitan cities of the world are responsible for

considerably as a result of increasing human activities

the addition of pollutants through chemical factories,

such as burning of fossil fuels, industrial and automobile

residential activities (Point sources) and vehicular traffic

exhaust

(non-point sources) which are the primary sources of soil

heavy metals from automobile sources is a serious

pollution. The objective of this study, is to investigate the

environmental issue. The majority of the heavy metals

effect of heavy metal pollution of soil along road sides.

emissions.

recent

The

years

pollution

has

soils

are toxic to the living organisms and even those

The present study reports the role of industrial and

considered as essential can be toxic if present in excess.

urban activities in the heavy metal contamination of the

The heavy metals can impair important biochemical

soils in the Madurai industrial area with the objectives:

processes posing a threat to human health, plant growth

To assess the extent of heavy metal pollution

and animal life (Jarup 2003; Michalke 2003; Silva et al.,

influenced by urban and industrial activities.

2005).

To predict the rate of heavy metals pollution in the The waste products from vehicles that ply

future if the activities are allowed with the same

highways contain some heavy metals inform of smokes.

pace.

Emissions from exhaust pipes of automobile engine and

To understand the variations in the behavior of

contacts between different metallic objects in machines

different heavy metal.

contain such heavy metals as Lead (Pb), Zinc(Zn), Iron (Fe), Copper (Cu), Chromium (Cr) and Cadmium (Cd)

METHODS

and are major sources of pollution among soils (Turer

Field Methodolgy

and Maynard, 2003).

To understand the state of environment of the

Soils are usually regarded as an ultimate sink.

Madurai area a detailed field survey was carried out and

For heavy metals discharged into the environment (Banat

after having identified possible sources of pollution a

et al., 2005) and sediments can be sensitive indicators for

part of Madurai area was selected. This area is under

monitoring contaminants in aquatic environment (Pekey

intense human interference in terms of growing

et al., 2004). Therefore the environmental problem of

urbanization (municipal sewage sludge, traffic pollution

soil and sediment pollution by heavy metals has received

in particular) and industrialization.

increasing attention in the last few decades in both

Selection of sampling site

developing and developed countries throughout the

In the present study stratified regular sampling

world (Zhang et al., 2007). Hence, inorder to monitor

method was adopted for soil sample collection as in

heavy metal pollution in an area, due to the

geo-assessment of the variables estimated, the stratified

anthropogenic activity (Sarala Thambavani and Vathana,

regular sampling is more suitable because this kind of

2011), the soil samples represent an excellent media

sampling draws homogenous error (Burgess et al., 1981).

because heavy metals are usually deposited in the top

Different sampling stations were selected and samples

soil (Govil et al., 2001; Romic and Romic, 2003) and

are collected from the top layer of the soil using plastic

help in knowing the sources of heavy metals and also

spatula after removing the debris, rock pieces and

controlling and optimizing their effects on the human

physical contaminants. In order to have the background

health.

concentration values of the heavy metal elements, three soil samples were collected, each from 100 cm below

790

Journal of Research in Biology (2013) 3(1): 789-796

Thambavani and Vathana, 2013 ground level, which are least affected by anthropogenic

RESULTS

activities (Table1). The samples were placed in the clean

The concentration of heavy metals Lead,

polythene bags, which were brought to the laboratory.

Copper, Chromium, Nickel and Cadmium in the soils

Laboratory Methodology

of Madurai industrial, traffic and residential area

The samples were brought to the laboratory

were analyzed, collected at six sampling stations during

where they are dried and mixed thoroughly to obtain the

May 2011- Oct 2011. The range of the concentrations

representative samples. Soon after drying the debris and

found in different sampling stations are (i) Pb industrial

other objects were hand picked up and the sample were

(24.81-42.37

grounds in a mortar to break up the aggregates or lumps,

and residential (20.42-2.66 mg/kg) (ii) Cu industrial

taking care not to break actual soil particles. Soil samples

(10.40-16.24

were then passed through a 2 mm sieve in order to

and residential (10.5 -18.16 mg/kg) (iii) Cr industrial

collect granulometric fraction. Since trace metals are

(17.0-34.50

often found mainly in clay and silt fractions of soil and

and residential (25.12 mg/kg) (13.60-18.52 mg/kg)

hence the size fraction <63 Âľm sieve (wet sieving) and

( i v)

was used to measure the concentration of the heavy

traffic (22.32-25.46 mg/kg) and residential (22.24- 25.12

metals Lead, Copper, Chromium, Zinc, Nickel and

mg/kg) (v) Ni industrial (11.85-14.0 mg/kg), traffic

Cadmium from all the samples collected.

( 11.52 -14.80 mg/kg) and residential (11.70-13.9 mg/kg)

For this purpose the clay and silt fraction were digested by acids to get the solution by taking 5 g of

(vi)

mg/kg), mg/kg), mg/kg),

traffic traffic traffic

in dustr ial

industrial

(26.80-5.32

mg/kg)

(10.69-18.20

mg/kg)

(14.56-21.60

(22.5-45.6

(1.24-4.32

mg/kg) mg/kg),

mg/kg),

traffic

(1.60-3.62 mg/kg) and residential (1.70-2.25 mg/kg).

sample into a 300 ml polypropylene wide-mouthed jar

The mean concentration for these heavy metals

and distilled water was added to make a total 200 ml.

from the surface soil have been calculated to be (i) Pb

Then it was acidified with 10 ml HF, 5 ml HClO4,

industrial (33.23), traffic (41.50) and residential (24.31).

2.5 ml HCl and 2.5 ml HNO3 in order to completely

(ii) Cu industrial (12.97), traffic (15.03) and residential

digest the soil. This jar was shaken on an orbital shaker

(14.98). (iii) Cr industrial (24.33), traffic (17.53) and

for 16 h at 200-220 rpm before being filtered through

residential (15.51). (iv) Zn industrial (29.78), traffic

whatman filter paper (No.42) into acid washed bottles.

(24.23) and residential (23.74). (v) Ni industrial (12.77),

The solution was stored and heavy metal contents were

traffic (13.72) and residential (12.99). (vi) Cd industrial

analyzed by Atomic Absorption Spectrophotometer as

(2.94), traffic (2.59) and residential (1.92) respectively at

per the method recommended by committee of soil

the confidence limits of 95%.

standard methods for analyses and measurement (1986).

The concentration of heavy metals in all the

The raw data obtained during the course of laboratory

sampling stations exhibit an increasing trend over a very

analyses were stored in Microsoft Excel software and

short period of monitoring from May 2011-Oct 2011

further processed to obtain various parameters required

(Figure 1). It was observed that the mean concentration

for interpretation.

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 Journal of Research in Biology (2013) 3(1): 789-796

791

Thambavani and Vathana, 2013 stations followed by Zinc, Chromium, Copper, Nickel

DISCUSSION

and Cadmium.

In order to evaluate the rate of accumulation of heavy metals in the soils the mean values for all heavy

Accumulative Signature of Heavy Metals An increasing trend has been found for the heavy

metals studied were considered along with Enrichment

metal elements Lead, Copper, Chromium, Zinc Nickel

factor values of all six metals (Table 2), which clearly

and Cadmium wherein the Lead and Cadmium are

indicate the highest enrichment of Cadmium followed by

getting accumulated with very rapid rate mainly due to

Lead, Zinc, Chromium, Copper and Nickel in all the

anthropogenic activities (Sayadi, 2009). In order to

three sampling stations of industrial, traffic and

assess the variations in the heavy metal accumulations in

residential area. The values of NSC for all six heavy

the soils, the calculated measures that is Enrichment

metal showed that Cadmium is increasing in soil

Factor and Normalized Scatter Coefficient were used.

environment of industrial area followed by Zinc,

The Enrichment Factor (EF) is a ratio of the

Chromium, Lead, Copper and Nickel. In traffic area

concentrations of the heavy metals in the soil samples to

Lead is increasing in soil environment followed by

the corresponding concentration of natural background

Cadmium, Chromium, Copper, Nickel and Zinc and in

concentration. EF is calculated with the help of the

residential area Copper is increasing in soil environment

formula given by Subramanian and Datta dilip (1998)

followed by Chromium, Cadmium, Lead, Nickel and

and presented in Table 2.

Zinc.

EF = Value of a given metal concentration found on soil (mg/kg) Natural local background concentration of the metal (mg/kg)

Normalized Scatter Coefficient (NSC) has been

It is observed that in all the sampling sites, Lead shows highest concentration in soil and also have high Table 2 Enrichment Factor for heavy metals in the soils

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). concentration in the last sampling â&#x20AC;&#x201C; concentration in first sampling NSC =

x 100 concentration in the last sampling + concentration in first sampling

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.

792

Pb 4.6 5.3 5.7 6.9 7.7 8.3 Pb 5.1 5.6 6.9 8.7 10.1 11.2 Pb 3.9 4.2 4.7 4.8 5.0 5.1

Cu 1.1 1.1 1.4 1.5 1.5 1.7 Cu 1.1 1.4 1.5 1.7 1.8 1.9 Cu 1.1 1.3 1.3 1.8 1.9 1.9

Industrial Area Cr Zn 1.7 1.9 1.8 2.1 2.2 2.3 2.7 2.5 2.9 2.9 3.5 4.0

Ni 1.1 1.1 1.0 1.1 1.2 1.2

Cd 3.8 4.1 8.8 11.8 13.1 13.5

Traffic Area Cr Zn 1.4 1.9 1.6 1.9 1.6 2.1 1.8 2.1 1.8 2.1 2.1 2.2

Ni 1.0 1.2 1.2 1.3 1.3 1.3

Cd 5.3 6.6 8.3 9.5 9.9 12.1

Residential Area Cr Zn 1.2 1.9 1.3 1.9 1.3 2.0 1.4 2.0 1.5 2.1 1.7 2.1

Ni 1.0 1.1 1.2 1.2 1.2 1.2

Cd 4.9 4.9 5.3 5.7 5.7 6.4

Journal of Research in Biology (2013) 3(1): 789-796

Thambavani and Vathana, 2013

Figure 1. Mean Concentrations of the heavy metals on different sampling stations

Figure 3. Traffic Area

Figure 2. Industrial Area

Figure 4. Residential Area

enrichment factor. Cadmium shows lowest concentration

cumulative activity in the region. Hence the Enrichment

in soil but is has quite high enrichment factor, while

factor should denote the total enrichment and or

Copper, Chromium, Zinc and Nickel shows higher metal

depletion of an element and cannot evaluate the trend for

concentration but rather low EF when compared to lead.

the short term accumulation.

The scatter plot of the mean concentration of

When the mean values of EF and NSC for all the

heavy metals was plotted against the EF for all the three

six heavy metals are studied at all the sampling stations

sampling sites (Figures 5,6,7). Per usual of the result

(Figures 8, 9,10) it can be stated that Cadmium has been

showed that Zinc is having high mean concentration but

enriched to a quite greater extent followed by Lead, Zinc,

it is not getting enriched in proportion to its mean

Chromium, Copper and Nickel at all the sampling sites.

concentration. On the other hand Cadmium though

On the other hand the Normalized Scatter Coefficient

having lowest mean concentration has higher rate of

value indicates that Cadmium has got enriched in faster

enrichment. Lead shows the highest mean concentration

rate at industrial area followed by Zinc, Chromium,

and also corresponding highest enrichment factor.

Lead, Copper and Nickel. In traffic area Lead is getting

The behavior of Zinc may be attributed to its

enriched in the faster rate followed by Cadmium,

source mainly from weathering of the parent rock while

Chromium, Copper, Zinc and Nickel. But in residential

that of Cadmium and Lead mainly due to anthropogenic

area, the NSC value indicate that Cu is quite enriched

activities. EF normally reveals the addition and or

with the faster rate followed by Chromium, Cadmium,

removal of metal under consideration which is a result of

Lead and Zinc.

Journal of Research in Biology (2013) 3(1): 789-796

793

Normalized Scatter Coefficient %

Enrichment Factor

Thambavani and Vathana, 2013

Enrichment Factor

Normalized Scatter Coefficient %

Figure 8 INDUSTRIAL AREA

Figure 5. Industrial Area

Enrichment Factor

Normalized Scatter Coefficient %

Figure 9 TRAFFIC AREA

Figure 6. Traffic Area

Figure 7. Residential Area CONCLUSION

Figure 10. RESIDENTIAL AREA by Lead, Zinc, Chromium, Copper and Nickel.

The variation assessment of heavy metal

Normalized Scatter Coefficient value indicate that Lead

pollution by using Enrichment Factor and Normalized

is getting accumulated in a faster rate followed by

Scatter Coefficient in the soil sample collected from the

Cadmium, Chromium, Copper, Zinc and Nickel. In

study area between May 2011-Oct 2011 has revealed

summary the soils in the Madurai industrial, traffic and

significant increase in the six heavy metals (viz Pb, Cu,

residential area are significantly contaminated by heavy

Cr, Zn, Ni and Cd). Enrichment Factor values shows

metals and hence more attention to be paid to heavy

that Cadmium has enriched to a greater extent followed

metal pollution particularly for Lead and Cadmium. In

794

Journal of Research in Biology (2013) 3(1): 789-796

Thambavani and Vathana, 2013 Govil Table 3 Normalized Scatter Coefficient (%)of the heavy metals in the soils of the study area Industrial Area 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 Pb 37.0 32.9 23.0 12.5 5.2 0 Pb 13.2 8.8 3.9 2.5 0.8 0

Cu 25.9 14.6 10.9 5.5 4.1 0

Traffic Area Cr Zn 19.5 6.6 15.8 4.9 14.0 1.9 8.0 0.9 7.3 7.3 0 0

Cu 26.4 17.9 16.8 1.6 0.6 0

Residential Area Cr Zn 15.3 6.1 13.1 4.7 11.9 3.4 8.7 2.6 5.4 0.4 0 0

PK,

Ni 8.9 7.2 3.2 2.5 0.6 0

GLN,

Krishna

AK.

2001.

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Ni 12.5 5.7 2.7 2.0 0.8 0

Reddy

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and

some

examples,

Cd 38.7 29.1 18.3 12.1 9.5 0

Ecotoxicology and Environmental Safety 56:122-139.

Cd 13.9 13.4 9.8 6.1 5.6 0

Romic M, Romic D. 2003. Heavy metal distribution in

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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