Journal of Research in Biology - Volume 2 Issue 5

Journal of Research in Biology An International Scientific Research Journal
Original Research
Studies on some biochemical effects of aqueous leaf

Extract of landolphia owerrience on albino rats
Journal of Research in Biology
Authors: ABSTRACT:
Friday O Uhegbu,
Amadike E Ugbogu and The effects of aqueous leaf extract (crude) of Landolphia owerrience on some
Kingsley C Nwoku. biochemical parameters in adult male and female Albino rats were investigated.
Administration of the plant leaf extract was by gavage at a dose level of 20 mg/kg,
30 mg/kg and 40 mg/kg body weight in 0.5 ml saline respectively, daily for 21 days.
Institution:
The concentration of ALT and AST were non-significantly increased as the dose
Department of Biochemistry,
increased. Alkaline phosphatase (ALP) level also increased non-significantly, but not
Faculty of Biological and
Physical Sciences Abia dose dependent. Protein level also significantly increased in a dose dependent
State University, PMB 2000, fashion. Hemoglobin concentration increased significantly, while lipid peroxidation
Uturu, Nigeria. level decreased non-significantly as the dose increased. The non-significant increase in
liver enzyme concentrations in this study showed that the Landolphia owerrience leaf
extract may not be toxic to the rat liver, while the low lipid peroxidation levels showed
that it may have antioxidant activity.
Corresponding author: Keywords:

Friday O Uhegbu. Ethno-medicine, hemoglobin, liver enzymes, medicinal plant, peroxidation,
protein.
Email: Article Citation:

fouhegbu@yahoo.com Friday O Uhegbu. Amadike E Ugbogu and Kingsley C Nwoku.
Studies on some biochemical effects of aqueous leaf Extract of landolphia owerrience
on albino rats.
Journal of Research in Biology (2012) 2(5): 410-417
Phone No:
+234(0)8035426500.
Dates:
Received: 02 May 2012 Accepted: 14 May 2012 Published: 16 Jun 2012
Web Address:
http://jresearchbiology.com/ This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
documents/RA0238.pdf. 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.
410-417 | JRB | 2012 | Vol 2 | No 5

An International
Scientific Research Journal www.jresearchbiology.com
Uhegbu et al., 2012
INTRODUCTION (Hwang et al., 2002).

Landolphia owerrience is a wild plant usually Alanine aminotransferase [ALT] activity rises in
found growing wild in Africa and Madagascar. The plant viral and toxic hepatitis and the degree and time course
belongs to the family Apocynaceae and it is of its elevation parallel to those of Aspartate
distinguished by a corolla tube which is usually aminotransferase [AST] activity. An elevated ALT and
thickened above the anthers. It produces glabrous fruits, alkaline phosphatase [ALP] activity also occurs in
which is characterized by dense inflorescence (Person, cirrhosis, extra hepatic biliary obstruction and hepatic
1962; Burkill, 1994). Other species of the genus include metastasis, while inconsistent elevations occur in acute
Landolphia hirsute and Landolphia kirki. The wild plant myocardial infarction and skeletal muscle disease
is rich in nutritional content as its composition parades (Jensen and Freese, 2009).
rare food nutrients especially the vitamins, carotene and Diseases like severe burns, severe renal disease
minerals (Ebi and Ofoefule, 1997). and chronic hepatitis and sarcoidosis cause rapid loss of
In South East of Nigeria, the extract from plasma protein and hemoglobin. Lipid peroxidation plays
Landolphia owerrience is used in the folklore treatment a role in carcinogenesis and arteriosclerosis and by
of venereal disease, colic diseases, teeth cleaning, curing extension prevents damage to cells that result into
and control of diabetes. The latex is drunk or used as an disease condition (Hsieh et al., 2009).
enema for intestinal worms, and in treatment of arrow Since the crude plant leaf extract of
poison. Preliminary photochemical tests on Landolphia owerrience is used for folklore treatment and
Landolphia owerrience have shown that it contains management of various ailments: venereal disease, colic,
active cardio glycosides, saponins, tannins, flavonoids diabetes, worm expulsion and teeth cleaning. This
and steroids and also shows antimicrobial activities present study was conducted to investigate the effect of
(Iwu 1993). The antimicrobial screening of the plant leaf extract on the stability of the liver, vis--vis
Landolphia owerrience among other plants has shown its effect on liver enzymes [ALT, AST and ALP], serum
that the extract inhibits Escherichia coli, protein levels, hemoglobin and lipid peroxidation.
Bacillus subtilis, Candida albicans and Aspergillus niger
and the activities were linked to the presence of steroids, MATERIALS AND METHODS
tannins and glycosides in the plants (Ebi and Ofoefule, Plant Material
1997; Okeke et al., 2001) The leaves of Landolphia owerrience were
The liver is the central organ that co-ordinate and collected from the University campus where it is grown
modulates most biochemical activities in the body. The as ornamental plant; in January 2012. The plant was
metabolic activities of the liver are essential for authenticated and taxonomically identified at the
providing fuel to the brain, muscle and other peripheral Department of Plant Science and Biotechnology, Abia
organs (Chang et al., 2004). Most compounds absorbed State University, Uturu, Nigeria. Voucher specimen of
by the intestine go through the liver, which enables it to the plant was deposited at the Departmental herbarium.
regulate the level of metabolites in the blood The plant leaves were washed with distilled water to
(Lehninger, 2005). The liver plays a central role in the remove dirt and contaminants, and then sun dried.
regulation of lipid metabolism, detoxification and Plant Extract
biotransformation of foreign compounds to more soluble The dry leaf samples were milled into a fine
forms for further breakdown and excretion powder using Arthur milling machine. 20g of the milled
411 Journal of Research in Biology (2012) 2(5): 410-417
Uhegbu et al., 2012
leaf sample was soaked in 200ml of distilled water, and catalyzed reaction. The assay procedure for ALT and
the mixture was left to stand for 48hrs with occasional AST are the same, except that for ALT, alanine citrate is
stirring. Filtration was done with a muslin bag. The added. The c o l o r a t io n produced w it h
concentration of the extract was determined to be 2, 4 dinitrophenyl-hydrazine is read at 540nm on a
27.2mg/ml. spectrophotometer.
Animals ALP
Sexually matured adult albino rats (32 males and Alkaline phosphatase was determined using
32 females) of 6 weeks old and weighing between 185 to Randox diagnostic kits following the principle described
200g were used for the investigation. The animals were by Neuman and Van Vreedenal, (1967). The assay is
kept in the laboratory under constant temperature based on the action of alkaline phosphatase (ALP) on
(244C) for at least one week before and throughout the phenyl phosphate to liberate phenol and phosphate.
experimental work. The animals were randomly divided The phenol released forms a chromogen with
into four groups of eight males and eight females each 4-aminoantipyrene in the presence of potassium
and housed in standard cages. The animals were allowed ferrocyanide that absorbs at 540nm.
for food and water ad libitum. Approval for animal Total Protein
studies was obtained from the Animal Ethics Committee Total serum protein was determined by the
of College of Health Sciences, Abia State University, Lowry method (1951). 1ml of each unknown protein
Uturu. solution (serum) was taken into 3ml of Biuret reagent.
Group one animals served as control, and The mixture was warmed for 15min at 37oC and allowed
received (0.5ml) of normal saline which is the vehicle for to cool. The absorbance of each test tube was read at
the extract, while groups two, three and four received 540nm.
20 mg/kg, 30 mg/kg and 40 mg/kg body weight of the Hemoglobin
plant extract respectively in 0.5ml sterile saline. The Hemoglobin was determined according to the
extract was administered once daily for 21 days. method described by Dacie and Lewis, 1990.
Collection of Blood Sample Lipid peroxidation
The animals were sacrificed under light Lipid peroxidation was determined by the
chloroform anesthesia, and blood collected from the method of Wallin et.al., (1993). Blood samples (0.5ml)
liver. The blood sample was centrifuged for 10min at were transferred to a test tube and 0.1ml of Triton X-100
3000rev. to obtain a clear supernatant (serum) which was was added and mixed properly. Next, 3ml of 30% TCA
stored at -20C until assayed for biochemical parameters. and 1ml of 0.6% TBA were added. The contents were
Biochemical effects shaken properly and the suspension was filtered. The
ALT and AST clear filtrate (3ml) was mixed with 2ml of 0.6% TBA
Assay of ALT and AST was done using Randox and heated for 15min in a boiling water bath. The
diagnostic kits. Determination was based on the principle mixture was allowed to cool and 2ml of chloroform was
described by Reitman and Frankel, (1957). The method added. The mixture was centrifuged at 10,000g for
is based on the determination of pyruvic acid as a 15min and the absorbance was read at 535nm. Lipid
product of the reaction catalyzed by alanine peroxidation products were extrapolated by a standard
aminotransferase or as a product of decarboxylation of curve.
the oxaloacetate produced by aspartate aminotransferase
Journal of Research in Biology (2012) 2(5): 410-417 412

Uhegbu et al., 2012
Statistical Analysis dose of the extract compared with the control (Table 1).
Data obtained were subjected to two-way The males had non-significantly (p0.01) higher alkaline
analysis of Variance (ANOVA) followed by Dennetts phosphatase activity compared to the females.
test to determine the statistical significance of the various Table 2 shows the variations in total protein level
changes in the parameters measured. SPSS software was in the test animals compared to control. Protein
used to analyze data. Values were considered significant concentration increased significantly (p0.05) compared
when p 0.01 to control. There is no significant difference (p0.05) for
the variation in protein concentration for the male and
RESULTS AND DISCUSSION female albino rats. The effect of the plant extract seems
ALT concentration significantly increased to be dose dependent as increase in protein concentration
(p0.01) in the test animals compared to control. is observed with increase in dose of extract administered.
(Table 1). There is also a non significant increase Hemoglobin concentration increased (p0.01)
(p0.01) in ALT concentration for the male animals significantly in the test animals compared to the control
compared to the female animals as the dose of (table 3). The increase is dose dependent. There was no
administered extract increased. The effect of the plant significant (p0.01) difference in hemoglobin
extract seems to be dose dependent as the enzyme concentration between the male and female test animals.
concentration increased with increase in dose of plant Lipid peroxidation levels decreased (p0.01)
extract administered. significantly in the test animals compared to the control.
Table 1 above also shows the variation in AST (Table 4). There are no significant (p0.01) difference in
concentration in the test animals compared to the control. values for the male and the female rats. As the dose of
A significant increase (p0.01) is observed in the administered extract increased there were significant
elevation of AST in test animals as the dose increased. (p0.01) reductions in lipid peroxidation values.
The effect of the plant extract seem to be dose dependent It is well known that the liver is the first target
as there was significant difference in enzyme organ in toxicological prospects regarding its role in
concentration as the dose increased. There were no detoxification, biotransformation and excretion of
significant (p0.01) differences in enzyme concentration xenobiotic. After enteric uptake of injurious materials, it
observed for the male and female test animals. is the first organ to be exposed to such hazards via the
The alkaline phosphatase activity in the test animals portal circulation (Ross and Kasum, 2002). The marker
increased (p0.01) non-significantly with increase in enzymes assayed are specifically located in some cell;
Table 1: Liver enzymes conc. (U/L)
Animal
Group 1 Group 2 Group 3 Group 4
(n=5)
Conc. of
extract
Control 20mg/kg 30mg/kg 40mg/kg
Sex M F M F M F M F
ALT 19.20.26 16.20.44 19.50.35 16.40.11 19.60.01 16.50.04 19.60.52 16.50.32
AST 16.30.05 13.30.21 16.40.25 13.50.13 16.50.06 13.50.71 16.50.04 13.60.46
ALP 23.40.06 20.50.29 23.60.02 20.60.16 23.60.24 20.60.93 23.50.16 20.70.46
*Values are mean SD of triplicate determinations M =Male, F=Female

Uhegbu et al., 2012
Table 2: Total Protein conc. (mg/dl)

Animal
(n=5)
Conc. of
extract
Sex M F M F M F M F
10.250.2 9.60.7 14.50.4 13.50.1 15.00.05 14.70.5 16.40.3 15.70.6
however, they can leak into the serum or other parts as a between 13.50.13 and 13.60.4 for females (Table 1).
result of injury to the cell where they are located The slight increase in AST concentrations seems to be
(Adesokan and Akanji, 2003). The measurement of the dose dependent, as elevations occurred as the plant
various enzyme activities in the tissues and body fluids extract dose increased. Normal activity of AST is
play a significant role in disease investigation, diagnosis 1-15u/l, but in this study AST elevation was observed to
and detection of tissue cellular damage (Malomo, 2000). be from 16.30.05u/l and 13.30.01u/l for male and
The ALT concentration was elevated in all the test female animals respectively between 16.4.00.25 and
animals (Table 1). The increase in ALT concentration 16.50.04 for males and between 13.50.13 and
were non-significant (p0.05) compared to the control. 13.60.04 for females administered the various
The effect of the plant extract also seems to be dose concentration of the plant extract. ALP showed
dependent, as the increase in plant extract concentration non-significant (p0.05) increase compared to control.
resulted in slight increase in enzyme concentration. Elevation of ALP is found in large number of disorders
There were significant (p0.05) differences in ALT such as biliary cirrhosis. Although it is found both in the
variation for the male and female animals, as dose of the liver and the bile, it leaks into the blood stream in a
plant extract increased. An elevated ALT level occurs in manner similar to that of ALT and AST. ALP is found in
cirrhosis, extra hepatic biliary obstruction and hepatic other organs such as bone, placenta and intestine
metases (Effraim et al., 2003). Inconsistent elevations (Ranjna, 1996).
also occur in acute myocardial infarctions and skeletal The plant leaf extract did not change
muscule disease. Normal activity of ALT is 1-20u/l, but significantly any of the liver enzymes including ALT,
in this study values ranged from 19.50.35u/l to AST and ALP. This result showed that the leaf extract of
19.60.52 for males and 16.40.11u/l to 16.50.03u/l for L .owerrience does not have any apparent toxicity on the
females liver of rats. Green leafy plants are usually associated
AST concentration was also non-significantly with hepatoprotective properties (Weber et al., 2003;
(p0.05) elevated from 16.30.05u/l and 13.30.01u/l Oboh, 2005; Roy et al., 2006, Iweala and Obidoa, 2010).
for the male and female control animals respectively,
between 16.40.25u/l and 16.50.04u/l for the males and
Table 3: Hemoglobin concentration (g/dl)
Animal
(n=5)
Conc. of
extract
Sex M F M F M F M F
9.80.50 9.50.41 11.70.54 11.00.31 12.11.05 12.41.12 12.80.73 12.50.65
Uhegbu et al., 2012
Table 4: Lipid peroxidation level [TBARS] (mg/ml)

Animal
(n=5)
Conc. of
extract
Sex M F M F M F M F
5.020.25 4.500.21 4.521.01 4.170.14 3.830.21 3.620.15 3.380.35 3.240.02
Total protein levels were also significantly (p0.01) in the test animals compared to control. (Table
elevated (p0.05) (Table 2) from 10.250.2mg/l and 4). The biological consequences of lipid peroxidation
9.60.7mg/l for the male and female animals include structural damage to membranes and essential
respectively between 14.50.4mg/l and 16.40.3mg/l for biomolecules. It is an underlying cause of degenerative
the males and between 13.50.1mg/l and 16.70.6mg/l diseases (Koba et al., 2002). In this study the plant
for the females. The effect of the plant extract is also extract had significant effect on lipid peroxidation and
dose dependent, and the increase in protein concentration may not alter essential functions of cells that may lead to
with increase in dose is significant (p0.05). Essentially, death (Hsieh et al., 2009). The reduction in lipid
all the albumin and fibrinogen of the plasma protein and peroxidation could be attributed to the presence of
about 50 to 80% of the globulins are formed in the liver. antioxidant phytochemicals including flavonoids and
The leaves of L. owerrience plant are rich in protein, anthocyanidins in the L. owerrience whose phenolic
essential amino acids and mineral elements (Ebi and structure favour their reaction with free radicals and
Ofoefule, 1997). The serum protein of the experimental reactive oxygen species (ROS) (Miller and Ruiz-Larrea,
rats was significantly increased which is a reflection of 2002). This reaction with ROS protects lipids from
the digestibility and hence availability of protein peroxidation and by extension prevents damage to cells.
co nst ituent s of leaves of L. owerrience The reduction of lipid peroxidation by flavonoid prevents
(Ross and Kasum, 2002).The elevation of serum protein DNA damage, a critical event in most disease. (Cai et al.,
could enhance the overall physiological function of the 1997; Subashree et al., 2009; Mohd-Esa et al., 2010;
animals (Iweala and Obidoa, 2010). MohanaSundaram et al., 2011).
Hemoglobin concentration significantly Ethno-medicine practitionersclaims that it is
increased (p0.05) in the animals (Table 3). The plant effective to use the plant extracts either crude aqueous
extract most probably had no adverse effect on the red extract or the alcoholic extract for the treatment and
blood cells. The phytochemical constituents of management of various diseases. They claim it as
L. owerrience which include flavonoids and phytosterol effective in treating and managing venereal disease, colic
(Iwu, 1993) are possible candidates that increase white disease, and control of diabetes and as enema for
blood cells. The mineral and vitamin contents of intestinal worms. The strong activity of the ethanolic
L. owerrience (Ebi and Ofoefule, 1997) include extracts against known etiologic agents of diseases
haematinic factors such as iron which play a major role traditionally treated with L. owerrience root of similar
in the synthesis of haemoglobin (Alada, 2000; Tindal, preparations provides scientific justification for the use
1965). This could explain the increase in haemoglobin of the herb in ethnomedical practice in Nigeria.
seen in the experimental rats.
Lipid peroxidation levels significantly decreased
Uhegbu et al., 2012
ACKNOWLEDGEMENT Hsieh YS, Kuo WH, Lin TW, Chang HR, Lin TH,
I wish to thank Mr. Uche Arukwe and Chen PN and Chu SC. 2009. Protective effects of
Mr. Clement Asumugha of the Biochemistry Berberine against Low-Density Lipoprotein (LDL)
Laboratories, Department of Biochemistry, Abia State oxidation and oxidized LDL- Induced cytotoxicity on
University, Uturu, Nigeria, who helped in some of the endothelial cells. J Agric Food Chem., 55:10437-1044
analysis.
Hwang JM, Wang CJ, Chou FP, Tseng TH, Hsieh
YS, Lin WL and Chu CY. 2002. Inhibitory effect of
REFERENCES
berberine on tert-butyl hydroperoxide-induced oxidative
Adesokan AA and Akanji MA. 2003. Effect of
damage in rat liver. Arch Toxicol., 76:664-670.
administration of aqueous extract of Enantia chlorantha
on the activities of some enzymes in the small intestine Iweala EEJ and Obidoa O. 2010. Studies on Some
of rats. Nigeria J. Biochem. Molec. Biol., 18(2):103-105. Biochemical and Histological Changes Associated with
Lo ng Ter m Co nsu mpt io n of Leaves of
Alada ARA. 2000. The haematological effect of
Ocimum gratissimum L. in Male Rats. Am. J. Food
Telfairia occidentalis diet preparation. Afr. J.Biomed.
Technol., 5:376-384
Res: 185-186.
Iwu MM. 1993. Handbook of African Medicinal Plants.
Burkill HM. 1994. The useful plants of tropical West
CRC Press, Boca Raton, Ann. Arbor and London,
Africa. (2nd edition). Royal Botanic Gardens, Kew,
Tokyo.
ISBN: 0947643567
Jensen JE and Freese D. 2009. Liver Function Tests.
Cai Q, Rahan RO and Zang R. 1997. Dietary
Colorado Center for Digestive disorders 205s.Suite A
flavonoids, quercetin, luteolin and genistein, reduce
Longmont Co. 80501. 151-163.
DNA damage and lipid peroxidation and quench free
radicals. Cancer Lett. 119:99-108 Koba K, Akahoshi A, Yamasaki M, Tanaka K,
Yamada K, Iwata T, Kamegai T, Tsutsumi K and
Chang JS, Chiang LC, Hsu FF and Lin CC. 2004.
Sugano M. 2002. Dietary conjugated linolenic acid in
Chemoprevention against hepatocellular Carcinoma of
relation to CLA differently modifies body fat mass and
Comus officinalis - invitro. Am. J. Chin. Med.,
serum and liver lipid levels in rats. Lipids, 37:343-350.
32(5):717-725
Lehninger AL, Nelson OL and Cox MM. 2005. Text
Dacie JV and Lewis SM. 1990. Practical Hematology.
Book of Biochemistry. (6th edition) Worth Publishers.
Churchill Livingstone New York 34.
NY USA 450.
Ebi GC and Ofoefule SI. 1997. Investigation into
Lowry OH, Rosebrough NJ, Farr AL and Randall
t he fo lk lo r ic ant im icr o bia l act iv it ie s of
RJ. 1951. Protein measurement with the folin phenol
Landolphia owerrience. Phyt. Res., 11:149-151.
reagent. J. Biol. Chem., 193:265-275
Effraim KD, Jacks TW and Sodipo OA. 2003.
Malomo SO. 2000. Toxicological implication of
Histopathological studies on the toxicity of
Ceftriaxome administration in rats. Nig. J. Biochem. Mol.
Ocimum gratissimum on some organs of Rabbits: African
Biol., 15(1):33-35
J. Biomed., Res. 6:21-25.

Uhegbu et al., 2012
Miller NJ and Ruiz-Larrea MB. 2002. Flavonoid and Ross JA and Kasum CM. 2002. Dietary flavonoids:
other plant phenols in diet: Their significance as Bioavailability, metabolic effects and safety. Ann. Rev.
antioxidants. J. Nutr. Environ. Med., 12:39-51. Nutr., 22:19-34
Mohana Sundaram, Sivakumar Karthikeyan, Roy CK, Kamath JV and Asad M. 2006.
Magesh Gopinath, Sheeladevi Thirumalai, and Hepatoprotective activity of Psidium guajava Linn leaf
Pennarasi Prasanna. 2011. Studies on phytochemicals, extract. Ind. J. Exp. Biol., 44:305-311
antibacterial and Antioxidant effects of leaf extracts of
Subashree B, Baskar R, Laxmi KR, Lijina SR and
lamprachaenium microcephalum.. Journal of Research in
Rajasekaran P. 2009. Evaluation of antioxidant
Biology 4: 279-284.
potential in selected green leafy vegetables. Food Chem.,
Mohd-Esa N, Hern FS, Ismail A and Yee CL. 2010. 115:1213-1220.
Antioxidant activity in different parts of roselle
Tindal HD. 1965. Fruits and vegetables of West Africa.
(Hibiscus sabdariffa) extracts and potential exploitation
(1st Edition). Food and Agriculture Organization, USA
of their seeds. Food Chem., 122:1055-1060.
Wallin B, Rosengren B, Shertzer HG and Camejo G.
Neuman HV and Vreedenal M. 1967. An improved
1993. Lipoprotein oxidation and measurement of
alkaline phosphatase determination with p-nitrophenol
thiobarbituric acid reacting substances formation in a
phosphate. Clinica Chimica Acta.17(2):183-187.
single microtiter plate: its use for evaluation of
Oboh G. 2005. Hepatoprotective property of ethanolic antioxidants Anal. Biochem., 208:10-15.
and aqueous extracts of Telfairia occidentalis (Fluted
Weber LW, Boll M and Stampfl A. 2003.
pumpkin) leaves against garlic-induced oxidative stress.
Hepatotoxicity and mechanism of action of
J. Med. Food 8:560-563
haloalkanes: carbon tetrachloride as a toxicological
Okeke MI, Iroegbu CU, Eze EN, Okoli AS and model. Crit. Rev. Toxicol., 23:105-136.
Esimone CO. 2001. Evaluation of extracts of the root of
Landolphia owerrience for antibacterial activity.
J. Ethnopharmacol., 78(2-3):119-27
Person JGM, Dilst FJH, Kuijpers RP, Leenwerberg

AJM, Vonk GJA and Van-Dilst FJH. 1962. The
African species of Landolphia. P.Beaux Series of
Revisions of Apocynaceae. Xxxiv Wageningen Submit your articles online at jresearchbiology.com
Agricultural University Paper No.92. 323. Advantages

Easy online submission
Ranjna O. 1996. Practical Clinical Biochemistry, Complete Peer review
Affordable Charges
Methods and Interpretations (2nd edition) Jaypee Quick processing
Brothers Medical Publishers. India 100-109 Extensive indexing
You retain your copyright
Reitman S and Frankel SL. 1957. Determination of submit@jresearchbiology.com
ALT and AST concentrations in serum. Am. J. Chem www.jresearchbiology.com/Submit.php.
Path., 28:56
Original Research
Effect of starvation period on growth performances and

survival of Cyprinus carpio (Linnaeus, 1758)
Authors: ABSTRACT:
Roya Assareh1, Majid
Mohammad Nejad Starvation is one of the problems that the fish may encounter in course of its
Shamoushaki 2, Hamid life. This study was conducted for six weeks at two groups and three replications as
Faghani Langroodi3 and follows: Group A: Starvation in six weeks and Group B: Feeding in six weeks with three
Ali Karbakhsh Ravari4. times a day at 08:00, 12:00 and 16:00 h. Feeding rate was equal to 10 % of body
weight for group A. The physical and chemical factors were so controlled through the
Institution:
1and 3.Department of experiment that the amount of dissolved oxygen was fixed up to 6 ppm, the
Fishery, Tonekabon Branch, temperature 28 2C and pH 7.8 to 8.1. Results showed significant effects in Specific
Islamic Azad University, Growth Rate (SGR), Body Weight Index (BWI %), Growth Rate (GR), Feed Conversion
Tonekabon, Iran. Ratio (FCR), Condition Factor (CF) and survival (P<0.05). The results showed that there
are significant differences with regard to the amount of body weight and body length
2 and 4. Department of of fingerlings in two groups (P<0.05). Result of this study showed that increasing of
Fishery, Bandar Gaz Branch, starvation period decrease growth and survival in Cyprinus carpio.
Islamic Azad University,
Bandar Gaz, Iran.

Roya Asareh. Cyprinus carpio, growth performances, survival, starvation.

aramiis_dream@yahoo.com. Roya Assareh, Majid Mohammad Nejad Shamoushaki , Hamid Faghani Langroodi and
Ali Karbakhsh Ravari.
Effect of starvation period on growth performances and survival of Cyprinus carpio
Web Address: (Linnaeus, 1758)
http://jresearchbiology.com/ Journal of research in Biology (2012) 2(5): 418-423
documents/RA0250.pdf.
Dates:
Received: 26 May 2012 Accepted: 01 Jun 2012 Published: 25 Jun 2012
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
418-423 | JRB | 2012 | Vol 2 | No 5

An International
Assareh et al., 2012
INTRODUCTION invertebrates, investigations on larval starvation or food

Understanding of natural foods and dietary limitation have mainly been on crustaceans (e.g., Paul
habits of fish culture could be an important factor in and Paul, 1980; Olson and Olson, 1989; Qiu and Qian,
providing effective method of nutrition (Mohammad 1997). There have also been many studies on mollusks
Nejad Shamoushaki, 2012). Starvation is one of the (Roberts et al., 2001; Takami et al., 2002; Moran and
problems that the fish may encountered in course of its Manahan, 2004), polychaetes (McEdward and Qian,
life. Starvation is an experienced and endured situation 2001; Pechenik and Cerulii, 1991) and echinoderm
by many fish species in the natural environment (Larsson larvae (Fenaux et al., 1994; Olson and Olson, 1989). In
and Lewander, 1973; McLeese and Moon, 1989; Navarro contrast, few studies investigated in vertebrates,
and Gutirrez, 1995; Olivereau and Olivereau, 1997; especially fish. However, the effect of experimental
Blanger et al., 2002; Furn et al., 2008). The condition starvation on red sea bream (Pagrosomus major),
of fish may affect the probability of their endurance since Japanese flounder (Paralichalmus olivaceus), Chinese
fish are more vulnerable to predation, disease and sturgeon (Acipenser sinensis) and yellow sea bream
unfavorable environmental conditions in poor condition. (Dentex tumifrons) larvae were investigated recently
In addition, they are less efficient in foraging (Amara (Bao et al., 1998; Zhuang et al., 1999; Xie et al., 2004;
and Galois, 2004; (Islam and Tanaka, 2005). Fish Sheng et al., 2007).
alternate periods of feeding and fasting in response to Cyprinus carpio belongs to Cyprinidae is one of
several factors (such as temperature, spawning the most economically important and valuable telostei in
migration, reproduction, etc., in their natural the Caspian Sea. No paper has described the effect of
environment. Cultured fish also experience such starvation or limited food on the growth and survival of
situations derived from these same factors and also Cyprinus carpio. Up to now, the effect of starvation on
imposed by routine aquaculture procedures. To the Cyprinus carpio has not been reported, but it is
overcome these food restrictions, fish mobilize their necessary and important for the aquaculture of these
energy reserves, which impose metabolic adjustments species. Therefore, the present study aimed to examine
that are species dependent. Intraspecific adjustments to the effect of starvation on growth performances and
these conditions also depend on different factors as fish survival of Cyprinus carpio. In this study, we examined
age, nutritional state, etc. (Navarro and Gutirrez, 1995; the effect of prolonged starvation on growth
Prez-Jimnez et al., 2007). In the wild, fish may have a performances and survival in the Cyprinus carpio.
mortality rate despondent on predation, starvation or
limited food resources and oceanographic conditions that MATERIALS AND METHODS
may transport fish to an unfavorable environment (Olson Fish supply and experimental design
and Olson, 1989; Anil et al., 1995; Alexander and This study has been carried out in Sijual bony
Roughgarden, 1996; Anil and Dattesh, 1997). Due to the fishes reproduce and cultivate center (Gorgan, Golestan,
spatial and temporal patchiness of food in nature, periods Iran) on 2011 summer. This study was operated as long
of food deprivation are common in the lives of many as six weeks and two groups with 90 numbers of fish in
animals (Mehner and Wieser, 1994). Since the rate of each group and three replicate as: Group A: Starvation
mortality of fish exists in natural conditions because of in six weeks and Group B: Feeding in six weeks with
poor endurance of starvation, many investigations have three times a day at 08:00, 12:00 and 16:00 h. Initial
focused on fish starvation or food limitation. In marine body weight and length average of Cyprinus carpio were
3.2430.132 gr and 5.80.303 cm respectively. Fishes in 2010; Mohammad Nejad Shamoushaki, 2012):
group A were fed SFC (starting food carp) commercial FCR= Total feed intake/ total biomass gain
food during (six week) of the study. The feed was SGR= [(In final weightln initial weight)/ rearing
administered only during daylight hours. Pertinent duration in days] 100
characteristics of this feed were: 32% crude protein; BWI= [(Body weight finalbody weight initial)/ body
10.5% crude fat; 11.2% ash and 8.7% moisture. Given weight initial] 100
the importance of the physical and chemical factors and GR= (Body weight finalbody weight initial)/ rearing
their impact on water supply and ultimately the fish duration in days
growth, these factors were so controlled through the BWI= [(Body weight /total length 3)] 100
experiment that the amount of dissolved oxygen was Survival= (Number of fish harvested/number of fish
fixed up to 6 ppm, the temperature 282C and stocked) 100
pH 7.8 to 8.1. Fish were sampled every week to evaluate Statistical analysis
growth in weight and length, for this purpose 10 numbers For all data analysis SPSS version 13 was used
of fish in each replicate were captured, weighed and and for drawing graphs. Excel 2003 was used. All data
measured. After each sampling period the amount of feed were ensured to normality with the Shapiro-wilk test.
given was adjusted according to mean weight in each Normal distribution of data using one-way analysis of
aquarium (Only for group B). Feeding rate which paid variance test (Oneway ANOVA) in the overall
attention to live weight and in different times and after differences between groups were determined at 95%
each biometry, equal 10 % of body weight is calculated confidence level.
and was intered to each aquariums of group B.
Growth performances assay RESULTS AND DISCUSSION
From results of the last sample fish performances Final weight and length of Cyprinus carpio in
were evaluated in terms of Feed Conversion Ratio two groups were shown in figures 1 and 2. The results
(FCR), Specific Growth Rate (SGR, gr d-1), Body showed that starvation and feeding have effects on
Weight Index (BWI %), Growth Rate (GR, gr d-1), increasing body weight and body length of
Condition Factor (CF, gr/Cm) and Survival (%). These Cyprinus carpio and there is a need full difference in this
performance indices were calculated as follows respect among two groups (p < 0.05). There were
(Hung et al., 1989; Ronyai et al., 1990; Biswas et al., significant difference among the starvation and feeding
Figure 1.The average of body weight of Figure 2.The average of body lenght of
Cyprinus carpio in two groups Cyprinus carpio in two groups
The Latin letters show that there are significant The Latin letters show that there are significant
differences among two groups differences among two groups

groups of Cyprinus carpio, and the body length in solution to this problem. Therefore, one way to
feeding group was the higher than starvation group. encounter starvation in fish is compensatory growth
Body weight in starvation group was less than the (not investigated in this study). Also, it should be
feeding group. Obtained results in this study showed that considered the mortality will increase if enough food is
increasing of starvation period decrease body weight and not provided after starvation. McEdward and Qian
body length but feeding increase body weight and body (2001) also suggested that starvation reduces growth
length of Cyprinus carpio. rates largely by increasing the duration of the fish period
Comparison of average growth performances on relative to the opportunities for acquiring food. Some
C. carpio growth during test period are shown in table 1. studies have reported the recovered growth rates of fish
Results showed that had significant effects in BWI, SGR, following short periods of starvation. Growth rates for
GR, FCR, CF and survival in two groups (P < 0.05) starvation groups of prosobranch gastropod (Crepidula
(Group A has not been feeding fish because the FCR was fomicata) appear to bounce when food was resumed
calculated for it). In the present research, the result (Pechenik et al., 2002). McEdward and Qian (2001)
showed that starvation period for Cyprinus carpio could suggested four possible explanations. Firstly, they
also influence their growth performances, and during the thought that larvae might grow efficiently on egg
periods of 1-6 weeks of starvation, the growth greatly reserve. Therefore, they will be larger and have a greater
reduced (Table 1). The survival in starvation group with capacity to feed when food is available compared to the
an increase of starvation period was significantly lower larvae offered food postpartum. Secondly, early
from that of the feeding group (Table 1). starvation in life might cause significant mortality and
The survey results showed that starvation select for very hardy larvae that have inherently greater
strongly affects the growth and survival of fish. capacity for feeding and growth. Thirdly, larvae might
Moreover, fish mortality will be increased during respond by feeding more vigorously (either at higher
starvation. Also, results showed that with increasing rates or more continuously, i.e. compensatory growth)
duration of starvation mortality have increased and fishes after initial starvation; therefore, acquire more food
were extremely thin and if the fish do not find to get during the same potential feeding period. Finally, fish
enough food may increase the mortality and must find a might respond to food density by altering the allocation
of resources between growth and development
Table1. Comparison of growth performances (Sheng et al., 2007).
in two groups at six weeks
Growth CONCLUSION
performances
Starvation Feeding
In conclusion, this investigation demonstrates that
FCR - 4.151.214 starvation affects the growth and survival rate of the
SGR (gr d1) -0.1950.0156b 0.8040.028a Cyprinus carpio. These results can help the conservators
b a
BWI (%) -7.763.0.588 40.181.677 of Cyprinus carpio to decide when they should release
b a
GR (gr d1) -0.00610.00046 0.0110.004 (Cyprinus carpio) to the sea. In addition, these results
b a
CF (gr/Cm) 1.60.062 1.0530.021 indicate the farmers' knowledge of feeding status of
Survival (%) 51.672.887b 88.332.7a Cyprinus carpio. However, we did not investigate the
The small Latin letters show that there are effects of compensatory growth on the Cyprinus carpio
significant differences among different treatments in this study and it is a part of our future work.

ACKNOWLEDGEMENTS 305:26-31.
This research was supported by the Sijual bony
Fenaux L, Strathmann MF and Strathmann RR.
fishes reproduce and cultivate center (Gorgan, Golestan,
1994. Five tests of food-limited growth of larvae in
Iran). We also thank Mr.Jabbare, Mr.Maleki,
coastal waters by comparisons of rates of development
Mr.Shakiba, Mr.Eree and Mr.Sancholi for their valuable
and form of echinoplutei. Limnol. Oceanogr. 39:84-89.
helps.
Furn M, Garca-Gallego M, Hidalgo MC, Morales
REFERENCES AE, Domezain A, Domezain J and Sanz A. 2008.
Alexander SE and Roughgarden J. 1996. Larval Effect of starvation and refeeding on digestive enzyme
transport and population dynamics of intertidal activities in sturgeon (Acipenser naccarii) and trout
barnacles: a coupled benthic/oceanic model. Ecol. (Oncorhynchus mykiss). Comparative Biochemistry and
Monogr, 66(3):259-275. Physiology, Part A, 149:420-425.
Amara R and Galois R. 2004. Nutritional condition of Hung SSO, Lutes PB and Storebakken T. 1989.
metamorphosing sole: spatial and temporal analyses. Growth and feed efficiency of white sturgeon (Acipenser
J. Fish Biol., 64:72-88. transmontanus) sub yearling at different feeding rates.
Aquaculture 80:147-153.
Anil AC, Chiba K, Okamoto K and Kurokura K.
1995. Influence of temperature and salinity on the larval Islam Md S and Tanaka M. 2005. Nutritional
development of Balanus amphitrite: implications in the condition, starvation status and growth of early juvenile
fouling ecology. Mar. Ecol., Prog. Ser, 118:159-166. Japanese sea bass (Lateolabrax japonicus) related to prey
distribution and feeding in the nursery ground. Journal of
Anil AC and Dattesh D. 1997. Starvation threshold of
Experimental Marine Biology and Ecology 323:172-183.
Balanus amphitrite larvae in relation to temperature.
Emerging Non-Metallic Materials for the Marine Larsson A and Lewander K. 1973. Metabolic effects of
Environment. Proceedings of US- Pacific Rim workshop, starvation in the eel, Anguilla anguilla L. Comp.
Hawaii, USA, section P, US Office of Naval Research Biochem. Physiol. A, 44:367-374.
(ONR). Asia Office, Tokyo, Japan: 12-23.
McLeese JM and Moon TW. 1989. Seasonal changes
Bao BL, Su JX and Yin MC. 1998. Effect of delayed in the intestinal mucosa for winter flounder,
feeding on feeding ability, survival and growth of red sea Pseudopleuronectes americanus (Walbaum), from
bream and olive flounder larvae during early Passamaquoddy Bay, New Brunswick. J. Fish Biol.,
development. J. Fish. Chin. 22(1):3338 (In Chinese). 35:381-393.
Blanger F, Blier PU and Dutil JD. 2002. Digestive McEdward LR, Qian PY. 2001. Effects of the duration
capacity compensatory growth in Atlantic cod and timing of starvation during larval life on the
(Gadus morhua). Fish Physiol. Biochem., 26:121-128. metamorphosis and initial juvenile size of the polychaete
Hydroides elegans (Haswell). J. Exp. Mar. Biol. Ecol.
Biswas G, Thirunavukkarasu AR, Sundaray JK and
261:185-197.
Kailasam M. 2010. Optimization of feeding frequency
of Asian seabass (Lates calcarifer) fry reared in net Mehner T and Wieser W. 1994. Energetic and
cages under brackishwater environment. Aquaculture, metabolic correlates of starvation in juvenile perch

(Perca fluviatilis). J. Fish Biol., 45:325-333. Roberts RD, Lapworth C and Barker RJ. 2001. Effect
of starvation on the growth and survival of post-larval
Mohammad Nejad Shamoushaki M. 2012. Effect of
abalone (Haliotis iris). Aquaculture 200:323-338.
feeding frequency on growth performances and survival
of Rutilus rutilus caspicus. Journal of research in Biology Ronyai A, Peteri A and Radics F. 1990. Cross breeding
2(3):184-189. of starlet and Lena river sturgeon. Aquaculture 6:13-18.
Navarro I and Gutirrez J. 1995. Fasting and Sheng J, Lin Q, Chen Q, Shen L and Lu J. 2007.
starvation. In: Hochachka, P.W., Mommsen, T.P. (Eds.), Effect of starvation on the initiation of feeding, growth
Biochemistry and Molecular Biology of Fishes. and survival rate of juvenile seahorses,
Metabolic Biochemistry, vol. 4. Elsevier, Amsterdam, Hippocampus trimaculatus Leach and Hippocampus
393-434. kuda Bleeker. Aquaculture 271:469-478.
Olivereau M and Olivereau JM. 1997. Long-term Xie S and Yang Y. 2004. Compensatory growth and
starvation in the European eel: general effects and food consumption in gibel carp, Carassius auratu
responses of pituitary growth hormone-(GH) and sgibelio, and chinese long snout catfish,
somatostatin-(SL) secreting cells. Fish Physiol. Leiocassis longirostris experiencingcycles of feed
Biochem., 17:261-269. deprivation and refeeding. Aquaculture 241:235-247.
Olson RR and Olson MH. 1989. Food limitation of Zhuang P, Zhang LZ, Zhang T, Zhang Z and Cao
planktrophic marine invertebrate larvae: does it control WX. 1999. Effects of delaying first feeding time on the
recruitment success?Ann. Rev. Ecolog. Syst, survival and growth of larval Chinese sturgeon,
20:225-247. Acipenser sinensis. Acta Hydrobiol. Sin. 23 (6):560-565
(In Chinese).
Paul AJ and Paul JM. 1980. The effects of early
starvation on later feeding success of king crab zoeae. J.
Exp. Mar. Biol. Ecol., 44:247-251.
Pechenik JA and Cerulii T. 1991. Influence of delayed

metamorphosis on survival, growth, and reproduction of
the marine polychaete Capitella sp. I. J. Exp. Mar. Biol.
Ecol., 151:17-27.
Prez-Jimnez A, Guedes MJ. Morales AE and Oliva- Submit your articles online at jresearchbiology.com
Teles A. 2007. Metabolic responses to short starvation
Advantages
and refeeding in Dicentrarchus labrax. Effect of dietary Easy online submission
composition. Aquaculture 265: 325-335. Complete Peer review
Affordable Charges
Qiu JW and Qian PY. 1997. Combined effects of Quick processing
Extensive indexing
salinity, temperature, and food concentration on the early You retain your copyright
development of the polychaete Hydroides elegans submit@jresearchbiology.com
(Haswell, 1883). Mar. Ecol., Prog. Ser, 152:79-88. www.jresearchbiology.com/Submit.php.

Original Research
Physical and chemical characteristics of Paraliyar river in

Kanyakumari district, Tamil Nadu, India
Authors: ABSTRACT:
Dyona L and Stella
Roslin A.
The physico-chemical parameters of the Paraliyar River in Kanyakumari
District, Tamilnadu were studied for a period of six months from July to December
2010. The pH of the water varied from 5.62 and 8.67. The dissolved Oxygen content of
Institution:
the river water ranges between 3.57 and 7.20. The nutrients such as Nitrate, Nitrite,
Department of Plant Biology
Phosphate, Calcium and Magnesium are also showed temporal variations. The
and Plant Biotechnology
Holy Cross College, Alkalinity and acidity of the river water showed the maximum value of
Nagercoil-4. 152 mg/l, 132mg/l and 292mg/l, 198mg/l respectively. In general, all the physical and
chemical parameters of the river water showed strong temporal variations in relation
to sampling months.

Stella Roslin A. water quality, hydrology, river ecosystem; western Ghats, water pollution.

asroslin@gmail.com. Dyona L and Stella Roslin A.
Physical and Chemical characteristics of Paraliyar river in Kanyakumari district,
Tamil Nadu, India.
Web Address: Journal of Research in Biology (2012) 2(5): 424-438
http://jresearchbiology.com/
documents/RA0221.pdf. Dates:
Received: 23 Mar 2012 Accepted: 22 Apr 2012 Published: 27 Jun 2012

424-0438 | JRB | 2012 | Vol 2 | No 5

An International
Dyona and Roslin, 2012
INTRODUCTION present study an attempt has been made to analyse the

In India ponds, rivers and ground water are used physical and chemical parameters of the Paraliyar river
for domestic and agricultural purposes. The quality of in Kanyakumari District, Tamilnadu.
water may be described according to their
physico - chemical and micro-biological characteristics. MATERIALS AND METHODS
For effective maintenance of water quality through The present study was carried out at selected
appropriate control measures, continuous monitoring of locations of Paraliyar river. The Paraliyar rises in the
large number of quality parameters are essential Mountain north of Mahendrahiri and flows generally in
(Bhandari and Nayal, 2008). The usual source of the south, south-westerly direction through Kalkulam
drinking water is from streams, rivers, wells and Vilavancode taluks of Kanyakumari district. Near
and boreholes which are usually not treated Ponmana, it is interouted by Perunchanidam. It receives
(Agbaire and Obi, 2009). water from Pechiparai reservoir through the left bank
Rivers are subjected to various natural processes channel, before the weir called Puthandam. Six stations
taking place in the environment, such as the hydrological Movattumukku (station I), Puthan Dam (station II),
cycle. As a consequence of unprecedented development, Villukuri station III), Aloor(Alanvilai) (station IV),
human beings are responsible for choking several lakes Vellamody (station V), Pillaithoppu (stationVI) were
to death. Storm water runoff and discharge of sewage selected for the collection of water samples. The water
into rivers are two common ways that various nutrients samples were collected at monthly intervals during early
enter the aquatic ecosystems resulting in the pollution hours of the day for a period of six months from July to
(Sudhira and Kumar, 2000; Adeyemo, 2003; December 2010.
Adeyemo et al., 2008). The pH of water sample was measured with a pH
Water quality deals with the physical, chemical meter previously calibrated with buffer solutions.
and biological characteristics in relation to all other Conductivity was measured with a conductivity meter
hydrological properties. Any characteristic of water that calibrated with potassium chloride solution. Temperature
effects the survival, reproduction, growth and production was measured with a thermometer. Alkalinity was
of aquaculture species, influences management decision, determined by titrating a known volume of water sample
causes environmental impacts or reduces product quality with 0.02 M HCl. Dissolved oxygen (DO) was
and safety can be considered a water quality variable determined by Winklers titration. Total dissolved solid
(Iqbal et al., 2004). Water quality provides current (TDS) was determined gravimetrically by evaporating a
information about the concentration of various solutes at known volume of water to dryness in a pre-weighed
a given place and time. Water quality parameters provide crucible on a steam bath. Total hardness was determined
the basis for judging the suitability of water for its by titrating with EDTA using Erichrome black T as
designated uses and to improve existing conditions. indicator. The salinity was determined using a
Rivers and lakes are very important part of our natural Refractometer (ATAGO). Flame photometer (Model
heritage. They have been widely utilized by mankind Systronic 128) was used for determination of metal ions
over the centuries, to the extent that very few; if any are Na+, K+ and Ca2+. Silver nitrate method was used to
now in a natural condition (Leonard, 1971). A estimate the chloride present in water samples. Sulphate
continuous monitoring of water quality is very essential was determined by turbidimetric method. Total hardness
to determine the state of pollution in our rivers. In the was calculated by complexometric titration using EDTA
(Vogel, 1978). Nesslers method was used for ammonia, from 25.4 C to 29.8C at station I. The lowest value was
brucine method was used for nitrate, nitrite and observed in July and the highest temperature was during
nitrate-nitrogen, reactive phosphate (by using October. At station II, the temperature was varied
spectrophotometric methods) and calcium ion content between 25.8C and 29.4C. The temperature was high
(by using titrimetric methods) were measured according during October and low in July at station II. The
to A.P.H.A (1998). temperature of the water samples collected from the
station III, varied from 25.6 (July) to 29.8 (December).
RESULTS At station IV, the temperature of the water samples
pH ranged between 25.6 and 29.6. The lower value was
The pH of the water samples varied from 5.65 to observed in July and higher during October. Temperature
8.64 at Station 1. The lowest value was observed in July value ranged between 25.8 and 28.9 were, observed at
and the highest pH was recorded during October. At station V. The lowest value was observed in July and
station II, the pH was varied between 5.62 and 8.29. The highest value was in October. The temperature of the
pH was high during October and low in July at Station II. water samples collected from station VI showed a
The pH of the water samples collected from the Station maximum temperature value of 29.4 (December) and
III, varied from 5.92 (July) to 8.67 (November). At minimum of 25.3 during July. In general, the temperature
station IV, the pH of the water samples ranged between of the water samples showed lower values in July and
5.72 and 8.54. The lower value was observed in July and higher values in October (Fig 2).
higher was during December. pH values ranged between EC (Electrical Conductivity)
5.68 and 8.27 were observed at Station V. The lowest The EC of the water samples varied from
value was observed in July and the highest value was 1.205 S to 3.502 S at station I. The lowest value was
observed in December. The pH of the water samples observed in October and the highest value was during
collected from station VI showed a Maximum pH value December. EC value ranged between 1.204 S and 3.602
of 8.64 (December) and minimum of 5.85 during July. In S were observed at station II. The lowest value was
general, the pH of the water samples showed lower observed in July and highest value was observed in
values in July (Fig 1). December. At station III, the EC was varied between
Temperature 1.206 S and 2.911 S. The EC value was high during
The temperature of the water samples varied December and low in October. The EC of the water
Fig2.Surface water temperature (oC) of the water

Fig 1. pH of the water samples collected from samples collected from the river Paraliyar during the
six selected stations of river Paraliyar. study period.

samples collected from station IV, showed a maximum Dissolved Oxygen

value of 3.234 S (December) and minimum of 1.205 S The DO of the water samples varied between
during October. At station V, the EC of the water 3.98 mg/l and 5.18 mg/l at station I. The lowest value
samples ranged between 1.204 S and 3.234 S. The was observed in July and highest value was observed in
low value was observed in October and high during October. The DO of the water samples collected from the
December. The EC of the water samples collected from station II, varied from 3.57 mg/l (July) to 5.06 mg/l
the station VI, varied from 1.573 S (July) to 3.921 S (October). At station III, the DO of the water samples
(December). In general, the EC of the water samples varied from 4.28 mg/l to 5.24 mg/l. The DO was high
showed lower values in October and higher values in during October and low in July. DO values ranged
December (Fig 3). between 4.52 mg/l and 6.54 mg/l were observed in
Total Dissolved Solids station IV. The DO was high during October and low in
At station I, the TDS of the water samples varied September. At station V, the DO was varied between
from 0.602 ppm to 0.745 ppm. The TDS value was high 4.59 mg/l and 5.22 mg/l. The DO was high during
during September and low in July. The TDS of the water October and low in September. At station V, the DO of
samples varied between 0.654 ppm and 0.798 ppm at the water samples showed a maximum value of
station II. The lowest value was observed in July and the 7.20 mg/l (October), minimum value of 4.27 mg/l during
highest value was during December.TDS value ranged July. In general, the DO of the water samples showed
between 0.611ppm and 0.755 ppm was observed in lower values in July (Fig 5).
station III. The TDS value was high during December Alkalinity
and low in August. At station IV, the TDS was varied The alkalinity of the water samples collected
between 0.608 ppm and 0.784 ppm . The TDS was high from station I, varied from 154 mg/l (November) to
during August and low in July. The TDS of the water 292 mg/l (December). At station II, the alkalinity of the
samples collected from the station V, varied from water samples varied from 162 mg/l to 282 mg/l. The
0.664ppm (July) to 0.792 ppm (September) At station alkalinity value was high during August and low in
VI, the TDS of the water samples showed a maximum October. The alkalinity of the water samples varied
value of 0.791ppm in November and minimum value of between 174 mg/l and 283 mg/l at station III. The lowest
0.638 ppm during December. In general the TDS of the value was observed in November and the highest value
water samples showed lower values in July. (Fig 4). was during December. At station IV, the alkalinity of the
Fig 3. Electrical conductivity (S) values observed in Fig 4. Total Dissolved Solids (ppm) values observed
Paraliyar river from July 2010 to December 2010. in Paraliyar river from July 2010 to December 2010.

water samples showed a maximum value of the acidity, of the water samples was low in October and
281 mg/l (December) and minimum value of 152 mg/l high during July (Fig 7).
during (October). Alkalinity value ranged between 162 Ammonia
mg/l and 277 mg/l were observed in system V. The At station I the ammonia of the water samples
alkalinity value was high during December and low in varied from 0.54 mg/l to 1.98 mg/l. The ammonia was
November. At station VI, the alkalinity was varied high during November and low in August. The ammonia
between 154 mg/l and 265 mg/l. The alkalinity was high of the water samples varied between 0.62 mg/l and
during August and low in November. In general, the 1.82 mg/l at station II. The lowest value was observed in
alkalinity of the water samples, showed lower values in August and highest value was observed in November.
November and higher values in December (Fig 6). The ammonia of the water samples collected from the
Acidity station III, Varied from 0.69 mg/l (August) to 1.92 mg/l
At station I, the acidity of the water samples (November). At station IV, the ammonia was varied
showed a maximum value of 187 mg/l (July) and between 0.47 mg/l and 1.45 mg/l. The ammonia was
minimum value of 134 mg/l during October. The acidity high during November and low in August. At station V,
of the water samples collected from station II, varied the ammonia of the water samples, showed a maximum
from 136 mg/l (October) to 177 mg/l (November). The value of 1.79 mg/l (September) and minimum value of
acidity of the water samples varied between 0.59 mg/l (August). Ammonia value ranged between
132 mg/l and 198 mg/l at station III. The lowest value 0.35 mg/l and 1.64 mg/l were observed in station VI. The
was observed in October and the highest value was ammonia concentration was high during November and
observed in July. At station IV, the acidity of the water low in October. In general, the ammonia concentration of
samples varied from 131 mg/l to 191 mg/l. the acidity the water samples showed lower values in August and
value was high during July and low in October. At higher value in November (Fig 8).
station V, the acidity was varied between 138 mg/l and Total Hardness
176 mg/l. The acidity was high during July and low in The total hardness of the water samples varied
October. Acidity value ranged between 135 mg/l and 181 between 238 mg/l and 547 mg/l at station I. The lowest
mg/l were observed in station VI. The acidity value was value was observed in July and highest value was
high during December and low in October. In general, observed in December. Total hardness value ranged
Fig 5. Dissolved Oxygen (mg/l) content of the water

Fig 6.Akalinity values (mg/l) observed in Paraliyar
samples collected from 6 sampling sites in
river from July 2010 to December 2010.
Paraliyar river.

Fig 7. Acidity Level(mg/l) of the water samples Fig 8. Ammonia Concentration(mg/l) of the water
collected from the river Paraliyar during the study samples collected from 6 sampling sites in Paraliyar
period. river.
between 236 mg/l and 545 mg/l were observed in station 6.4 mg/l were observed in station V. The nitrate content
II. The total hardness was high during December and low was high during November and low in September. At
in July. At station III, the total hardness of the water station VI, the nitrate of the water samples showed a
sa mp le s sho wed a ma x i mu m va lu e of maximum value of 3.2 mg/l (September) and minimum
564 mg/l (December) and minimum value of 240 mg/l value of 7.3 mg/l (November). In general, the nitrate of
during (July). At station IV, the total hardness was varied the water samples showed lower values in September
between 225 mg/l and 554 mg/l. The total hardness was and higher values in November (Fig 10).
high during November and low in July. At station V the Sulphate
total hardness of the water samples varied form The sulphate of the water samples collected from
247 mg/l to 527 mg/l. The total hardness was high during the station I, varied from 23 (July) to 37 mg/l (October).
November and low in July. The total hardness of water At station II, the sulphate was varied between 29 and 35
samples collected from station VI, varied from 258 mg/l mg/l, The sulphate was high during November and low
(July) to 549 mg/l (December). In general, the total in July. The sulphate of the water samples varied
hardness of the water samples was low in July and high between 26 and 38 mg/l. at station III. The lowest value
in December (Fig 9). was observed in July and highest value was observed in
Nitrate October. At station IV, the sulphate of the water samples
The nitrate of the water samples varied between varied from 25 to 47 mg/l. The sulphate was high during
2.1 and 5.4 mg/l at station I. The lowest value was November and low in July. At station V, the sulphate of
observed in September and highest value was observed the water samples showed a maximum value of 37
in August. At station II, the nitrate of the water samples (November) and minimum value of 21 mg/l (July).
varied from 2.8 to 5.7 mg/l. The nitrate was high during Sulphate value ranged between 29 and 31 mg/l were
November and low in September. At station III, the observed in station VI. The sulphate was high during
nitrate was varied between 2.5 and 4.8 mg/l. The nitrate November and low in July. In general, the sulphate of
content was high during November and low in July. The the water samples showed lower values in July and
nitrate concentration of the water samples collected from higher values in November (Fig 11).
the station IV, varied from 6.8 (August) to 2.8 mg/l Nitrite
(September). Nitrate value ranged between 3.9 mg/l and At station I, the nitrite of the water samples
Fig 9. Total Hardness(mg/l) of the water samples Fig 10. Nitrate concentration (mg/l) of the water
collected from six selected stations of river Paraliyar. samples collected from the paraliyar river.
varied from 0.51 to 0.92 mg/l. The nitrite was high Sulphite
during December and low in September. The nitrite of The sulphite of the water samples varied
the water samples varied between 0.49 and 0.85 mg/l at between 19.1 and 23.4 mg/l at station I. The lowest value
station II. The lowest value was observed in September was observed in July and highest value was observed in
and highest value was observed in December. At station September. At station II, the sulphite of the water
III, the nitrite of the water samples showed a maximum samples showed a maximum value of 25.6 (September)
value of 0.97 (December) and minimum value of 0.49 and minimum value of 19.8 mg/l (July). The sulphite of
mg/l during (July). Nitrite value ranged between 0.31 the water samples collected from the station III, varied
and 0.69 mg/l were observed in station IV. The nitrite from 19.1 (November) to 20.9 mg/l (December). At
was high during December and low in August. At station station IV, the sulphite was varied between 17.6 and 24.3
V, the nitrite was varied between 0.38 and 0.72 mg/l. mg/l. The sulphite was high during September and low in
The nitrite was high during October and low in November. At station V, the sulphite of the water
November. The nitrite of water samples collected from samples varied from 17.2 to 21.7 mg/l. The sulphite was
station VI, Varied from 0.45 (September) to 0.98 mg/l high during September and low in November. Sulphite
(December). In general, the nitrite of the water samples value ranged between 17.3 and 22.4 mg/l were observed
showed lower in September and higher in December in station VI. The sulphite was high during September
(Fig 12). and low in November. In general the sulphite of the
Fig 11. Sulphate level of the water samples collected Fig 12. Nitrate level (mg/l) observed in Paraliyar
from the river Paraliyar during the study period. river from July 2010 to December 2010.

water samples showed lower values in November and 3.2 mg/l at station III. The lowest value was observed in
higher values in September. (Fig 13). July and highest value was observed in December. At
Chloride station IV, the fluoride concentration of the water
The chloride of water samples collected from samples showed a maximum value of 3.9 (December)
station I, varied from 154 (August) to 281 mg/l and minimum value of 0.7 mg/l (July). At station V, the
(December). Chloride value ranged between 162 and 254 fluoride was varied between 0.8 and 3.7 mg/l. The
mg/l were observed in station II. The chloride was high fluoride was high during December and low in July.
during December and low in August. At station III, the Fluoride value ranged between 0.5 and 2.9 mg/l were
chloride was varied between 139 and 287 mg/l. The observed in station VI. The fluoride content was high
chloride was high during December and low in August. during December and low in July. In general, the
At station IV, the chloride of the water samples varied fluoride of the water samples showed lower values in
from 137 to 289 mg/l. The chloride was high during July and higher values in December. (Fig 15).
December and low in July. The Chloride of the water Phosphate
samples varied between 142 and 265 mg/l at station V. At station I, the phosphate of the water samples
The lowest value was observed in August and highest varied from 0.54 to 2.4 mg/l. The phosphate was high
value was observed in November. At station VI, The during October and low in September. The phosphate of
chloride of the water samples showed a maximum value the water samples varied between 0.17 and 2.6 mg/l at
of 254 (November) and minimum value of 131 mg/l station II. The lowest value was observed in September
during (August). In general, the chloride of the water and highest value was observed in October. The
samples showed lower in August and higher in phosphate of water samples collected from station III,
December. (Fig 14). varied from 0.15 (September) to 2.8 mg/l (October).
Fluoride Phosphate value ranged between 0.32 mg/l and 3.2 mg/l
The fluoride content of the water samples were observed in station IV. The phosphate was high
collected from the station I, varied from 0.4 (July) to 3.5 during October and low in September. At station, V, the
mg/l (December). At station II, the fluoride content of phosphate of the water samples showed a maximum
the water samples varied from 0.6 to 3.1 mg/l. The value of 3.1 mg/l during August and minimum value of
fluoride was high during December and low in July. The 0.40 mg/l during September. At station VI, the phosphate
fluoride of the water samples varied between 0.8 and was varied between 0.41 mg/l and 2.7 mg/l. The
Fig 13. Sulphite level (mg/l) of the water samples Fig 14. Chloride level (mg/l) of the water samples
collected from 6 sampling sites in Paraliyar river. collected from the paraliyar river.

phosphate was high during October and low in the water samples varied between 254 and 652 mg/l at
September. In general, the phosphate of the water station II. The lowest value was observed in July and
samples showed lower in September and higher in highest value was observed in December. Sodium value
October (Fig 16). ranged between 255 mg/l and 654 mg/l were observed in
Silicate station III. The sodium was high during December and
At station I, the silicate of the water samples low during July. The sodium of water samples collected
varied from 15 mg/l to 38 mg/l. The silicate was high from station IV, varied from 247 mg/l (July) to 649 mg/l
during December and low during August. The silicate of (December). At station V, the sodium of the water
the water samples collected from the station II, varied samples varied from 282 mg/l to 651 mg/l. The sodium
from 13 mg/l (August) to 32 mg/l (December). The was high during December and low during July. At
silicate of the water samples varied between 18 mg/l and station VI, the sodium was varied between 295 mg/l and
42 mg/l at station III. The lowest value was observed in 632 mg/l. The sodium was high during December and
August and highest value was observed in December. low during July. In general, the sodium of the water
Silicate value ranged between 11 and 37 mg/l were samples showed lower in July and higher in December
observed in station IV. The silicate was high during (Fig 18).
December and low during August. At station V, the Potassium
silicate of the water samples showed a maximum value The potassium of the water samples varied
of 41 (December) and minimum value of 19 (August). At between 23.5 mg/l and 73.9 mg/l at station I. The lowest
station VI, the silicate was varied between 17 mg/l and value was observed in September and highest value was
39 mg/l. The silicate was high during December and low observed in December. The potassium of the water
during August. In general, the silicate of the water samples collected from the station II, varied from 24.9
samples showed lower values in August and higher mg/l (September) to 77.6 mg/l (December). At station
values in December. (Fig 17). III, the potassium of the water samples showed a
Sodium maximum value of 78.3 mg/l (December) and minimum
At station I, the sodium of the water samples value of 24.3 mg/l (September). Potassium value ranged
showed a maximum value of 647 (December) and between 26.7 mg/l and 78.7 mg/l were observed in
minimum value of 267 mg/l during (July). The sodium of station IV. The potassium was high during December
Fig 15. Fluoride concentration (mg/l) of the water Fig 16. Phosphate level (mg/l) of the water samples
samples collected from six selected stations of collected from the river paraliyar during the
river Paraliyar. study period.

and low in September. At station V, the potassium was COD

varied between 24.7 mg/l and 77.5 mg/l. The potassium At station I, the COD of the water samples
was high during December and low in September. At showed a maximum value of 3.1 mg/l (October) and
station VI, the potassium of the water samples varied minimum value of 1.7 mg/l (November). COD value
from 25.2 mg/l to 77.6 mg/l. The potassium was high ranged between 1.5 and 2.9 mg/l . The COD was high
during December and low during September. In general, during October and low in November. At station III, the
the potassium of the water samples showed lower values COD was varied between 1.6 and 3.2 mg/l. The COD
in September and higher values in December (Fig 19). was high during October and low in November. The
Calcium COD of water samples collected from station IV, varied
The calcium of water samples collected from from 1.5 (November) to 3.8 mg/l (October). At station V,
station I, varied from 41.9 (September) to 61.9 mg/l the COD of the water samples varied from
(October). Calcium value ranged between 42.9 mg/l and 1.2 and 3.7 mg/l. The COD was high during October and
62.7 mg/l were observed in station II. The calcium was low in November. The COD of the water samples varied
high during August and low in September. At station III, between 1.5 and 3.6mg/l at station VI. The lowest value
the calcium of the after samples varied from was observed in November and highest value was
42.8 to 63.5 mg/l. The calcium was high during August observed in October. In general, the COD of the water
and low in September. At station IV, the calcium was samples showed lower value in November and higher
varied between 47.8 mg/l and 67.8 mg/l. The calcium values in October (Fig 21).
was high during October and low in September. At BOD
station V, the calcium of the water samples showed a At station I, the BOD of the water samples varied
maximum value of 67.9 mg/l (October) and minimum from 1.92 and 3.87 mg/l. The BOD was high during
value of 45.4 mg/l during September. The calcium of the December and low in September. At station II, the BOD
water samples varied between 44.6 mg/l and 65.4 mg/l at was varied between1.09 and 3.75 mg/l. The BOD was
station VI. The lowest value was observed in September, high during December and low in September. BOD value
and highest value was observed in October. In general, ranged between 1.99 and 3.98 mg/l. The BOD was high
the calcium of the water samples showed lower in during December and low in September. At station IV,
September and higher in October (Fig 20). the BOD of the water samples showed a maximum value
of 3.92 mg/l (December) and minimum value of
Fig 17. Silicate level (mg/l) observed in Paraliyar Fig 18. Sodium value (mg/l) of the water samples
river from July 2010 to December 2010. collected from 6 sampling sites in Paraliyar river.
Fig 19. Potassium value (mg/l) of the water samples Fig 20. Calcium value (mg/l) of the water samples
collected from the paraliyar river. collected from six selected stations of river Paraliyar.
1.87 mg/l (September). The BOD of water samples fluxes (Thayer, 1971) Nutrient concentrations and
collected from station V, varied from 1.54 mg/l distributions have therefore been documented as having
(September) to 3.87 mg/l (December). The BOD of the seasonal patterns (Baird and ulanowicz, 1989;
water samples varied between 1.75 and 3.82 mg/l at Morris, 2000). Monthly variations are evident in all the
station VI. The lowest value was observed in September physico-chemical parameters examined in this study.
and highest value was observed in October. In general, Seasonal cycles are due to imbalances in the process of
the BOD of the water samples showed lower values in mineralization and consumption (Morris, 2000). Raw
September and higher values in December. (Fig 22). sewage is the source of nitrates and phosphates in rivers
(Aggarwal et al., 2000, Adeyemo, 2003).
DISCUSSION The nitrate concentration of the water samples
In the present study pH of the water samples varied between 2.1 and 7.3mg/l. Nitrate is a form of
ranged between 5.14 and 8.9. Generally, aquatic nitrogen and a vital nutrient for growth, reproduction,
organisms are affected by pH because most of and the survival of organisms. High nitrate levels
their metabolic activities are pH dependent (1 mg 1-1) are not good for aquatic life. The high level of
(Wang et al., 2002). Optimal pH range for sustainable nitrate observed during this study is in agreement with
aquatic life is pH 6.5 - 8.2 (Murdock et al., 2001). Wolfhard and Reinhard (1998) who concluded that
Sreenivasan (1976) has demonstrated that a large nitrates are usually built up during dry seasons and that
variation in pH of water is an indication of a highly high levels of nitrates are only observed during early
productive nature of the water body. Free carbon dioxide rainy seasons. This is because initial rain flush out
liberated during respiration and decay of organic matter deposited nitrate from near-surface soils and nitrate level
are highly soluble in natural waters. The carbon dioxide reduces drastically as rainy season progresses.
content of water depends upon the water temperature, The phosphate concentration of the water
depth, rate of respiration, decomposition of organic samples varied between 0.15 and 3.2mg/l. There are
matter, chemical nature of the bottom and geographical various sources of phosphate to rivers, such as firm rock
features of the terrain surrounding the water body deposit, run off from surface catchments, and interaction
(Sakhare and Joshi, 2002). between the water and sediment from dead plant and
Temporal variations in aquatic systems can have animal remains at the bottom of rivers. Phosphate is
direct and indirect effects on factors influencing nutrient considered to be the most significant among the nutrient

Fig 21.Chemical Oxygen Demand (mg/l) observed in Fig 22. Biological Oxygen Demand (mg/l) of the
Paraliyar river from July 2010 to December 2010. water samples collected from the river paraliyar
during the study period.
responsible for eutrophication of lakes, as it is the knowledge of total dissolved solids concentration and
primary initiating factor. The result reveals that the salinity of water sample (Odum, 1971).
nutrient load in the water shed is high; because of Sawyer (1960) classified water on the basis of
nutrient enrichment, productivity, decay and hardness in to three categories that is, soft (0.00-75 mgl-1)
sedimentation (Adeyemo, 2003). moderately hard (75.00- 150.00 mgl-1) and hard
In the present study, dissolved oxygen content of (151.00-300.00 mgl-1). The hardness of the water varied
the water ranged from 3.57 to 7.2 mg/l. Analysis of DO between 225 and 564. Brown (1993) reported that total
is a key test for the monitoring of pollution such as for hardness act as limiting factor for alkalinity. Calcareous
treatment process of water wastages. The presence of DO water with alkalinity more than 50 ppm is most
in water may be due to direct diffusion from air and productive, 0 20 ppm for low production, 20-40 ppm
photosynthetic activity of autotrophs. The addition of a for medium production and 40-90 ppm for higher
variety of biodegradable pollutants from domestic and production. Carbonates and bicarbonates in hydroxides
industrial sources stimulates the growth of of Ca, Mg, Na, K, NH4 and Fe generally cause alkalinity
microorganisms which consume the DO of the water. of natural fresh water. Carbonates and bicarbonates are
DO is a good indicator of water quality and its relation to the major components of alkalinity; they have positive
the distribution and abundance of various algal species correlation with alkalinity.
along with the degree of pollution by organic matter and In natural unpolluted waters, the acidity is
level of self purification of water. mainly contributed by dissolved Co2. In polluted waters
Conductivity measures the capacity of a weak acids like CH3 COOH may contribute significantly
substance or solution to conduct electrical current. to the total acidity. In some organic waters, Organic
Olsen (1950) classified the name of water bodies having acids also contribute to acidity (Brown,1 993). The
conductivity volume greater than 500.00 s/cm as transparency of water is mainly affected by factors such
eutrophic. The Electrical conductivity of the river water as biological productivity, suspended particles and water
depends on the concentration of dissolved solutes. The colour. Clay, silt, organic matter, plankton and other
EC values varied between 1.204 and 3.92s. Several microscopic organisms cause turbidity in natural waters
factors influence the conductivity including temperature, (Kishor et al., 2005). This has been recognized as a
ionic mobility and ionic valencies. In turn conductivity valuable limiting factor in the biological productivity of
provides a rapid mean of obtaining approximate the water bodies.

In natural waters, dissolved solids are composed (COD) is a measure of the Oxygen equivalent of the
mainly carbonates, bicarbonates, chlorides, sulphates, organic matter content of water that is susceptible to
phosphates, nitrates, calcium, magnesium, sodium, oxidation by a strong chemical oxidant.
potassium, iron and manganese etc. (Esmaeili and Johal, Sewage or residential waste, consisting largely of
2005) The salts of sodium, Potassium and Calcium phosphate containing detergents, in a major source of
contribute Chlorides in waters. Large contents of nutrients in bodies of water. The concept nutrient
Chloride in fresh water is an indicator of organic overloading has a great impact on all subsequent
pollution (Venkatasubramani and Meenambal, 2007). eutrophication research and lake management. Its fair to
In aquatic environment, calcium serves as one of state that nitrates and phosphates are probably the key
the micro nutrients for most of the organisms. On the nutrients in controlling aquatic plant growth. The Nitrate
basis of calcium richness, Ohle (1934) classified water and phosphate are two important constituents that
bodies into (i) poor (ii) Medium and (iii) rich water immensely help in the growth of the plants where they
body. Magnesium is often associated with calcium in all are present. If they are present in lake and ponds they are
kinds of waters, but its concentrations remains generally excessively promote the growth of aquatic weeds and
lower than the calcium (Venkatasubramani and polluting our aquatic resources. International studies on
Meenambal, 2007). Magnesium is essential for the nitrates and phosphates in the surface waters of
chlorophyll growth and acts as a limiting factor for the various bodies of water have expressed their concern and
growth of Phytoplankton (Dagaonkar and Saksena, drawn the attention of scientists around the globe. These
1992). Therefore, depletion of Magnesium reduces the constituents are immensely help in the growth of the
phytoplankton population. Dwivedi et al., (2000) macrophytes like water hyacinth (Eichhornia crassiper)
-1
recorded Magnesium content upto 3.27 mg l ) in which is the most troublesome aquatic weed in the
Naktara reservoir. Like, Sodium, Potassium is also a world. The major sources of nitrate in lakes and ponds
naturally occurring element, but the Concentration in are from the catchment area by rainfall, sewage effluents,
fresh water bodies remain quite louder than the sodium agro waste, suspended organic matter when algae and
and calcium. Under was potassium concentration the other suspended micro organisms die and settle down to
growth rate and photosynthesis of algae especially blue the bottom. They carry their, Nitrogen and phosphorous
green algae become, poor and respiration in increased with them, during decomposition. This Nitrogen is
(Wetzel, 1983). released and becomes available for subsequent growth of
The concentration of ammonia in the water aquatic biota (Singh and Mahajan, 1987). Presence of
samples varied between 0.35 and 1.98 NU. Ammonia nitrate in water indicates the final stage of mineralization
higher concentration is harmful to fishes and other life. (Nema et al., 1984). Phosphorous is present in many
The toxicity of Ammonia increases with the pH because forms among them orthophosphate plays an important
at higher pH most of the Ammonia remains in the role in the aquatic ecosystem. Orthophosphate is the
gaseous form. At low pH due to conversion of Ammonia soluble reactive phosphorous which is also termed as
in to Ammonium ions (which are much less toxic than inorganic phosphate. It plays a dynamic role in aquatic
the gaseous form) decreases its toxicity. eco system which is taken up widely by phytoplankton
Bio-chemical oxygen demand (BOD) in the (Goldman, 1965).
amount of oxygen utilized by microorganism in
stabilizing the organic matter. Chemical organic demand

CONCLUSION: Husbandry and Medicine. 242-5. Pergamon Press

In conclusion results of the present study Oxford, U.K.
revealed that most of the physical and chemical

Dagaonkar A and Saksena DN. 1992.
parameters of the Paraliyar River are within the
Physico-chemical and Biological characterization of a
permissible limit reported for the potable waters. temple tank, Kaila Sagar, Gwalior, Madhya Pradesh.
However, in some months, the nutrients and other J. Hydrobiol. 8(1):11-19
parameters showed higher values than the permissible
Dwivedi RK, Khan MA, Singh HP, Singh DN and
limits. The deterioration in the physicochemical quality
Tyagi RK. 2000. Pro-duction Dynamics and fisheries
and rise in the nutrient level observed in this study is
development in Naktara Reservoir, Madhya Pradesh,
alarming, and periodic monitoring and preventative India. J. Inland Fish. Soc. India 32:81-86.
measures are required to save the aquatic system from
eutrophication. Esmaeili HR and Johal MS. 2005. Study of
physico-chemical parameters of water of Gobindsagar
reservoir, India. In Proceeding of National Seminar on
REFERENCES
New Trends in Fishery Development in India. Punjab
Adeyemo OK. 2003. Consequences of pollution and University, Chandigarh, India.
degradation of Nigerian aquatic environment on fisheries
resources, The Environmentlist,23(4):297-306. Furhan Iqbal M, Ali Abdus Salam BA, Khan S,
Ahmad M,Qamar and Kashif Umar. 2004. Seasonal
Adeyemo OK, Adedokun OA, Yusuf RK and Adelaye Variations of Physico Chemical Characteristics of
EA. 2008. Seasonal Changes in Physico - Chemical River Soaan Water at Dhoak Pathan Bridge
Parameters and Nutrient load of River sediments in (Chakwal),Pakistan. Int J Agr Biol., 6:89-92.
Iboldan City, Nigeria. Global Nest J,10:326-336.
Goldman CR. 1965. Primary productivity and limiting
Agbaire PO and Obi CG. 2009. Seasonal Variations of factors in the lake of the Alaska peninsula.Ecol.Manogr.
some Physico Chemical Properties of River 30:207-230.
Et h io p e Wat er in n Abr ak a. , Nig er ia.
J.Appl.Sci.Environ.Manage.March.Vol.13(1)55-57. Kishor K and Joshi BDD. 2005. Physico-chemical
characteristics of pond water at Khanpur village in
Aggarwal TR, Singh KN and Gupta AK. 2000. Bareilly district (U.P.). Him. J. Environ. Zool. 19:89-92.
Impact of sewage containing domestic water and heavy
metals on the chemistry of Varunq river water, Pollution Leonard LC. 1971. Water and Water Pollution.
Research,19(3),491-494. Vol .1:256-63. Marcel Dekkar, Inc. New York, USA.
American Public Health Association (APHA). 1998. Morris JT. 2000. Effects of sea level anomalies on
Standard methods for Examination of water and estuarine processes In : Estuarine science : A synthetic
Wastewter, 20th Edition. A.P.H.A.,1015 Fifteenth street, approach to research and practice, Hobbie J.E. (ed.),
NW,Washington,DC. Island Press, Washington, DC, 107-127.
Baird D and Ulanowicz RE. 1989. The seasonal Murdock T, Cheo M and OLaughlin k. 2001.
dynamics of the Chesapeake Bay ecosystem. Ecology Streemkeepers Field Guide : Watershed Inventary and
Monograph, 59:329-364. Stream Monitoring methods. Adopt-A-Stream
Foundation, Everett, WA.207.
Brown L. 1993. Aquaculture for Veterinarians Fish

Nema P, Rajgopalan S and Mehta CG. 1984. Quality Science, 12:240-253.

and treatment of Sabarmati river water Ahemedabad.
J.I.W.W.A. 16(I):99-107. Vogel AI. 1978. A text book of Quantitative Inorganic
Analysis Including Elementary Instrumental Analysis 4th
Narendra Singh Bhandari and Kapil Nayal. 2008. Ed. The English Language Book Society and Langman.
Correlation Study on Physico-Chemical Parameters and Co(a) P 834 (b) P 328-32(c) 504-506(d) 499-500
Quality Assesment of Kosi River Water, Uttarkhand. (e) 830-831.
E-J.Chem., 5(2):342-346.
Venkatasubramani R and Meenambal T. 2007. Study
rd
Odum EP. 1971. Fundamentals of Ecology. 3 Ed. 278- of sub-surface water quality in Mattupalayam Taluk of
84. Toppen Company, Ltd., Japan. Coimbatore district Tamil Nadu. Nat. Environ. Poll.
Tech. 6:307-310.
Ohle W. 1934. Die Bedeutung der Anstanschvorgange
Zwischen Schlam und Wasser fur den Stoffkreislauf der Wang W, Wang A, Chen L, Liu Y and Sun R. 2002.
Gewasser, "Vom Wasser". Effects of pH on Survival, Phosphorus Concentration,
Adonylate Energy Charge and Na+ - K+ ATP are
Olsen S. 1950. Aquatic plants and hydrospheric factor, I. Activities of Penacus chinensis Osbeck Juveniles,
Aquatic plants in Switzerland, Arizona. J. Sevensk. Aquatic Toxicology, 60:75-83.
Botanisk Tidskriff 44:1-34.
Wetzel RG. 1983. Limnology. Saunders College
Sakhare VB and Joshi PK. 2002. Ecology of Palas- Publishing, Philadelphia.
Nilegaon Reservoir in Osmanabad District, Maharastra.
J. Aquat. Biol., 18(2):17-22. Wolfhard S and Reinhard B. 1998. The heterogeneity
of runoff and its significance for water quality problems,
Sawyer CH. 1960. Chemistry for sanitary Engineers. Hydrological Sciences Journal des Sciences
McGraw Hill Book Co., New York. Hydrologiques, 43:103-113.
Singh R and Mahajan I. 1987. Phytoplankton and

water chemistry of Rawalsar and Renuka lake. H.P.India.
J.Ecol., 14(2):273-277.
Sreenivasan A. 1976. Limnological studies and primary

production in temple pond ecosystem. Hydrobiol.
48:117-123.
Sudhira HS and Kumar VS. 2000. Monitoring of lake

water quality in Mysore city. In : International Submit your articles online at jresearchbiology.com
Symposium on Restoration of lakes and Wetlands : Advantages
Proceedings of Lake 2000, Ramachandra,T.V., Easy online submission
Rajasekara, M.C. and Ahalya, N.(Eds), Bangalore, Complete Peer review
India : Centre for Ecological Sciences, Indian Institute of Affordable Charges
Quick processing
Science 1-10.
Extensive indexing
Thayer G. 1971. Phytoplankton production and the
submit@jresearchbiology.com
distribution of nutrients in a Shallow, unstratified
www.jresearchbiology.com/Submit.php.
estuarine system near Beaufort, NC, Chesapeake

Original Research
Radio-protective and anti-clastogenic effects of Barleria lupulina Lindl. extract against

(gamma)-ray (1.2 Gy) induced mitotic chromosomal aberrations of laboratory mice
Mus musculus and its effect on fish tumor induced after - irradiation.
Authors: ABSTRACT:
Pradip Kumar Sur1,
Pranab Kumar Das 2. Cancer and tumor formation are a common problem to mankind all over the
globe. Cancer is a life threatening disease. In this study, we have investigated for
Institution:
radio-protection by using Barleria lupulina Lindl. plant extract (BLPE) on mice, one
1. Associate Professor,
hour pre and one hour post treated with -ray (1.2 Gy dose). Adult healthy mice were
Cytogenetics Laboratory,
Dept. of Zoology, subjected to whole body -irradiation (1.2 Gy). Different types of mitotic
Kanchrapara College, chromosomal aberrations were studied. Out of the two sets, maximum protection was
Kanchrapara-743145, obtained with BLPE post treated animals (72.22%). Analysis of the data revealed that
Dist-(N)24 Pgns ,West BLPE can give significant protection to mice against chromosomal damage induced by
Bengal, India. -ray. Moreover, thin layer chromatography and phytochemical analysis revealed the
presence of steroid, terpenoid, glycosides, flavonoid, tannins and carbohydrate, in the
2. Research Scholar, BLPE. BLPE was also found to reduce -ray induced tumor in fish
Cytogenetics Laboratory, (Oreochromis mossambicus). Thus we report for the first time potent anti-clastogenic
Dept. of Zoology, and anti tumor activity of BLPE in mice and fish, respectively.
Kanchrapara College,
Kanchrapara-743145,
Dist-(N)24 Pgns ,West
Bengal, India.
Keywords:
Corresponding author: Barleria lupulina Lindl; (gamma)-ray; chromosomal aberrations; mice bone
Pradip Kumar Sur. marrow cells; radiation protection; anti-clastogenic; anti-tumor; anti-cancer.

drpksur@rediffmail.com Pradip Kumar Sur, Pranab Kumar Das.
Radio-protective and anti-clastogenic effects of Barleria lupulina Lindl. extract against
(gamma)-ray (1.2 Gy) induced mitotic chromosomal aberrations of laboratory mice
Mus musculus and its effect on fish tumor induced after - irradiation.
Dates:
Web Address: Received: 14 Jun 2012 Accepted: 27 Jun 2012 Published: 30 Jun 2012

439-447 | JRB | 2012 | Vol 2 | No 5

An International
Sur and Das, 2012
INTRODUCTION identified from Central National Herbarium, Botanical

Cancer is a broad term in medical biology where Survey of India, Howrah-711103, West Bengal, India
uncontrolled cell growth takes place leading to tumor (Ministry of Environment & Forests, Govt. of India).
formation. Malignant tumors are called cancer Leaves of this plant were cleaned, washed and dried in
(Kumar V et al., 2006). About ten million people are shade. Leaf extract was obtained from the dried leaves
diagnosed with cancer and six million dies every year all by running 20 cycles of Soxhlet Extraction (continuous
over the world due to cancer (Park K 2011). hot extraction) using ethanol (99.9%). A part of this
Chromosomal aberrations lead to tumor formation and Barleria lupulina Ethanol Extract (BLEE) was used for
cancer (Donna GA et al., 2003). The pioneering TLC and phytochemical analysis.
discovery by Muller (1927) on artificial mutagenesis in Then the BLEE was dried to remove the ethanol,
Drosophila opened a new horizon to the cytogenetic and dissolved in distilled water to make
study of chromosomal aberrations. Sur PK et al., (1989, Barleria lupulina Plant Extract (BLPE). 1% of this stock
2004, 2010) studied the effects of different doses of solution was used for injection into mice and fish.
X-rays on grasshopper chromosomes. Kim HK et al., Experimental Protocol with animals:
(2012) stated that cytotoxic chemotherapy remains the The animals were divided into following sets:
primary treatment option for many cancer patients. SET I (BLPE pre-treated Mice injected with BLPE
Search for a radio-protective agent had been intensely Series): at 1ml per 100 gm body
weight, one hour before
carried out by workers like Patt et al., (1949), whole body -irradiation
Mozdarani and Nazari (2009), Jagetia et al.,(2003) etc. (1.2 Gy) from Cobalt (Co60)
In traditional medicine, Barleria lupulina plant is SET II (CONTROL) Mice exposed to whole
body -irradiation (1.2 Gy)
externally used as an anti-inflammatory agent from Cobalt (Co60)
(Kanchanapoom T et al., 2001) and even against snake
SET III (BLPE post-treated Mice injected with BLPE
bites, dog bites, swelling due to fall, insect bite Series): at 1ml/100 gm body
weight, one hour after
(Doss A et al., 2011), herpes simplex and herpes zoster whole body -irradiation
(Wanikiat P et al., 2008). Even, anti-diabetic potential of (1.2 Gy) from Cobalt (Co60)
this plant extract in rats had been reported SET IV (Fish): Fresh water tilapia fish
Oreochromis mossambicus
(Suba V et al., 2004). exposed to whole body
But anti-clastogenic, anti tumor, anti-cancer and -irradiation (1.2 Gy) from
Cobalt (Co60)
radiation protection activities of this plant extract had not
been reported hitherto. In the present study, we have Mitotic chromosomes from femur bone marrow
reported for the first time, potent anti clastogenic activity cells of mice (SET I, SET II, SET III) were studied for
of Barleria lupulina Lindl. Plant Leaf extract (BLPE) in each set of animals after 1 hr, 16 hr, 48 hr, 1 week, 2
mice, and anti tumor activity in fish, up to a significant weeks and 4 weeks (1 month) of treatment.
level. The study is cleared by Animal Ethical
Committee, Dept of Zoology, Kalyani University, West
MATERIALS AND METHODS Bengal, India.
Preparation of the plant extract : Preparation of metaphase plates and data scoring:
Barleria lupulina of Acanthaceae family The mice were injected intraperitonially with
(common name hophead Philippine violet) (Fig. (i), was 0.03% colchicine solution at 1ml/100 gm body weight
Table 1: Various chromosomal aberrations for mice: SET I, SET II and SET III at different time intervals
Types of aberrations
Percentage
Time Animal No. of No. of iso iso sub Total
rabbit ear of Total
interval SET cells chromosomes chromatid centromeric ring rabbit ear Aberrations
chromatid chromatid chromatid translocation chromosome Aberrations
break dissociation chromosome chromosome with gap
gap break gap
Sur and Das, 2012
SET I 300 12000 35 30 18 15 82 16 18 36 36 286 2.38 %

1 hour SET II 300 12000 73 36 20 40 53 81 27 51 40 421 3.51 %
SET III 300 12000 36 35 5 33 52 7 22 33 38 261 2.18 %
SET I 300 12000 182 60 64 121 263 15 13 167 183 1068 8.90 %
16 hour SET II 300 12000 257 143 155 170 186 28 80 214 128 1361 11.34 %
SET III 300 12000 133 100 25 135 20 82 74 202 68 839 6.99 %
SET I 300 12000 152 102 62 101 351 14 42 61 18 903 7.53 %

48 hour SET II 300 12000 327 220 164 328 491 55 109 191 111 1996 16.63 %
SET III 300 12000 86 4 2 301 80 10 20 9 42 554 4.62 %
SET I 300 12000 252 15 13 102 52 105 55 203 101 898 7.48 %
1 week SET II 300 12000 272 190 28 163 355 80 27 81 82 1278 10.65 %
SET III 300 12000 89 51 25 127 27 23 30 72 60 504 4.20 %
SET I 300 12000 257 128 42 171 43 41 86 126 14 908 7.57 %
2 weeks SET II 300 12000 330 80 26 217 245 109 27 28 54 1116 9.30 %
SET III 300 12000 142 21 5 62 65 25 17 100 62 499 4.16 %
SET I 300 12000 67 30 33 68 66 34 25 100 8 431 3.59 %
4 weeks SET II 300 12000 121 50 15 135 56 41 15 20 10 463 3.86 %
SET III 300 12000 33 2 5 28 121 8 18 35 9 259 2.16 %
SET I 1800 72000 945 365 232 578 857 225 239 693 360 4494 6.24 %
Total
SET II 1800 72000 1380 719 408 1053 1386 394 285 585 425 6635 9.22 %
Aberrations
SET III 1800 72000 519 213 67 686 365 155 181 451 279 2916 4.05 %
SET I 1.31 % 0.51 % 0.32 % 0.80 % 1.19 % 0.31 % 0.33 % 0.96 % 0.50 %
Percentage of Total
SET II 1.92 % 1.00 % 0.57 % 1.46 % 1.93 % 0.55 % 0.40 % 0.81 % 0.59 %
Aberrations
SET III 0.72 % 0.30 % 0.09 % 0.95 % 0.51 % 0.22 % 0.25 % 0.63 % 0.39 %
441
Sur and Das, 2012
after the six different time intervals as mentioned above. (Table (i), Fig (ii)) and the study was done at various
After 1 hr of injection, the mice were chloroformed and time intervals, viz: 1 hr, 16 hr, 48 hr, 1 week, 2 week and
sacrificed. The femur bone was dissected out and 4 weeks (Table (i)). In case of SET II mice, the
bone marrow was suspended in 1% sodium citrate frequency of chromosomal aberrations increased from 1
solution and fixed in aceto-alcohol (acetic acid: hour to 48 hours, was maximum at 48th hour (16.63%)
alcohol 1:3 v/v). Slide preparation was done by and then decreased thereafter. On the other hand, less
dropping fixed cells on clean grease free chilled slides frequency of aberrations was observed for SET I and
(which were maintained at -5C), slides were heat fixed SET III mice. At 48th hour, SET III mice showed much
and air dried. Chromosomes were stained with Giemsa less chromosomal aberrations (4.62%) than SET I mice
staining solution. Scoring of data was done by using 300 (7.53%) (Table (i), Fig (iii)). Moreover, when compared
well-spread metaphase plates for each SET and each with types of aberrations, much higher percentage of
hour. Nine different types of structural chromosomal aberrations was observed with SET II mice (as 1.93%
aberrations were studied. with centromeric dissociation, 1.92% with chromatid
Preparative Thin Layer Chromatography (TLC): break, etc). But much less aberrations had been scored
Preparative TLC was performed to identify with SET I animals (as 0.31% with translocation, etc.)
active component in BLEE. For TLC, commercially and even lesser were scored with SET III animals
available 60F254 (pore size; 60 , uv fluorescence at (as 0.22% with translocation, etc.) (Table (i)).
254 nm, Merck, Germany) silica gel plates were used as Statistical Analysis (Comparison between SET I and
the stationary phase whereas chloroform: ethyl acetate: SET III mice (Table (ii))
ethanol (4: 3.5: 2.5) was used as the mobile phase. To evaluate the effectiveness of pre-treatment
The mobile phase was chosen by trial-and-error method. (SET I) and post-treatment (SET III) of BLPE on the
A drop of BLEE was put on the silica gel plate and TLC mice, statistical analysis was done according to methods
was run using the mobile phase. mentioned by Snedecor GW (1967).
Phytochemical analysis: Analysis of the data reveals, that, in the pooled
Phytochemical analysis was carried out for data, the Standard Error (SE), Critical Difference (CD) at
further identification of active component in BLEE. 5%, CD at 1% levels for SET I animals are 127.60,
Standard phytochemical tests to study the presence of 250.10 and 308.57 respectively. And that for SET III are
steroid, terpenoid, glycoside, flavonoid, alkaloid, tannin, 87.92, 171.96 and 226.39 respectively. Therefore, the
saponin, carbohydrate, protein, fixed oil and pH were values shown by SET III are always less than the SET I.
carried out by methods discussed by Trease GE and The t-Values, Chi-square values and r-Values are 7.57**,
Evans WC (1997). 51.58** and 0.90** respectively (significant at 1% level
(highly significant)). Therefore, Post Treatment of BLPE
RESULTS (SET III) reflects significantly high protection than the
Comparison between SET I, SET II and SET III mice Pre Treated series (SET I) (Table (ii). Whereas, in SET II
(Table (i)): animals, chromosomal aberrations were much higher.
300 cells for each hour for each SET were Therefore it may be inferred that BLPE provided
studied in these series (total 1800 cells and therefore protection against -ray (1.2 Gy) induced mitotic
72000 chromosomes in each SET). Different types of chromosomal aberrations in mice to a significantly high
structural chromosomal aberrations were studied level.
Table 2: Statistical analysis to compare between SET I and SET III mice
Aberration NC with Chromatid Isochromatid Isochromatid Subchromatid Centromeric Ring Rabbitear Rabbitear
Statistics aberration break gap break gap dissociation Translocation Chromosome chromosome chromosome Pooled
with gap
Sur and Das, 2012
SET I
11.06 37.71 18.45 8.79 21.33 53.41 14.26 11.23 25.79 28.27 127.60
+/- S.E.
SET III
27.87 18.85 14.97 4.40 41.73 15.16 11.67 8.97 28.57 8.91 87.92
+/- S.E.
SET I
21.68 73.91 36.16 17.22 41.82 104.69 27.95 22.01 50.54 55.40 250.10
C.D. at 5%

SET III
54.51 36.87 29.29 8.61 81.62 29.65 22.83 17.54 55.88 17.43 171.96
C.D. at 5%
SET I
28.48 97.10 47.50 22.62 54.94 137.54 36.72 28.92 66.40 72.79 308.57
C.D. at 1%
SET III
71.76 48.54 38.55 11.33 107.45 39.04 30.05 23.09 73.56 22.95 226.39
C.D. at 1%
t - Values 12.12** 6.27** 2.57 5.98** 1.18 3.70* 1.63 1.52 4.31** 1.57 7.57**
Chi-Square
68.72** 29.96** 197.20** 58.65** 168.52** 259.34** 111.27** 101.52** 92.66** 96.45** 51.58**
(2)Values
r - Values 0.08 0.84** -0.24 -0.07 0.25 -0.10 -0.12 -0.48 0.64** 0.64** 0.90**
* Significant at 5% level
** Significant at 1% level
Statistical analysis of the data were undertaken as Standard Error ( S.E ) ; Critical Difference ( C.D) at 1% and 5 % level; t- test ; chi- square( ) analysis
and correlation coefficient (r-value ) of both the series. NC= Number of cells.
443
Sur and Das, 2012
Preparative TLC:
In p r e p a r at ive TLC, six spots
(retention factor (Rf) values as 0.089, 0.196, 0.34, 0.46,
0.53, 0.84) were obtained (Fig. (iv)). No extra spot was
obtained under uv (254 nm) fluorescence.
Phytochemical analysis:
Phytochemical analysis of BLEE reveals the
presence of steroid, terpenoid, glycoside, flavonoid,
tannin and carbohydrate. This result corresponds to the
appearance of six spots in TLC. The pH of BLEE had
been found to be 5 (acidic).
Experiments with SET IV fishes
(Oreochromis mossambicus)
In freshwater tilapia fish
(Oreochromis mossambicus), tumor (ulcer proliferative
growth) was occurred near the right nostril of the fish
Fig 1: Barleria lupulina plant with flower pod after 13 days of -ray (1.2 Gy) treatment. Thereafter,
after post treatment with BLPE, the tumor subsided
completely by nine days (Fig: (v)).
DISCUSSION
It was reported that three genes - MYC,
EGFR and FGFR2 predicts poor survival in
fluorouracil-treated metastatic gastric cancer
patients (Kim HK et al., 2012). In previous studies,
Mantena et al., (2008) reported radiation-induced
aberrant metaphases and micro nucleated erythrocytes at
24 hr post exposure to 4 Gy radiations.
Fig 2: Photomicrographs (PM) showing various types

of structural chromosomal aberrations in mice
Post-Treated with BLPE (SET III)
PM 1: rabbit-ear chromosome, ring chromosome,
rabbit-ear chromosome with gap.
PM 2: translocation, chromatid break.
PM 3: iso chromatid gap, chromatid break Fig 3: Comparative analysis of chromosomal
PM 4: centromeric dissociation, sub chromatid gap, damage for SET I, SET II and SET III mice with
translocation. respect to six different time intervals
Sur and Das, 2012
Fig 5: Reduction of tumor in fish, Oreochromis

Fig 4: Preparative TLC showing six different spots, mossambicus (induced by 1.2 Gy gamma ray) treated
with corresponding Rf values. (SAMPLE indicates by BLPE (1gm%), in 9 days and compared with
the two spots where the same BLEE were spotted CONTROL fish.
Lemon et al., (2008) reported 2 Gy radiation DNA plant extract had reduced chromosomal aberrations up to
damage in whole body exposed normal and transgenic a significant level i.e. 72.22%, showing a great role in
mice. Even Jagetia et al., (2003) proved that naringin, a radiation-protection.
citrus flavonone, protects mice from irradiated cellular Moreover, BLPE proved to reduce -ray induced
DNA damage. Doss A et al., (2011) stated the tumors in fresh water fish, Oreochromis mossambicus
antibacterial effects of extracts from Barleria lupulina within 9 days (Fig v).
and also evaluated its phyto-constituents. Repeated experiments on other doses of
In this study, it had been observed that in our gamma-rays (0.5Gy, 0.8Gy, 1 Gy, 1.5Gy, 2Gy and
mice exposed only to -ray (1.2 Gy) (SET II), maximum 2.4Gy) + BLPE also supported this view. Whole plant
th
percentage of aberration was obtained at 48 hr (16.63%) extract has already been patented in regard to its anti-
(Table (i), Fig (iii)). But on Pre-treating with BLPE, the clastogenic, anti tumor, anti-cancer and radiation
th
percentage of aberration at 48 hr is reduced to 7.53% protection activities.
(SET I) and in Post-treated series it was 4.62 % (SET III)
(Table (i), (Fig (iii). ACKNOWLEDGEMENTS
Therefore, it may be concluded that the extract The authors are highly thankful to University
from leaves of Barleria lupulina (BLPE) provided as an Grant Commission, BahadurShah Zafar Marg, New
ameliorating agent against -ray induced structural Delhi-110002, for providing financial support in form of
chromosomal damage, in mice, and post treatment of the UGC Major Research Project in Zoology, 2011 entitled

Sur and Das, 2012
Assessment of protective role of some ayurvedic Kumar V, Abbas AK and Fausto N. 2006. Robbins
formulations on artificial mutagenesis in some and Cotran Pathologic Basis of Disease, 7th Edition,
mammalian models mice Mus musculus and shrew Reed Elsevier India Pvt. Ltd., New Delhi, India.
Suncus murinus) (F. No: 39-579/2010 (SR)). We also
Lemon JA, Rollo CD, Boreham DR. 2008. Elevated
extend our sincere thanks to Prof. Indranil Manna
DNA damage in a mouse model of oxidative stress:
(Director) & Dr. Debabrata Basu and Mr. Biswanath
impacts of ionizing radiation and a protective dietary
Kundu, Bioceramics & Coating Division, Central Glass
supplement. Mutagen 23 (6):473-482.
& Ceramic Research Institute, 196 Raja S C Mullick
Road, Kolkata-700032 (INDIA), for providing the Mantena SK, Unnikrishnan MK, Uma Devi P. 2008.
necessary radiation facility. Radioprotective effect of sulfasalazine on mouse bone
Moreover, the authors highly acknowledge Prof. marrow chromosomes. Mutagen 23(4):285-29.
Pandey (The Principal) and Ms. Moulisha Biswas,
Mozdarani H, Nazari E. 2009. Cytogenetic damage in
Bengal Institute of Pharmaceutical Sciences, Kalyani,
preimplantation mouse embryos generated after paternal
Nadia, West Bengal, India, for the work on extraction of
and parental -irradiation and the influence of vitamin C.
plant extract, TLC and Phytochemical analysis.
Reproduction Res., 137:35-43.
REFERENCES Muller HJ. 1927. Artificial transmutation of the gene.

Donna GA, Colin Co, Frank McC, Joe WG. 2003. Sci., 66:84-87.
Chromosome aberrations in solid tumors. Nat Genet
Park K. 2011. Parks text book of Preventive and Social
34:369-376.
Medicine. Bhanot Publishers, Jabbalpur. 21:353
Doss A, Parivuguna V, Vijayasanthi M and Sruthi
Patt HM, Tyree EB, Straube RL, Smith DE. 1949.
Surendran. 2011. Antibacterial evaluation and
Cysteine protects against X-irradiation. Sci.,
phytochemical analysis of certain medicinal plants,
110:213-214.
Western Ghats, Coimbatore. Journal of Research in
Biology. 1:24-29. Snedecor GW, Cochran WG. 1967. Statistical
Methods. Iowa St Univ Press, Ames, Iowa, USA,
Jagetia GC, Venkatesha VA, Reddy TK. 2003.
Naringin, a citrus flavonone, protects against Suba V, Murugesan T, Bhaskara Rao R, Ghosh L,
radiation-induced chromosome damage in mouse bone Pal M, Mandal SC. 2004. Antidiabetic potential of
marrow. Mutagen 18(4):337-343. Barleria lupulina extract in rats. Fitoterapia 75(1):1-4.
Kanchanapoom T, Kasai R, Yamasaki K. 2001. Sur PK and Manna GK. 1989. A preliminary study of
Iridoid glucosides from Barleria lupulina. Phytochem X-ray induced male meiotic chromosome aberration of
58:337-341. grasshopper, Oxya velox. Perspective in cytology and
genetics (End.- G.K. Manna and U. Sinha). 6:895-899.
Kim HK, Choi IJ, Kim CG, Kim HS, Oshima A,
Yamada Y. 2012. Three-gene predictor of clinical Sur PK and Pandey BN. 2004. A comparative study of
outcome for gastric cancer patients treated with X-ray and chemical induced marker chromosome
chemotherapy. The Pharmaco Jrnl 12:119-127. aberration in grasshoppers, Oxya velox and
Gesonula punctifrons and their alterations by chemicals.
Sur and Das, 2012
Ph. D. Thesis, Magadh University, Bodh Gaya.
Sur PK, Das PK, Thakur A, Roy S. 2010. A

comparative study of X-ray (160r) induced male meiotic
chromosome aberrations in two short-horned
grasshoppers, Oxya velox and Gesonula punctifrons.
Proc Zool Soc India 9(1):79-87.
Trease GE, Evans WC. 1997. Pharmacognosy. Ed.XIV,

Mac Millian Publ. Ltd., London.
Wanikiat P, Panthong A, Sujayanon P, Yoosook C,

Rossi AG. 2008. The anti-inflammatory effects and
the inhibition of neutrophil responsiveness by
Barleria lupulina and Clinacanthus nutans extracts.
J Ethnopharmacol 116(2):234-244.
Submit your articles online at Ficuspublishers.com

Advantages
Complete Peer review
Affordable Charges
Quick processing
Extensive indexing
Open Access and Quick spreading
You retains your copyright
submit@ficuspublishers.com
www.ficuspublishers.com/submit1.php.

Original Research
Impact of landuse transformation on arbuscular mycorrhizal fungal

diversity in the Kerala part of Nilgiri Biosphere Reserve, India
Authors: ABSTRACT:
Baiju EC1,
Chandrashekara UM2 and The study was conducted to analyze the effect of transformation of paddy
Sankaran KV2. fields into perennial crop dominant landuse systems on diversity, distribution and
abundance of arbuscular mycorrhizal fungi (AM fungi). When the basal area of tree
and palm community in different landuse systems derived from paddy fields did not
differ significantly, density was low in coconut and rubber plantation. Among different
landuse systems, polyculture homegarden showed significantly high value for tree
species diversity index (2.31). Polyculture homegardens also differ from paddy field
Institution: and other landuse systems studied with significantly more AM fungal spore
1. Sree Krishna College, abundance in the soil. Even though significantly low spore abundance in the soil was
Guruvayur, Thrissur, Kerala. recorded, percentage of root colonization value was significantly more in the roots
collected from arecanut mixed with perennial cropping system. In the present study,
2. Kerala Forest research fifty six AM fungi species were recovered from paddy field and landuse systems
Institute, Peechi, Thrissur, derived from it. About 55%-74% of AM fungal species were found to be common in
Kerala. paddy fields and a given landuse system derived from it. AM fungal species diversity
index values did not vary among landuse systems, with exceptions being in paddy
fields and polyculture farms, where significantly low values were recorded. These
findings highlight the fact that due to landuse transformation, aboveground plant
species composition changed drastically while the changes in AM fungal species
composition and spore abundance remained a slow process.

Baiju EC. Aboveground plant diversity, AM fungal diversity, AM fungal spore
abundance, Landuse transformation, Mycorrhizal root colonization, Paddy fields,
Perennial cropping systems.

easybaiju@gmail.com Baiju EC, Chandrashekara UM and Sankaran KV.
Impact of landuse transformation on arbuscular mycorrhizal fungal diversity in the
Kerala part of Nilgiri Biosphere Reserve, India.
Phone No:
0487 2600443. Dates:
09447750443. Received: 06 Apr 2012 Accepted: 14 May 2012 Published: 30 Jun 2012
Web Address:
448-459 | JRB | 2012 | Vol 2 | No 5

An International
Baiju et al., 2012
INTRODUCTION in managing soil health (Mohankumar and Mahadevan

In tropical countries, due to socioeconomic and 1987; Ragupathy et al., 1990; Sengupta and Chaudhuri
cultural changes, several landuse systems are being 1990; Muthukumar and Udaiyan 2000; Dhar and Mridha
transformed into some other landuse systems. For 2007; Shi et al., 2007). It is reported that landuse and
instance, in rural Kerala agricultural land has been going land management can influence the diversity and
through transformation due to sprawls in effectiveness of AM fungi (Strzemska 1975; Ocampo
agriculturisation, industrialization and globalization and Hayman 1980; Mulligan et al., 1985). According to
(Thampi 1995). Despite the fact that increase in area Giller (1996), in an ecosystem landuse change can bring
under cash crops help to increase farm income, changes more changes in belowground biodiversity than in
in cropping pattern which favour perennial crops that aboveground biodiversity. However, detailed studies on
have immediate and direct impact on the staple food changes in density, distribution and diversity of
security of State (Panikar 1980). Several efforts have AM fungi are due to the transformation of landuse
been made to analyze the root causes and consequences systems which were once dominated by annual crops to
of transformation of food crop based systems to cash those dominated by perennial crops are lacking. In the
crop based system of the State (Kannan and present study, we examined abundance, diversity and
Pushpangadan 1988; Narayanan 1995 Baiju and distribution pattern of AM fungi in landuse systems
Chandrashekara 2007). Such studies also highlighted the derived from paddy fields in comparison with paddy
need for more site specific studies which would help to fields in the Kerala part of Nilgiri Biosphere Reserve.
identify strategies, policy interventions and programmes
for reviving the rural landscape of Kerala that was once MATERIALS AND METHODS
dominated by paddy fields. Thus, though information on Study area
the impact of landuse transformation on the The study was conducted in Vazhikadavu
socioeconomic and cultural aspects of rural Kerala is Panchayat (a Panchayat is a group of villages which
available on biodiversity in general, belowground constitute the basic unit of rural administration. Each
biodiversity in particular is lacking. Panchayat generally covers between two and twenty
Among belowground biota, Arbuscular villages, depending on the size of the villages),
Mycorrhizal fungi (AM fungi) which form obligate, Malappuram District, Kerala located between 7619 to
mutualistic, symbiotic relationship with the roots of trees 7623 E longitude and 1123 to 1125 N latitude.
and crop plants facilitate host plant nutrient uptake. Here, an area of 2.6 x 1.4 km was selected for detail
Similarly, AM fungi can also enhance tolerance or studies. The area was divided into 200 m x 200 m grids
resistance to root pathogens or abiotic stress, such as and the grid intersection points were marked using a
drought and metal toxicities. In addition, AM fungi may Geographical Positioning System. Out of the 72 grid
play a vital role in the formation of stable soil intersection points, 23 points represented land cover
aggregates, building up a macroporous structure of soil types other than agriculture/forestry and another 20
that allows penetration of water and air and prevents points represented landuses that were not derived from
erosion (Miller and Jastrow 1992; Smith and Read 1997; paddy fields. In the remaining 29 points, four were paddy
Meharg and Cairney 2000; Borowicz 2001). Quite a few fields while 25 points were agroecosystems/agroforestry
efforts have been made in the tropical region to systems that were once as paddy fields (Figure 1).
characterize the diversity of AM fungi and also their role Salient characters of paddy fields and landuse systems
Baiju et al., 2012
Figure 1. Paddy fields and landuse systems derived from paddy fields in the Kerala part of Nilgiri Biosphere Reserve.
Non sample point

Landuse systems derived from other than paddy field
Agroecosystems/agroforestry systems derived from paddy field
(AM: arecanut mixed with perennial crops, AP: arecanut plantation, AV:arecanut mixed with annual crops,
CO: coconut plantation, HG: homegardens PA: paddy field, PF: polyculture farm and RP: rubber plantation).
derived from paddy fields are given in Table 1. Age each of 10 m X 10 m size were marked. All trees and
since the transformation of paddy fields into other type palms present in each quadrat were identified, counted
of croplands ranges from 5 to 25 years. and the Gbh (girth measured at 1.37 m above the ground
Climate level) was recorded. To estimate the density and basal
The climate in the study area is typically area of shrub community, four sub-quadrats, each of
monsoonic with annual rainfall varying from 5 m x 5 m of size, nested in each of the quadrats laid for
1621mm to 3271 mm (mean over 1990-2008: 2312mm). tree enumeration were used. On the other hand, for
More than 65% of annual rainfall is drawn from the estimating herb density, four sub-quadrats, each of
southwest monsoon during June- August period. The 1 m x 1 m, nested in each of the quadrats laid for tree
northeast monsoon, which sets in October and lasts till enumeration were used. Since herb density was more and
the end of November, accounts for much less rainfall measurement of girth of individual plant of herbs,
(hardly 25% of annual rainfall). The mean annual particularly of trailing herbs were tedious and time
maximum and minimum temperatures are 35C and consuming, their biomass was estimated. All herbs
15C respectively. Soils are acidic (pH 5.6- 6.2) and within each sub-quadrat were uprooted, sorted into
gravelly clay loam. In general, soils are poor in total different species and weighed after air drying for getting
nitrogen (0.05-1.2%), available phosphorous constant weight. Species diversity index values were
(7. 4-14. 0 ppm ), e xch a n gea bl e p ot a s si um calculated separately for tree, shrub and herb
(0.15- 0.23 Cmol (+)/kg) and organic carbon (1.0-2.0%) communities using the following equation
(Chandrashekara et al., 2008). (Shannon and Wiener 1963):
Phytosociological analysis H= -(ni/N) x ln (ni/N)
For each landuse type derived from paddy fields, Where, ni= Density of a species and N= Density of all
three plots were selected. In each plot, twelve quadrats, species

Baiju et al., 2012
Leaf area index (LAI) is an important parameter observed under a stereoscope and spores of fungi
to understand the canopy cover of landuse systems. enumerated from each soil sample.
The LAI of landuse systems derived from paddy fields Percent colonization of AM fungi
were analyzed after the south-west monsoon Feeder roots (1cm long) collected from the soil
(September-October) using canopy analyzer (LI COR, sample were stained using the method of Philips and
USA). Hayman (1970) and colonization by AM fungi was
Composition and diversity of AM fungi assessed. A total of 100 root pieces were examined from
Sampling technique each soil sample collected from different landuse
In each of the four plots selected for a given systems. Root bits showing vesicles/arbuscules were
landuse type, a quadrat (40 x 20m) was laid and divided considered as being colonized by AM fungi. The percent
into five equal blocks. From each block, 12 soil cores of mycorrhizal colonization was computed using the
(0-20cm) were obtained and all samples from a given following formula:
plot were mixed together to get a composite sample. The % AM colonization = (Total number of root bits
samples were air dried for 24 hrs in shade, sieved positive for AM colonization/ total number of bits
through 2 mm sieve and were stored at 4C until they observed for AM colonization) x 100.
were analyzed for spore abundance. Estimation of infective propagules of AM fungi
Enumeration of AM fungi Infective propagules in the soil consist of
Isolation of AM spores was done using wet 1) spores, 2) dead roots with AM colonization and
sieving and decanting technique (Gerdemann and 3) network of AM fungi. Estimation of the number of
Nicolson 1963). To start with, 10 g of the soil samples infective propagules would indicate the capability of
were suspended in water and stirred thoroughly. The soil mycorrhization of each type of soil (Porter, 1979). The
suspension was allowed to stand undisturbed for one procedure employed for estimating the number of
minute and then passed through 750, 500, 250, 100 and infective propagules was as follows. 30 g of the test soil
45 m sieves arranged one below the other in the same taken in a polythene bag was added with 270 g of
order. The contents from the last three sieves were sterilized sand soil mixture (1:1). This mixture was
filtered through filter papers and the filtrate was shaken thoroughly to get 10-1 dilution. From this
Table 1. Characteristics of paddy fields and landuse types transformed from
paddy fields in the Kerala part of the Nilgiri Biosphere Reserve
Land Use Type Characteristic features of landuse systems
Paddy cultivation from June-December. Vegetable cultivation or leaving the
1. Paddy fields (PA)
field fallow from January to May
2. Tree based systems Tree crops are dominant. The system may be monoculture or polyculture
2.1. Polyculture farm (PF) Land cultivated away from the farmer's dwelling place with annual, biennial and
tree crops, sometimes integrated with animal husbandry.
2.2.Polyculture homegardens Land cultivated around the farmer's dwelling place with annual, biennial and tree
(HG) crops, mostly integrated with animal husbandry.
2.3. Plantations of a tree crop Area is dominated by one tree species, along with some annual/perennial crops.
with some other associated 2.3.1. Arecanut plantation integrated with cultivation annual crops (AV)
crops (annual, biennial or 2.3.2. Arecanut plantation integrated with cultivation of some perennial crops
perennial) (AM)
Mono-specific tree plantations.
2.4.1. Coconut plantation (CP)
2.4. Monoculture plantations
2.4.2. Rubber plantation (RP)
2.4.3. Arecanut plantation (AP)
Baiju et al., 2012
dilution, 30 g of soil was transferred to another polythene H= - (ni/N) x ln (ni/N)

bag and added with 270 g of sterilized sand soil mixture Where, ni = density (number of spores in 10 gm
-2
(1:1) to get 10 dilution. This procedure was repeated to of soil) of the ith species and N= density of all species.
get dilutions of 10-3, 10-4, 10-5 and 10-6. Each dilution Frequency distribution of individuals AM fungal
was replicated for five times. Seeds of sorghum were species in a given landuse type was calculated as the
sown in the polythene bags and the plants were number of plots (in that landuse type) where the species
maintained for six weeks in the glass house. Presence or was encountered divided by total number of plots in that
absence of colonization was determined by staining landuse type.
technique (Philips and Hayman 1970). MPN number was Similarity index value for a given landuse system
determined referring to MPN table (Fischer and and paddy fields were calculated using the following
Yates 1963). formula:
Estimation of diversity of AM fungi Similarity index value =2C/ (A+ B)
Trap plant method was used to estimate the Where, A=number of species recorded in paddy
diversity of AM fungi in different landuse systems. In fields, B=number of species recorded in a given landuse
this method, 400 g of test soil was mixed with 400 g of system derived from paddy fields, C=number of species
sterilized sand; soil mixture (1:1 ratio) was taken in pots common in paddy fields and the given landuse system
and seeds of sorghum, maize/cow pea were sown. The Statistical Analysis
plants were maintained in the glass house by periodic The significance of differences between paddy
watering up to a period of three months after which the fields and landuse systems derived from paddy fields for
soil in each pot was wet sieved and the spores are each parameter such as number of AM fungal spores,
observed under a compound microscope. Identification percent colonization of AM fungi in roots, number of
of the spores was done using the Manual for the infective propagules in soil, species diversity index value
identification of AM fungi by Schenck and Perez (1990) of AM fungi were tested separately by Analysis of
and INVAM website by Joe Morton. Species diversity Variance (ANOVA). Differences were deemed to be
index (H) of AM fungal species was determined for significant when P<0.05 according to Least Significant
each land use system using the formula Difference (LSD) test. Similarly, significance of
Table 2. Density and basal area of tree and shrub community and density and biomass of herb community in
different landuse systems derived from paddy field in the Kerala part of Nilgiri Biosphere Reserve. Values
are Mean SE. In a column, means with same alphabet in the superscript are not significantly different at 5%
level.
Trees Shrubs Herbs
Landuse
systems Density Basal area Density Basal area Density Biomass
(individual ha-1) (m2 ha-1) (individual ha-1) (cm2 ha-1) (individual ha-1) (gm m-2)
PF 954224a 13.74.3a 609299 a
350180a 7811a 67686a
HG 885316a 12.15.7a 469115a 29883a 5616a 78270a
AV 1058293a 14.44.6a 610250 a
857387b 411345c 1450743b
AM 928367a 19.612.2a 36191029 b
1044309b 14965b 2222623c
CP 20050b 18.810.9a 29191918b 23451635c 8733a 1433499b
RP 43362c 10.53.0a 684397 a
1161749ab 12241b 1082283ab
AP 956294a 12.14.7a 4223664 b
4484476c 12715b 2414377c
PF: Polyculture farms, HG: Polyculture homegardens, AM: Arecanut with perennials, AV: Arecanut with annuals,
CP: Coconut plantations, RP: Rubber plantations and AP: Arecanut plantations.

Baiju et al., 2012
differences among different landuse systems derived low basal area in homegardens and polyculture farms of
from paddy fields for each parameter such as plant the present study area is thus an indication of young age
density, basal area/biomass (in case of herbs) and species of these landuse systems.
diversity index value were tested separately using Density and basal cover/biomass of understorey
ANOVA and LSD test. components (herbs and shrubs) seem to be primarily
determined by management practices adopted by the
RESULTS AND DISCUSSION farmers. For instance, due to regular weeding in
Phytosociological analysis polyculture farms, homegardens and rubber plantations
In landuse systems transformed from paddy low values for parameters like density and basal area
fields, density of trees and palms ranged from 200-1058 (biomass in case of herbs) were recorded. Since majority
-1
individuals ha with significantly low density in coconut of shrub species recorded from arecanut and coconut
plantation followed by rubber plantation (Table 2). Trees based cropping systems have multi-purpose values, high
and palm density in other landuse systems such as density and basal cover of the understorey community is
polyculture farmlands, homegardens, arecanut with maintained.
annual crops, arecanut mixed with perennial crops and Among different landuse systems, homegardens
monoculture of arecanut did not differ significantly are species rich with mean tree species diversity index
(P>0.05). The wide variation noticed here between value of 2.31 followed by polyculture farmlands
coconut plantation, rubber plantation and other croplands (Table 3). These may be attributed to the cultivation of
in terms of tree and palm density can be attributed to several multipurpose trees apart from dominant cash
factors like composition and space requirement of crops like coconut and arecanut. A positive correlation
different crop species. However, mean basal area of tree between tree basal area and tree species diversity in
and palm components among different landuse systems homegardens and polyculture farms were observed. The
did not vary significantly. It is reported that in fully packages of practice given by the Rubber Board stipulate
established homegardens the mean basal area of tree that no trees other than rubber shall be managed or
2 -1
component can vary from 31 to 63 m ha , depending grown in rubber plantation. Since the farmers are
upon the species composition (Sankar and adopting this package of practice so that they get
Chandrashekara 2002; Das and Das 2005). The estimated subsidies and other benefits from the Rubber Board, the
Table 3. Shannon-Weiner Species diversity index and leaf area index of plant communities in different
landuse systems derived from paddy field in the Kerala part of Nilgiri Biosphere Reserve. Values are
Mean SE. In a column, means with same alphabet in the superscript are not significantly different at 5%
level.
Tree species Shrub species Herb species Leaf area index
Landuse systems
diversity diversity diversity (LAI)
PF 1.430.09c 1.920.26ab 3.140.93a 2.500.25b
HG 2.310.12d 2.260.11b 3.690.45a 3.190.23c
AV 0.870.26b 1.940.23a 3.380.72a 1.590.04a
AM 0.940.39b 2.110.09b 2.530.98a 3.260.11c
CP 0.630.06b 1.900.32a 3.370.99a 2.110.32b
RP 0.160.04a 2.460.07c 3.291.01a 4.350.06d
b a a
AP 0.460.25 1.500.45 3.231.22 1.250.44a
Baiju et al., 2012
lowest tree diversity index value was recorded for rubber Table 4. Spore density (number of spores per 10 g of
plantations. Generally species diversity index values soil) of AM fungi in soils of paddy field and landuse
systems derived from paddy fields in the Kerala part
recorded for herb and shrub communities in different of Nilgiri Biosphere Reserve. Values are Mean SE.
landuse systems were not significantly different In a column, means with same alphabet in the
superscript are not significantly different at 5%
(Table 3). This can be attributed to weeding and other level.
management practices adopted in these plots. Number of
LAI value ranged from 1.25 - 4.35 with higher Landuse systems spores per
10 g of soil
value in rubber plantation (Table 3). In arecanut based Paddy fields 50 7bc
cropping system, space maintained between plants is Polyculture farms 50 5bc
Polyculture homegardens 67 3d
more and crown area per palm is also less. Thus, LAI
Arecanut mixed with annual crops 61 3cd
value obtained for this cropping system was lesser than Arecanut mixed with perennial
35 1a
that in other systems. crops
Coconut plantations 56 6cd
Composition and diversity of Arbuscular Rubber plantations 56 3cd
Mycorrhizal fungi Arecanut plantations 41 1ab
The mean AM fungal spore density in paddy mixed with perennial cropping system percentage of root
fields and landuse system derived from paddy fields colonization value of AM fungi was significantly more
ranged from 50-67 spores per 10g of soil (Table 4). than that in paddy fields (P<0.05; Table 5). Therefore, it
These values are within in the range of AM fungal spore can be concluded that due to intensive management
density reported for natural forests of South India adopted in arecanut mixed with perennial cropping
(Visalakshi 1997). Comparison of paddy fields and other system spore abundance may be decreased. On the other
landuse systems for AM fungal spore density revealed hand, when favourable conditions are available, AM
that the values are not significantly different (P>0.05); fungal species may propagate well as we observed in
exception being in polyculture homegardens and root colonization studies conducted by collecting roots
arecanut mixed with perennial cropping system. The AM from the arecanut mixed with perennial cropping system.
fungal spore density in polyculture homegardens was According to Oehl et al., (2003) the AM root
more than that in paddy fields (P<0.05). On the other colonization in the trap cultures established from
hand, significantly low spore density was recorded for different field sites can exhibit the pattern similar to
arecanut mixed with perennial cropping system (P<0.05). spore abundance in different agroecosystems. However,
It is also interesting to note that in arecanut in the present study correlation between percentage
Table 5. Percentage colonization and number of infective propagules of AM fungi in soils of paddy fields and
landuse systems derived from paddy fields in the Kerala part of Nilgiri Biosphere Reserve. In a column,
means with same alphabet in the superscript are not significantly different at 5% level.
% of colonization of AM Number of infective
Landuse systems
fungi in roots propagules per g of soil
Paddy fields 476a 7323b
Polyculture farms 526a 12134a
Polyculture homegardens 6611a 13332a
Arecanut mixed with annual crops 564a 18343a
Arecanut mixed with perennial crops 818b 17045a
Coconut plantations 557a 12344a
Rubber plantations 4615a 13285a
Arecanut plantations 666a 16340a

Baiju et al., 2012
Table 6. Mean spore abundance (spores per 10 g of soil) of AM fungi in paddy field and landuse systems
derived for paddy field in the Kerala part of Nilgiri Biosphere Reserve.
AM fungi PA PF HG AV AM CP RP AP
Acaulospora appendicula 0.7 1.7 3.2 3.3 0.8 2.0 3.0 -
Acaulospora bireticulata 1.7 2.7 5.0 2.0 1.6 2.0 1.0 1.3
Acaulospora denticulata 1.0 1.0 1.6 2.0 0.6 1.0 3.3 1.0
Acaulospora dilatata 0.7 - 1.4 3.3 1.0 - 0.8 1.0
Acaulospora elegans - 1.3 0.4 1.0 0.8 1.0 - 1.0
Acaulospora lacunose 2.7 - 0.6 1.3 1.4 1.0 2.0 2.0
Acaulospora laevis - 1.0 - 2.3 0.2 1.5 1.8 0.3
Acaulospora longula 1.0 0.7 0.8 1.0 2.2 0.5 0.5 3.0
Acaulospora mellea 2.3 0.7 1.8 3.0 1.4 - - 2.0
Acaulospora morrowae - 1.3 - 1.0 0.5 1.5 1.0 -
Acaulospora myriocarpa 0.7 - 0.6 2.0 1.4 - 1.0 1.3
Acaulospora rehmii - 1.3 0.2 0.7 - 2.0 - -
Acaulospora rugosa 0.7 2.0 4.4 1.0 1.4 - - 1.0
Acaulospora scrobiculata 6.0 - - 1.3 0.2 4.0 3.5 -
Acaulospora spinosa - 1.0 0.8 1.0 0.8 2.0 1.0 1.0
Acaulospora tuberculata 0.7 - 0.8 - - - 1.8 -
Gigaspora albida 0.7 0.7 0.4 - 0.8 2.0 0.3 0.7
Gigaspora decipiens - 1.3 1.2 1.0 0.4 - 0.8 -
Gigaspora gigantean - - - - 0.6 1.1 - 0.3
Glomus albidum - - 0.6 - - 0.5 0.8 -
Glomus aggregatum 3.0 1.7 2.6 3.3 2.6 - - 4.3
Glomus ambisporum 2.0 1.7 1.6 - - 1.0 - -
Glomus botryoides - - - 0.7 0.6 - 0.5 -
Glomus canadense - 2.0 1.6 2.0 0.8 3.0 - 1.3
Glomus citricolum 1.0 - 0.6 - - 2.0 1.0 -
Glomus claroideum - 0.3 0.4 2.7 - - - 1.3
Glomus clarum 2.3 2.0 2.2 2.0 1.0 0.5 1.0 -
Glomus constrictum - - - - - 2.0 - 0.7
Glomus convolutum - - 0.6 0.7 - - 0.8 -
Glomus delhiense 0.7 - 0.6 2.0 1.2 1.5 - 2.0
Glomus diaphanum - 1.7 1.0 2.0 1.4 - - 2.0
Glomus etunicatum 1.7 - - - 0.2 - 1.0 -
Glomus fasciculatum 1.7 3.7 4.2 2.0 - 1.0 - 0.7
Glomus fragile - - - - 0.4 2.0 0.5 0.3
Glomus geosporum 0.7 1.3 1.8 0.7 1.0 - 2.0 -
Glomus halonatum - - 0.4 - 0.8 1.0 - -
Glomus heterosporum 1.3 1.0 1.0 1.0 - - 0.5 0.3
Glomus hoi - - - 1.3 - 2.0 - 1.7
Glomus intraradices 3.3 2.0 2.4 1.0 0.6 - 2.8 0.3
Glomus invermaium - - 0.4 - - 0.5 - -
Glomus leptotichum - - - 1.0 0.6 0.5 0.3 -
Glomus macrocarpum 0.7 2.0 2.0 - - 2.0 - -
Glomus maculosum 7.0 4.7 9.6 1.3 1.6 4.5 5.5 4.0
Glomus monosporum - - - 1.3 - - - -
Glomus mosseae 1.7 2.0 2.0 - - 1.0 3.5 -
Glomus multicaule 0.7 0.7 1.2 1.3 1.4 2.0 2.5 2.7
Glomus multisubstensum - - - - 0.4 - - 0.7
Glomus occultum - 2.0 0.8 1.3 0.8 - 2.0 -
Glomus pallidum 0.7 0.7 1.2 1.3 - 2.0 - -

Baiju et al., 2012
Glomus pansihalos - - 0.8 - 0.2 - 2.0 -

Glomus pulvinatum - 2.0 1.2 2.0 1.2 2.0 - 2.0
Glomus pustulatum 1.3 - 0.2 - 0.4 - 2.5 -
Glomus radiatum - 1.0 0.8 1.0 0.4 2.0 2.5 -
Glomus reticulatum 0.7 0.3 0.8 - 0.8 - 1.3 1.0
Glomus scintillans - 0.3 1.2 1.7 0.2 0.5 1.5 -
Glomus segmentatum 0.3 0.3 - - - 1.0 - -
colonization of AM fungi in roots and number of authors (Thapar and Khan 1985; Ragupathy and
infective propagules per gram of soil is not significant Mahadevan 1993; Muthukumar and Udaiyan 2000;
(r = 0.61; P>0.05). At the same time, the values obtained Mohanan 2003) can be linked to acidic nature of the soil
for number of propagules per g of soil in different the landuse systems studied. It may also be pointed out
landuse systems are not significantly different (Table 5; here that the genus Glomus is of rare occurrence in
P>0.05). This pattern observed for number of infective Western Australia due to high soil pH (Porter et al.,
propagules can be attributed to the fact that a short 1987). As in the present study, rare occurrence of
period (about two months in this case) of trap culturing Gigaspora in Indian soil has been reported elsewhere
may not allow most of the species to sporulate. Thus, (Ragupathy and Mahadevan 1993; Sankaran et al., 1993;
further studies by prolonging the trap culture period may Muthukumar and Udaiyan 2000; Mohanan 2003). It is
show the actual relationships between percentage interesting to note that six out of 30 AM fungal species
colonization and number of infective propagules in each recorded from the natural forests of Kerala
landuse system. (Chandrashekara et al., 2008), were also recorded from
Fifty six species belonging to three genera paddy fields.
namely, Acaulospora, Gigaspora and Glomus were Comparison of AM fungal species composition
recovered from the soils of paddy fields and landuse in different landuse systems in the study area indicated
systems derived from it (Table 6). Glomus and that there are a few generalist species and also highly
Acaulospora showed dominance in the present study specialist species. For instance, species like
with 37 and 16 species respectively. The preponderance Acaulospora bireticulata, A. denticulata, A. longula,
of these two genera in Indian soils reported by several Glomus maculosum and G. multicaule can be regarded as
Table 7. Species diversity index of AM fungi of generalist species as they are found in all landuse
paddy fields and landuse systems derived from systems in the present study. On the other hand,
paddy field in the Kerala part of Nilgiri Biosphere
Reserve. Values are Mean SE. In a column, means Glomus monosporum can be considered as a highly
with same alphabet in the superscript are not specialist species due to its occurrence only in soils of
significantly different at 5% level.
arecanut mixed with annual crops.
Landuse systems Species diversity
Paddy fields 2.10 0.07a Out of 30 species recorded from paddy fields
Polyculture farms 2.13 0.01a seven species namely Acaulospora lacunose, A. mellea,
Homegardens 2.88 0.08b A. scrobiculata, Glomus aggregatum, G. clarum,
Arecanut mixed with perennial
2.86 0.05b G. intraradices and G. maculosum are contributing to
crops
b
Arecanut mixed with annual crops 2.67 0.15 more than 50% of total spore abundance. However, when
Coconut plantations 2.71 0.16b
these species are present in other landuse systems, their
Rubber plantations 2.83 0.15b
Arecanut plantations 2.56 0.15b spore abundance was lesser than that in paddy fields.

Baiju et al., 2012
Thus, due to landuse change the dominance of above 19 t h Kerala Sci ence Congress, KSCSTE,
mentioned species seems to decrease and at the same Thiruvananthapuram. 388-390.
time contribution to total spore abundance by the
Borowicz VA. 2001. Do arbuscular mycorrhizal fungi
constituent species becomes uniform. These two changes
alter plant-pathogen relations? Ecology 82:3057-3068.
lead to comparatively high species diversity index value
in majority of the landuse systems derived from paddy Chandrashekara UM, Balasundaran M, Sankaran
fields (Table 7). KV, Sujatha MP, Varma RV, Senapati BK, Sahgal
Similarity index value estimated for paddy field M. 2008. Conservation and sustainable management of
and landuse systems derived from it ranged from belowground biodiversity in the Kerala part of Nilgiri
0.55-0.74 with following order : homegardens (0.74)> Biosphere Reserve - Phase I. KFRI Research Report No.
polyculture fams (0.66)> rubber plantations (0.63)> 316. Kerala Forest Research Institute, Peechi, Kerala.
arecanut mixed with perennial crops (0.61)> arecanut
Das T, Das AK. 2005. Inventorying plant biodiversity in
with annual crops (0.59)> arecanut plantation (0.58)>
homegardens: a case study in Barak Valley, Assam,
coconut plantation (0.55). Thus, it is clear that
North East India. Curr Sci., 89:155-163.
transformation of paddy fields into different landuse
systems did not alter drastically the AM fungal species Dhar PP, Mridha MAU. 2007. Biodiversity of
composition. The present study also revealed that arbuscular mycorrhizal fungi in different trees of
changes in the aboveground plant species composition is Madhpur forest of Bangladesh. J For Res, 17:201-205.
drastic, as it is mainly triggered by farmers activities.
Fischer RA, Yates F. 1963. .Statistical Tables of
On the other hand, changes in the AM fungal species
Biological, Agricultural and Medical Research. Oliver
composition and spore abundance due to landuse change
and Boyd, UK.
appear to be a slow process.
Gerdemann JM, Nicolson TH. 1963. Spores of
ACKNOWLEDGEMENTS mycorrhizal endogone species extracted from soil by wet
We are grateful to Dr. R, Gnanaharan and sieving and decanting. Trans Br Mycol Soc., 46:235-240.
Dr. J. K. Sharma, former Directors, Kerala forest
Giller PS. 1996. The diversity of soil communities, the
Research Institute (KFRI) for their keen interest and
poor mans tropical rainforest. Biodivers. Conserv,
encouragement. Dr. K.G. Saxena, Jawaharlal Nehru
5:135-168.
University, New Delhi and K.S. Rao, University of
Delhi, New Delhi are gratefully acknowledged for their Kannan KP, Pushpangadan K. 1988. Agriculture
constant support. This study was supported by the Stagnation and Economic Growth in Kerala: An
Conservation and Sustainable Management of Explanatory Analysis. Working Paper No. 227, Centre
Belowground-Biodiversity (CSM-BGBD) Project of for Development Studies, Trivandrum.
TSBF Institute of CIAT, Nairobi.
Meharg AA, Cairney JWG. 2000. Co-evolution of
mycorrhizal symbionts and their hosts to metal-
REFERENCES
contaminated environments. Adv Ecol Res., 30:69-112.
Baiju EC, Chandrashekara UM. 2007.
Transformation of paddy fields to different landuse Miller RM, Jastrow JD. 1992. The application of VA
systems in Vazhikadavu Panchayat. In: Proceedings of mycorrhizae to ecosystem restoration and reclamation.
Baiju et al., 2012
In: Allen MF (ed) Mycorrhizal Functioning. Chapman arbuscular mycorrhizal fungi for rapid assessment of
and Hall, Ltd., London, England, 438-467. infection. Trans Br Mycol Soc., 55:158-161.
Mohanan C. 2003. Mycorrhizae in forest plantations: Porter WM. 1979. The most probable number method
association, diversity and exploitation in planting stock for enumerating infective propagules of vesicular
improvement. KFRI Research Report No. 252, Kerala arbuscular mycorrhizal fungi in soil. Aust J Soil Res.,
Forest Research Institute, Peechi, Kerala. 17:515-518.
Mohankumar V, Mahadevan A. 1987. Survey of Porter WM, Robson AD, Abbot LK. 1987. Field
vesicular arbuscular mycorrhizae in mangrove survey of the vesicular-arbuscular mycorrhizal fungi in
vegetation. Curr Sci., 55:936. relation to pH. J Appl Ecol., 24:659-662.
Mulligan ME, Smucker JM, Safir JF. 1985. Tillage Ragupathy S, Mahadevan A. 1993. Distribution of
modifications of dry edible bean root colonization by vesicular-arbuscular mycorrhizae in plants and
VAM fungi. Agron J, 77:140-144. rhizosphere soils of tropical plains, Tamil Nadu, India.
Mycorrhiza. 3:123-136.
Muthukumar T, Udaiyan K. 2000. Arbuscular
mycorrhizas of plants growing in the Western Ghats Ragupathy S, Mohankumar V, Mahadevan A. 1990.
region, Southern India. Mycorrhiza, 9:297-313. Occurrence of vesicular arbuscular mycorrhizae in
tropical hydrophytes. Aqua Botanica. 36:287-291.
Narayanan NC. 1995. Issues in sustainable landuse: a
micro-level study in Madakkathara area, Trichur District. Sankar S, Chandrashekara UM. 2002. Development
In: Pillai PP and Nair RP (eds) Understanding and testing of sustainable agroforestry models in
Ecologically Sustainable Economic Development. different agroclimatic zone of Kerala with emphasis on
Institute of Planning and Applied Economic Research, socio-cultural, economic, technical and institutional
Thrissur, Kerala, 87-103. factors affecting the sector. KFRI Research Report 234.
Kerala Forest Research Institute, Peechi, Kerala.
Ocampo JA, Hayman DS. 1980. Effect of pesticides on
mycorrhiza in field grown barley, maize and potatoes. Sankaran KV, Balasundaran M, Thomas TP, Sujatha
Trans. Br. Mycol Soc., 74:413-416. MP. 1993. Litter dynamics, microbial associations and
soil studies in Acaia auriculiformis plantations in Kerala,
Oehl F, Sieverding E, Ineichen K, Mder P, Boller T,
KFRI Research Report No. 91, Kerala Forest Research
Wiemken A. 2003. Impact of landuse intensity on the
Institute, Peechi, Kerala.
species diversity of arbuscular mycorrhizal fungi in
agroecosystems of Central Europe. Appl. Environ. Schenck NC, Perez Y. 1990. Manual for the
Microbiol., 69:2816-28240. Identification of Mycorrhizal Fungi. Synergistic
Publications, Gainesville.
Panikar PGK. 1980. Recent trends in area under
production of rice in Kerala. Working Paper No. 116, Sengupta A, Chaudhuri S. 1990. Vesicular-arbuscular
Centre for Development Studies, Trivandrum. mycorrhizal fungi in pioneer salt marsh plants in the
Ganges River Delta in West Bengal (India). Plant and
Philips JM, Hayman DS. 1970. Improved procedures
Soil, 122:111-113.
for clearing and staining parasitic and vesicular

Baiju et al., 2012
Shannon CE, Wiener W. 1963. The Mathematical Thampi CJ. 1995. Sustainable landuse; farming systems
Theory of Communication. University of Illinois Presss, and land policy. In: Pillai PP and Nair RP (eds)
Urbano. Understanding ecologically sustainable economic
development. Institute of Planning and Applied
Shi ZY, Wang FY, Wei YL, Chen YL. 2007.
Economic Research, Thrissur, Kerala. 75-86.
Observations of arbuscular mycorrhizas on
Dipterocarpaceae grown in tropical rainforest in China. Thapar HS, Khan SN. 1985. Distribution of
J Agri and Environ Sci., 2:247-254. mycorrhizal fungi in forest soils of India. Ind J For.
8:5-7.
Smith SE, Read DJ. 1997. Mycorrhizal Symbiosis, 2nd
edn. Academic Press Ltd., London, England. Visalakshi N. 1997. Dynamics of vesicular-arbuscular
mycorrhizae in two tropical dry evergreen forests, South
Strzemska J. 1975. Mycorrhiza in farm crops grown in
India. Int J Ecol Environ Sci., 23:25-36.
monoculture. In: Sanders et al., (eds) Endomycorrhizas.
Academic Press, London. 527-537.
Submit your articles online at jresearchbiology.com

Advantages
Affordable Charges
Quick processing
Extensive indexing

Original Research
Avian diversity in Barna Wetland of Narmada basin in Central India

Authors: ABSTRACT:
Satish Balapure, Sumana
Dutta and Vipin Vyas.
Observations were made on the occurrence and diversity of waterbirds in
Barna reservoir from March 2009 to February 2011. A total 63 species of water birds
Institution:
belonging to 7 order and 12 families were recorded from the wetland during the
Department of
Environmental Science and study. Migratory birds also visit the reservoir during winter season. Anatidae was the
Limnology, Barkatullah most dominant family recorded during winter, in terms of species richness and
University, Bhopal- 462026, population. The present paper deals with various types of habitats available in the
India. reservoir and its surrounding area. The mosaic of habitats makes it an unique
avifaunal refuge. Protected catchment area is also a supportive feature for the
conservation of bird species.
Corresponding author:
Vipin Vyas.
Email: Keywords:
secvip@yahoo.co.in Avian diversity, Barna wetland, Migratory birds.
Web Address: Article Citation:

http://jresearchbiology.com/ Satish Balapure, Sumana Dutta and Vipin Vyas.
documents/RA0253.pdf. Avian diversity in Barna Wetland of Narmada basin in central India.
Dates:
Received: 06 Jun 2012 Accepted: 21 Jun 2012 Published: 02 Jul 2012

460-468 | JRB | 2012 | Vol 2 | No 5

An International
Balapure et al., 2012
INTRODUCTION study has been conducted on the water birds of the Barna
Wetlands are defined as lands transitional reservoir, but a preliminary survey in the winter of
between terrestrial and aquatic eco-systems where the 2001- 2002 revealed a huge congregation of more than
water table is usually at or near the surface or the land is 20,000 birds on t he main wat er bo d y
covered by shallow water (Mitsch & Gosselink 1986). (Islam & Rahmani, 2004). This site has not been covered
The various reservoirs, shallow ponds and numerous during the Asian Waterfowl Census, and needs regular
tanks support wetland biodiversity and add to the monitoring of water birds.
countrys wetland wealth. It is estimated that freshwater
wetlands alone support 10 percent of the known range of MATERIALS AND METHODS
biodiversity in India (Deepa & Ramachandra 1999) Barna is one of the major irrigation projects of
including 2400 birds species and sub species of birds. Madhya Pradesh constructed by damming river Barna in
Wetlands in India occupy 58.1 million hectares, Raisen district near Bari village. This is located about
including areas under wet paddy cultivation 100 kms from Bhopal. Narmada basin is located at the
(Prasad et al., 2002) latitude 2250 13.5 N and longitude 7750 78.10 E.
Wetlands and waterbirds are inseparable This reservoir is an important source of fish production
elements and support a rich array of waterbird in the area. Regular fish stoking is done in this reservoir
communities (Grimmett and Inskipp, 2007). Waterbirds every year. The water of the reservoir is mainly used for
are an important component of most of the wetland the fisheries and irrigation purpose. The reservoir
ecosystem as they occupy several trophic levels in the supports a rich biodiversity and provide habitat for
food web of wetland nutrient cycles. Activities of wildlife including migratory birds. A major part of the
waterbirds are considered as indicator of quality of the reservoir basin falls under Singhori Wild Life Sanctuary.
wetland ecosystem and form the terminal links in many Wetland habitats:
aquatic food chains, and as a result they reflect changes Study of avifaunal diversity of Barna wetland
originating in several different ecosystem components was conducted between March, 2009 and February,
(Custer and Osborne, 1977). Parish and Prentice (1989), 2011. Seasonal observations were made during the study
suggested various direct and indirect factors that causes but they were clubbed into four observations for further
deterioration of wetlands all over the world. Factors that analysis. Birds were observed within the transect of
cause wetland deterioration like vegetation changes, 300m. Nikon Binoculars of 1050 were used for
deterioration of water quality, siltation, cattle grazing and observations. The field book of Ali and Ripley (1986),
poaching were recorded in India by Vijayan (1986), Ali (1996) were used to identify bird species. The
Anjaneyulu (1991), Sampath (1993) and Trisal (l993). checklist was prepared using the standardized common
No in-depth study of ecology of wetlands as and scientific names of the birds of the Indian
birds habitat has been conducted in Madhya Pradesh. subcontinent by Manakkadan and Pittie (2001).
Some sporadic references are available that too limited Data analysis:
to check listing of birds. Wetland ecology has been The statistical analysis was carried out using
studied in isolation lacking avian ecology. Barna wetland software Paleontological Statics (PAST) version 2.04
of Madhya Pradesh is an identified wetland under (Hammer et al., 2001) to find out the significance of
National Wetland Conservation Programme by Ministry spatial variation on biological parameters. Margalefs
of Environment and Forests (Govt. of India). No detailed species richness (d), Shannon-Wiener diversity (H) and
Simpsons index (D) was calculated. Jaccards similarity winged stilt. Rathore and Sharma (1999) also reported
cluster was constructed based on bird species richness. Anatidae to be dominating family with 11 species in
Sarsai Nawar in UP. Surana (2007) recorded Anatidae to
RESULTS AND DISCUSSION: be most dominant family with 11 species, Ardeidae with
In the present study 63 bird species were nine species and Charadriidae with eight species in
recorded from Barna reservoir belonging to 12 families Chimdi lake of Nepal. Tak et al., (2010) reported 31
and seven orders (Table: 1). Das and Saikia 2011 water birds from Hathnikund wetland of Haryana with
recorded 39 species of water birds from 16 different maximum richness of Anatidae (35%) followed by
families from Deepor beel (Ramsar site) of Assam. Ardeidae (19.6%). Rajashekara and Venkatesha (2011)
Family Anatidae was found to be the most dominant recorded Ardeidae to be the most dominating family
family represented by 16 species followed by Ardeidae having nine species followed by Anatidae family having
with nine species and Charadriidae with eight species five species from Bangalore lake.
(Fig:1). Family Recurvirostridae had only one species Site-1, Site-4 and Site-7 harbored maximum bird
Himantopus himantopus commonly known as Black species richness compared to the other sites. The
Map of Barna reservoir in Narmada basin of central India were as study sites are
located zone wise

maximum diversity of water birds was noted at Site-4 as Koradi lake of Nagpur among which 54 species were
47 species followed by Site-7 having 45 bird species, recorded as resident; 09 species as seasonal local migrant
Site-1 with 31 species (Fig:2). These sites were and 13 species as winter migrant. There was a gradual
characterized as shallower zones of the reservoir. decline in species richness in the reservoir as the weather
Minimum water bird assemblage was noted at Site-8, condition changes from colder to warmer. A sharp
Site-9 and Site-2. Family Anatidae, Phalacrocoracidae, decline was recorded during summer 35 species of water
Rallidae and Alcedinidae were the abundant families of birds in the reservoir belonging to 12 families and 7
Barna reservoir which were observed at all the sites orders. With the onset of monsoon in June and July, the
comprised of both shallower zone as well as deeper zone. field conditions are unsuitable for birds and the bird
Family Ardeidae, Ciconiidae, Threskiornithidae, occurrence was significantly low in all the study areas.
Gruidae, Recurvirostridae, Charadriidae and Laridae The diversity of birds vary according to the weather
were restricted only in shallower zone of the reservoir. conditions like rainfall, temperature, relative humidity
Kurup (1991) attributed it to the larger mudflat areas and precipitation changes have a profound effect on
which attract shorebirds in large numbers. Cormorants water level, cover ratio and the quality of the habitat for
were noted both in shallow and deeper zone of the the water birds (Rao et al., 1997). Anatidae family which
reservoir. Waders were restricted to only shallow waters was the most dominant family during winter period was
having a depth of 2 mt. Deep water has been reported to represented by sixteen species. This indicates that most
reduce the availability and accessibility of invertebrates of the wintering water birds belong to Anatidae family.
to feeding waders (Murkin and Kadlec, 1986). Vyas et al., (2010) observed that Anatidae was the most
Murkin et al., (1997) found that diving ducks as a group dominating family in Bhoj wetland. The second most
selected habitat that consisted of deeper water and less dominating family was Charadriidae represented by 10
vegetation. species followed by Ardeidae represented by nine
There was a temporal variation in bird diversity species. Vijayan (1988) while working on Bharatpur
(Fig:3). The maximum diversity of birds was noted in wetland also recorded similar observations. Members of
winter season. Similar observation was noted by Anatidae family were found to dominate among the
Rajashekara and Venkatesha (2011) in 15 major lakes of winter migratory birds. Coot was the only dominant
Bangalore during 2008. Chinchkhede and Kedar (2012) migratory bird belonging to family Rallidae which could
reported 76 species belonging to 15 orders in and around not be recorded after Monsoon season in Barna reservoir.
50 Alcedinidae
45 Laridae
Charadriidae
40
Recurvirostridae
35
Species
Rallidae
30
Gruidae
25 Anatidae
20 Threskionithidae
15 Ciconidae
Ardeidae
10
Phalacrocoracidae
5
Podiciptidae
0
S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9
Fig 1: Family wise percentage composition of Fig 2: Spatial variation in species richness of
wetland birds in Barna reservoir wetland birds in Barna reservoir

100%
Alcedinidae
Laridae
80%
Charadriidae
Species percentage
Recurvirostridae
60% Rallidae
Gruidae
Anatidae
40% Threskionithidae
Ciconidae
Ardeidae
20%
Phalacrocoracidae
Podiciptidae
0%
Winter Summer Monsoon Post Monsoon
Fig 3: Temporal variation in species composition of Fig 4: Variation in site specific diversity and richness
wetland birds of Barna reservoir. indices among sites at Barna reservoir.
Family Ardeidae comprised of Egrets and herons showed Site-4 (6.14), followed by Site-7 (5.87) and Site-1 5.05).
an increasing population trend from the beginning of the Lowest species richness was recorded at Site-3, Site-6
summer season. Vijayan (1987, 1988) recorded increase and Site-9 having a depth of 4-6 mt.
in egrets population during monsoon period due to their The dendogram constructed showed the
breeding season. similarity of the sites according to the species richness of
The various diversity indices for water birds are the water birds present at nine sites of Barna reservoir
shown in Figure 4. The higher value for Shannons index (Fig: 5). Two main clusters were formed; first cluster
was observed at Site-4 (2.554) followed by Site-5 (2.4). was formed with Site-1, Site-4 and Site-7. Among the
Simpsons diversity index was highest at Site-6 followed first cluster Site-4 and Site-7 forms a subgroup. These
by Site-5 and Site-4. Species richness was highest at two sites exhibit maximum species diversity as compared
S-5
S-6
S-9
S-8
S-2
S-3
S-4
S-7
S-1
0.96
0.88
0.8
0.72
Similarity
0.64
0.56
0.48
0.4
0.32
0.24
0 1 2 3 4 5 6 7 8 9
Fig 5: Cluster analysis showing similarity in species richness of different sites of Barna reservoir.

Table 1: List of birds recorded in Barna reservoir during March 2009- February 2011
S. No Zoological Name Common Name Scientific Name
1.Order : Podicipediformes
1 1. Family- Podiciptidae Little Grebe Tachybaptus ruficollis
2 Great Crested Grebe Podiceps cristatus
2. Order: Pelecaniformes
3 2. Family - Phalacrocoracidae Great Cormorant Phalacrocorax carbo
4 Indian Shag Phalacrocorax fuscicollis
5 Little Cormorant Phalacrocorax niger
6 Darter Anhinga melanogaster
3. Order : Ciconiformes
7 3. Family Ardeidae Large Egret Casmerodius albus
8 Little Egret Egretta garzetta
9 Median Egret Mesophoyx intermedia
10 Cattle Egret Bubulcus ibis
11 Grey Heron Ardea cinerea
12 Purple Heron Ardea purpurea
13 Little Green Heron Butorides striatus
14 Black-Crowned Night-Heron Nycticorax nycticorax
15 Indian Pond Heron Ardeola grayii
16 4. Family Ciconidae Painted Stork Mycteria leucocephala
17 Asian Openbilled Stork Anastomus oscitans
18 White-Necked Stork Ciconia episcopus
19 5. Family Threskionithidae Black Ibis Pseudibis papillosa
20 Oriental White Ibis Threskiornis melanocephalus
21 Eurasian Spoonbill Platalea leucorodia
4. Order : Anseriformes
22 6. Family Anatidae Greyleg Goose Anser anser
23 Bar headed Goose Anser indicus
24 Brahminy Shelduck Tadorna ferruginea
25 Common Shelduck Tadorna tadorna
26 Comb Duck Sarkidiornis melanotos
27 Lesser Whistling -Duck Dendrocygna javanica
28 Northern Pintail Anas acuta
29 Common Teal Anas crecca
30 Spottbill Duck Anas poecilorhyncha
31 Gadwall Anas strepera
32 Eurasian Wigeon Anas penelope
33 Northern Shoveler Anas clypeata
34 Red carested Pochard Rhodonessa rufina
35 Common Pochard Aythya ferina
36 Tufted Pochard Aythya fuligula
37 Cotton Teal Nettapus coromandelianus
5. Order : Gruiformes
38 7. Family- Gruidae Common Crane Grus grus
39 Sarus Crane Grus antigone
40 8. Family- Rallidae Whitebreasted Waterhen Amaurornis phoenicurus
41 Watercock Gallicrex cinerea
42 Common Moorhen Gallinula chloropus
43 Purple Moorhen Porphyrio porphyrio
44 Common Coot Fulica atra
6. Order : Charadriiformes
45 9. Family- Recurvirostridae Black-winged Stilt Himantopus himantopus

46 10. Family Charadriidae Red-wattled Lapwing Vanellus indicus

47 River Lapwing Vanellus duvaucelii
48 Little Ringed Plover Charadrius dubius
49 Kentish Plover Charadrius alexandrinus
50 Marsh Sandpiper Tringa stagnatilis
51 Wood Sandpiper Tringa glareola
52 Common Sandpiper Actitis hypoleucos
53 Curlew Sandpiper Calidris ferruginea
54 Little Stint Calidris minutus
55 Dunlin Calidris alpine
56 11. Family Laridae Brown-headed Gull Larus brunnicephalus
57 Yellow-Legged Gull Larus cachinnans
58 Common Tern Sterna hirundo
59 River Tern Sterna aurantia
60 Little Tearn Sterna albifrons
7. Order : Coraciiformes
61 12. Family Alcedinidae Whitebrested Kingfisher Halcyon smyrnensis
62 Small Blue Kingfisher Alcedo atthis
63 Lesser Pied Kingfisher Ceryle rudis
to the other sites having shallow depth (0-2 mt) with rich spatial and temporal segregation among the water bird
marophytic vegetation which serves food for water reflected the different requirements that are met by these
birds. According to Mitchell & Prepas (1990) and limnologically variables. Higher values of Shannons and
Clifford (1991) shallow lakes with broad littoral zones Margalef indices indicated rich water bird occurrence
and abundant macrophytes and macroinvertebrates and species richness at Site 1, Site 4 and Site 7. Water
provide excellent habitat for aquatic birds. Second bird abundance status was calculated according to Kumar
cluster was formed with Site-2, Site-3, Site-5, Site-6, & Gupta (2009). Sarus Crane which was declared as
Site-8 and Site-9. In the second cluster, Sites 6 & 9 vulnerable species in Important Bird areas of India
formed sub cluster because of low richness of bird (Islam & Rahmani, 2004) recorded with a total
species may be due to their similar depth profile. These number of 25 from the shoreline of the wetland. Three
sites come under deeper zone having a depth of 4-6 n e ar t h r e at e n e d s p e c ie s n a me l y Dar t er
meters. Maximum depth was the another morphometric (Anhinga m e l a n o g a s t e r) , Pa int e d Stork
feature that showed strong correlations with guild (Mycteria leucocephala) and Oriental white Ibis
composition and richness of water birds (Paszkowski and (Threskiornis melanocephalus). We have recorded nearly
Tonn, 2006). 57 Darter, 53 Painted stork and 12 Oriental white Ibis
during the entire study period from the wetland.
CONCLUSION: Conservation of this wetland is very essential to sustain
It is revealed that overall, 63 species belonging to migratory bird populations along with the threatened and
12 families and 7 orders were documented which vulnerable birds of that area.
included 36% residents, 35% local migrants and 29% as
migratory species. Among these 24.5% were considered ACKNOWLEDGEMENT:
to be common, 23.81% fairly common, 31.75% The authors are thankful to Dr. Pradeep
uncommon and 19.05 % rare species. From the present Shrivastava, Professor, Department of Environmental
study it was revealed that existence of various patterns of Science and Limnology, Barkatullah University, Bhopal

(India) for his support and encouragement throughout the and data analysis. Paleontologia Electronica. 4:9.
study period.
Islam MZ and Rahmani AR. 2004. Important Bird
Areas in India priority sites for conservation. Ed Bombay
REFERENCE
Natural History Society. Oxford University Press. 1152.
Anjaneyulu M. 1991. Status of wetland and survery of
avifauna of Kollern lake in Andhra Pradesh, India. Ph.D. Kumar P and Gupta SK. 2009. Diversity and
Thesis submitted to Osmania University. Hyderabad. abundance of wetland birds around Kurukshetra, India.
Our Nature. 7:212-217
Ali S. 1996. The book of Indian birds. 11thEdition,
Bombay Natural History Society and Oxford University Kurup DN. 1991. Migrant shore birds in estuarine
Press, Bombay. habitats with special reference to Kadalundi and
Bharathapuzha estuaries. In Proceedings of the third
Ali S and Ripley SD. 1986. Hand book of the birds of
Kerala Science Congress, Feb-March, 1991, Kozhikode.
India and Pakistan. Compact ed., Oxford University
31-32.
Press, New Delhi.
Manakkadan R and Pittie A. 2001. Standardized
Custer TW and Osborne RG. 1977. Wading birds as
common and scientific names of the birds of the Indian
biological indicators. Long survey, U. S. Fish and
subcontinent. Buceros 6(1):1-37.
Wildlife Service, Washington, DC.
Mitsch WI and Gosselink IG. 1986. Wetlands. Van
Chinchkhede KH and Kedar GT. 2012. Avifaunal
Nostrand Reinhold, New York.
diversity of Koradi Lake in Nagpur District of central
India. Journal of Research in Biology 2:070-076. Mitchell PA and Prepas EE. 1990. Atlas of Alberta
Lakes. University of Alberta Press, Edmonton.
Clifford H. 1991. Aquatic Invertebrates of Alberta.
University of Alberta Press, Edmonton. Murkin HR and Kadlec JA. 986. Relationship between
waterfowl and macroinvertebrate densities in a northern
Das J and Saikia PK. 2011. Conservation threats
prairie marsh. J. Wildl. Manage, 50(2):212-217.
to the water birds in Deepor Beel, Assam
Journal of Research in Biology. 6:435-439. Murkin HR, Murkin EJ and Ball JP. 1997. Avian
habitat selection and prairie wetland dynamics: a 10-year
Deepa RS and Ramachandra TV. 1999. Impact of
experiment. Ecol. Apps. 7:1144-1159.
Urbanization in the Interconnectivity of Wetlands. Paper
presented at the National Symposium on Remote Sensing Paszkowski CA and Tonn WM. 2006. Foraging guilds
Applications for Natural Resources: Retrospective and of aquatic birds on productive boreal lakes:
Perspective (XIX-XXI 1999), Indian Society of Remote environmental relations and concordance patterns.
Sensing, Bangalore. Hydrobiologia 567:19-30
Grimmett R an Inskipp T. 2007. Birds of southern Parish D and Prentice R. 1989. Wetland and Waterfowl
India. Om Books International, New Delhi, India. Conservation in Asia, Asian Wetland Bureau, Kuala
Lumpur, Malaysia and International Waterfowl Research
Hammer O, Harper DAT and Ryan PD. 2001. PAST:
Bureau, Slimbridge, U.K.
Paleontological Statics Software Package for education

Prasad SN, Ramachandra TV, Ahalya N, Sengupta T, Vijayan VS. 1986. On conserving the bird fauna of
Kumar A, Tiwari AK, Vijayan VS and Vijayan L. Indian Wetlands. Proc. Indian Acad Sci. (Suppl)
2002. Conservation of wetlands of India-a review. 91-101.0000555.
Tropical Ecology 43(1):173-186.
Vijayan VS. 1987. Keoladeo National park ecology
Rajashekara S and Venkatesha MG. 2011. study. Annual report 1986. Bombay Natural Histry
Community composition of aquatic birds in lakes of Society.
Bangalore, India. Journal of Environmental Biology.
Vijayan VS. 1988. Keoladeo National park ecology
32(1):77-83.
study. Annual report 1987. Bombay Natural Histry
Rao VV, Srinivavasulu C, Nagulu V and Reddy MC. Society.
1997. Population fluctuation species variation and habitat
Vyas V, Vishwakarma M and Dhar N. 2010. Avian
utilization of egrets and herons at selected water boodles
Diversity of Bhoj Wetland: A Ramsar Site of Central
of Nalgonda district, Andhra Pradesh. Pavo,
India. Our Nature 8:34-39.
35(1& 2):101-110.
Rathore and Sharma. 1999. Avifauna of a lake in

district Etawah, Uttar Pradesh, India. Zoos Print
15(6): 275-278.
Sampath K. 1993. Ecological e'faluation of irrigation

tanks in the Thiruvanamallai Sambuvaryar, south district
of Tamil Nadu. In: Bird conservation: Strategies for the
Ninenties and beyond, A.Varghese, SSridhar, and A.K.
Chakaravarthy (eds.),OrnithologicaI society of India.
Bangalore. 142-144.
Surana R. 2007. Avian diversity during rehabilitation

stage of Chimdi lake, Sunsari, Nepal. Our Nature
5(1):75-80.
Tak PC, Sati JP and Rizvi AN. 2010. Status of

waterbirds at Hathnikund Barrage wetland,
Yamunanagar District, Haryana, India. Journal of Submit your articles online at jresearchbiology.com
Threatened Taxa. 2(4):841-844. Advantages

Trisal CL. 1993. Conservation of wetland in India and Complete Peer review
Affordable Charges
international treaties. In . proc. Int.symp.on wetland and Quick processing
waterfowl conservation in south and west Asia, Karachi, Extensive indexing
Pakistan. M.Moser, and J.Van Vessen(Eds.), Asian
wetland Bureau, Kuala Lumpur, Malaysia, 41-49. submit@jresearchbiology.com

Original Research
Nutritional evaluation of Moringa oleifera leaves using three drying

methods
Authors: ABSTRACT:
Glover-Amengor M and
Mensah F. Moringa oleifera, is a tropical plant with many useful parts. Nutritionally, it is
noted for high protein and vitamin A content. In recent times a lot of interest has been
generated in the nutritional benefits of this plant, so there is a need to process it in a
cost effective manner that will conserve the nutrients and ensure its availability as a
Institution:
food supplement. The objective of this study is to determine the optimal conditions
CSIR-Food Research
Institute, P. O. Box M20, (method, temperature and humidity) for drying Moringa oleifera leaves for maximum
Accra, Ghana. nutrient conservation. Leaves of M. oleifera were either solar, mechanical or room
temperature dried and milled into powder. The powders were analysed for moisture
and protein by proximate method; vitamin C by indophenol method; vitamins A,
Corresponding author: vitamin E, and luthein/zeaxanthin using HPLC. pH was measured with a pH meter;
Glover-Amengor M. water activity by dielectric & conductivity method; mould and yeast by ISO 7954
(1987) and mycotoxins by HPLC. The fresh leaves were also analysed. The results
showed that drying decreased protein levels in the leaves up to 19%. Vitamin levels
decreased (63% to 85%) depending on vitamin type, with all the drying methods used.
Email: Although beta-carotene and vitamin C levels were less affected by drying at room
mayamen11@yahoo.com temperature, this method did not offer convenient moisture content and water
fostermensah@hotmail.com activity for good storage of powder. Both solar and mechanical drying offered
products with good moisture and water activity levels that are convenient for storage
as well as appreciable nutrient levels.
Phone No:
Keywords:
+233-302-760474.
0243488814. Moringa oleifera, drying methods, beta carotene, vitamin C, water activity.
Web Address: Article Citation:

http://jresearchbiology.com/ Glover-Amengor M and Mensah F.
documents/RA0258.pdf. Nutritional evaluation of Moringa oleifera leaves using three drying methods.
Dates:

469-473 | JRB | 2012 | Vol 2 | No 5

An International
Glover-Amengor and Mensah, 2012
INTRODUCTION Proximate Analysis of M. oleifera fresh (raw)

Moringa oleifera (M. oleifera) is one of about leaves and leaf powder (dried and milled leaves) showed
thirteen species in the Moringaceae family. The plant, the following per 100g. of edible portion (Fuglie, 1999):
originating from the southern foothills of the Himalayas For the fresh leaves - Moisture 75.0%, Calories 92.0,
in northwestern India, is now cultivated in Africa, Protein 6.7g, Fat 1.7g, Carbohydrate 13.4g, Vitamin
Central and South America and other tropical, A - B carotene 6.8 mg, Vitamin B1 - thiamin 0.21 mg,
sub-tropical and semi-arid regions of the world. Vitamin B2 - riboflavin 0.05mg, Vitamin B3 - nicotinic
M. oleifera is a plant with many useful parts. Every part acid 0.8 mg, Vitamin C - ascorbic acid 220.0 mg,and for
of the plant is useful as food, medicine, or for industrial the leaf powder - Moisture 7.5%, Calories 205.0, Protein
purposes (Ramachandran et al., 1980; Babu, 2000; 27.1g, Fat 2.7g, Carbohydrate 38.2g, Vitamin A - B
Fahey, 2005; Khalafalla et al., 2010; Saint Saveur et al., carotene 18.9mg, Vitamin B1 thiamin 2.64mg, Vitamin
2010). Humans use its leaves, flowers and young pods B2 - riboflavin 20.5mg, Vitamin B3 - nicotinic acid
as vegetables whilst others use it as fodder 8.2mg, Vitamin C - ascorbic acid 17.3mg, Vitamin
(Moyo et al., 2011). M. oleifera is used to treat E - tocopherol acetate 113.0mg.
malnutrition, anaemia and to correct vitamin A Emphasis, however, had been on room
deficiency in several countries (Fuglie, 1999). temperature drying of leaves for maximum nutrient
The plant, which produces leaves throughout the conservation (Fuglie, 1999), without due consideration
year, is reported to be tolerant to bacteria, drought, for storage conditions of the final product. Alternative
fungus and mycobacteria and could therefore be easily drying methods have not been thoroughly investigated.
cultivated in the tropics. It is a readily available backyard M. oleifera should be processed in a cost effective
tree in homes where it will provide the much-needed manner that will remove drudgery and increase the
nutrientrich leaves for household nutrition volume of production to meet the increasing demand for
(Luu et al., 2005; Saint Saveur et al., 2010). Nutrients in the product (room drying takes about 96 hours as against
M. oleifera are found mainly in the leaves and tender 4-5 hours by solar and mechanical drying). The
pods. M. oleifera leaves contain high levels of vitamins processing method should also ensure a very good
A, B and C (Fuglie, 1999) as well as high calcium and product in terms of nutrient conservation. The current
iron, but are low in phosphorus. The leaves also contain study aims at determining the optimal conditions
appreciable amounts of magnesium, selenium and zinc. (method, temperature and humidity) for drying
Additionally the leaves are high in protein content but M. oleifera leaves in order to ensure a good product and
are low in fat and carbohydrates. M. oleifera leaves are conserve appreciable nutrient levels.
also rich in the sulphur-containing amino acids
methionine and cystine that are often not found in MATERIALS AND METHODS
abundance. The leaves have negligible amounts of M. oleifera leaves were sampled from the coastal
tannins. Trypsin and amylase inhibitors, lectins, belt of Ghana. Three different samples from the same
cyanogenicglucosides and glucosinolates were not field were tested. Samples were collected at two week
detected in the leaves (Makkar and Becker, 1997). If the intervals, very early in the morning, and transported in an
benefits of this plant resource are well tapped, it has the ice chest to the laboratory at CSIR-Food Research
potential to improve food and nutrition security and Institute. Samples were then washed first with potable
promote rural development. water to remove loose dust, followed by 1% saline for
three minutes (FDGS 998), and finally rinsed with
Aflatoxin
0
0
0
0
0
0
0
0
0
0
0
0
potable water.
Samples were either mechanically dried at 50oC
2.3 x 103
1.6 x 104
8.0 x 101
6.3 x 103
2.6 x 104
7.0 x 101
2.5 x 104
5.5 x 102
3.2 x 103
1.9 x 105
1.4 x 105
3.8 x107
mould count
Yeast and
and 55C using an electric dryer, or solar dried
(cfu/g)
(35C -55C) with a GIZ constructed solar dryer or dried
at room temperature (28C -31C). Three replicates were
5.27
5.46
6.10
5.35
5.56
5.84
5.69
6.01
5.89
5.70
5.70
5.40
pH
run for each drying method to ensure that variation was
due to the drying method and not the sample itself. The
0.607/27.8C
0.425/28.0C
0.385/29.0C
0.599/28.1C
0.435/28.2C
0.468/28.0C
0.723/27.3C
0.637/27.6C
0.612/28.0C
NA
NA
NA
activity
samples were milled with a stainless steel hammer mill
Water
into powder of particle size 0.3mm (FDGS 998) which
was put into clean polythene bags.
Table 1: Various drying methods and nutrient levels in M. oleifera leaves
1978.3
1483.4
1400.0
1620.6
2796.5
2289.9
2494.7
2527.0
1828.7
1637.0
1598.2
1688.0
4635.0
3272.7
2043.8
3317.2
Lutein+zeaxanthin
For each drying method, the following tests were
(g/100g DW)
performed: moisture, protein, vitamin C, vitamin A,
vitamin E, pH, water activity, mould and yeast and
mycotoxins. Tests were also performed on the fresh
128
160
172
153
74
101
131
102
98
108
125
111
974
545
555
692
(mg/100g DW)
a-tocopherol
leaves. Crude protein was determined by the Kjeldahl
method of AOAC 984.13 (1990); Moisture by
AOAC 925.10 (1990). Vitamin C was determined by the
Indophenol method and vitamins A, and E, were
27
31
34
31
27
40
22
30
57
38
36
43
168
240
135
181
(mg/100g DW)
Beta-caroten
determined by HPLC (AOAC 992), Water activity was

measured by dielectric & conductivity method, using the
Rotronic HygroLab water activity measuring instrument,
16
12
14
14
10
18
17
15
23
19
19
21
47
52
71
57
Vitamin C
(mg/100g
DW)
while pH was measured with Lab Ph Meter

(Radiometer, Copenhagen). Mould and yeast were
26%
23%
27%
26%
23%
28%
27%
23%
29%
32%
31%
30%
determined by ISO 7954 (1987). Mycotoxins were
25%
26%
26%
31%
Proteins
(g/100g
DW)
determined by HPLC Methods (Pons, W.A (1979)

JAOAC 62, 586-594 (extraction procedure).
8.0
3.6
5.0
5.5
8.1
4.8
6.2
6.4
13.6
10.2
9.9
11.2
72.6
72.5
72.6
72.6
Moisture
(g/100g)
RESULTS AND DISCUSSION

3,5h
4h
5h
4h
6h
6h
48h
96h
96h
-
-
-
Time
The results of the analysis is presented in Table 1

Drying conditions
Leaf drying caused a mean reduction of 19% of

35-55C
35-55C
35-55C
55C
50C
50C
31C
31C
31C
-
-
-
Temperature
protein with solar drying and a mean of 16% with

mechanical drying; while vitamin C reduction ranged
from 63% with room temperature drying to 75% with
1
2
3
Mean
1
2
3
Mean
1
2
3
Mean
1
2
3
Mean
Batch
solar drying. Mechanical drying recorded a mean

reduction of 73.7%. Beta carotene levels reduced by
temperature
Solar drying
Fresh leaves
82.9% with solar drying, 83.4% with mechanical and

Mecanical
76.2% with room temperature drying. Reduction in

drying
Room
vitamin E levels ranged from 77% with solar to 85%

with mechanical. Beta carotene and vitamin C levels storage. The beta carotene content of the dry leaves are
were least affected by room temperature drying, high enough to meet the RDAs of children. Ten (10)g
however, room temperature drying did not offer leaf powder will meet the RDA of children 1-13 years
convenient moisture and water activity levels for good and 50-100% of all other age groups.
storage (moisture >8%; water activity >0.7 (FDGS 999).
Leaf drying did not affect pH. Mould and yeast levels CONCLUSION AND RECOMMENDATIONS
were also not affected by the type of drying method; they Both solar and mechanical dryers could be
were highly variable with each sample, but were below satisfactorily used for Moringa leaf powder production as
acceptable levels (FDGS 999). Aflatoxin was not they both offer convenient storage conditions in terms of
detected in any of the dried samples, indicating that all moisture, water activity and acceptable nutrient levels.
methods offered satisfactory conditions with regard to However, since solar energy is readily available in the
aflatoxin content. Mechanical drying offered an tropics, solar dryers would have an advantage in that the
advantage over solar with regards to lutein/zeaxanthin cost of production will be minimised. Hence there is a
(23.8% reduction as against 51.1% with solar drying), potential of preparing good quality, nutrient dense
but this nutrient is likely to be lost during storage Moringa leaf powder at minimal cost in the tropics and
because it is highly unstable. sub-tropics, using solar dryers, that could be a readily
The protein level in the leaves ranged from 25% available supplement for preventing and fighting
for solar drying to 26% for mechanical and room malnutrition. It could also serve as a source of income
temperature drying with the protein content of fresh generation for rural folks who could process these leaves
leaves being 31% (DW). The protein content of the dried for the big markets.Simple solar dryers could be
leaves compares favourably with the value of 27.1% constructed from local materials and used for this
reported by Fuglie (Fuglie,1999) and 27.51 reported by purpose (Saint Saveur et al., 2010). Studies on the
Oduro et al., (2008). Ogbe and Affiku, however, reported shelf-life of the leaf powder is recommended as well as
a value of 17.01% in leaves from Nigeria, whilst supplementation trial with Moringa leaf powder.
Jongrungruangchok et al., (2010) reported 23.29% in
leaves cultivated in Thailand and (Moyo et al., 2011) ACKNOWLEDGEMENT
reported 30.29mg/100g for leaves from South Africa. This research was conducted with support from
Therefore, although leaf drying in the current study Moringanews.
decreased the protein level by up to 19%, values
obtained were still high for the production of a protein REFERENCES
concentrate that could be stored and used in treating Babu Suresh Chamba. 2000. Rural nutrition
protein - energy malnutrition. intervention with indigenous plant foods a case study
The vitamin C levels obtained for fresh of vitamin A deficiency in Malawi. Biotechnol.Agron.
M. oleifera leaves in this study were lower than values Soc. Environ., 4(3):169-179.
reported in literature (120- 200mg/100g fresh weight or
Fahey JW. 2005. Moringa oleifera: A review of the
360- 600mg/100g DW -Saint Saveur et al., 2010). The
Medical Evidence for Its Nutritional, Therapeutic and
comparatively low vitamin C values could not make the
Prophylactic Properties. Part 1. Trees for Life Journal.
powder a good source of this vitamin, but vitamin C
www.TFJournal.org.
itself is very unstable and is likely to be lost during
Fuglie LJ. 1999. The Miracle Tree: Moringa oleifera: Oduro I, Ellis WO and Owusu D. 2008. Nutritional
Natural Nutrition for the Tropics. Church World Service, Potential of two leafy vegetables: Moringa oleifera and
Dakar. 68 pp.; revised in 2001 and published as The Ipomoea batatas leaves. Scientific Research and Essay
Miracle Tree: The Multiple Attributes of Moringa, 3(2):059.
172 pp. http://www.echotech.org/
Ogbe AO and Affiku JP. 2011. Proximate Study,
G hana Standar d B oar d. 2009. Gh ana Mineral and Anti-Nutrient Composition of
Standard..Medicinal Plants Specifications for Moringa Moringa oleifera: Potential Benefits for Poultry
Leaf Products. FDGS 998. Nutrition and Health. Journal of Microbiology,
Biotechnology and Food Sciences 1(3):299.
G hana Standar d B oar d. 2009. Gh ana
Standard.Medicinal Plants Code of Practice for the Ramachandran C, Peter KV and Gopalakrishnan
production Moringa leaf products FDGS 999. PK. 1980. Drumstick (Moringa oleifera): A
multipurpose Indian Vegetable. Economic Botany
Jongrungruangchok S, Bunrathep S and Songsak T.
34(3):276-283.
2010. Nutrients and Minerals Content of Eleven
Different Samples of Moringa oleifera Cultivated in Saint Saveur, Armelle (de), Broin, Melanie (eds).
Thailand.J Health Res 24(3):125. 2010. MORINGA Growing and processing moringa
leaves. Moringanews/ Moringa Association of Ghana
Khalafalla MM, Abdellatef E, Dafalla HM,
15-29.
Nassrallah AA, Aboul-Enein KM, Lightfoot DA, El-
Deeb FE and El-Shemy HA. 2010. Active principle
from Moringa oleifera Lam leaves effective against two
leukemias and a hepatocarcinoma. African Journal of
Biotechnology 9(49):8467.
Luu Huu Manh, Nguyen NhutXuan Dung and Tran

Phung Ngoi. 2005. Introduction and evaluation of
Moringa oleifera for biomass production and feed for
goats in the Mekong Delta. Livestock Research for Rural
Development 17(9).
Makkar HPS and Becker K. 1997. Nutrient and

antiquality factors in different morphological parts of the Submit your articles online at jresearchbiology.com
Moringa oleifera tree. The Journal of Agricultural Advantages

Science 128:311-322.
Affordable Charges
Moyo B, Masika JP, Hugo A and Muchenje V. 2011. Quick processing
N ut r i t i on a l Ch a r a ct er i sa t i on of M o ri ng a Extensive indexing
(Moringa oleifera Lam.) leaves. African Journal of
Biotechnology. 10(60):12927. submit@jresearchbiology.com

Original Research
Studies on ground water pollution in industrial areas of Chennai

Authors: ABSTRACT:
Abdul Kadhar M, Arun
Kumar MS, Sivaraj S and In the present study an attempt has been made to evaluate the
Jawahar Ali A. physicochemical parameters of ground water samples collected from 10 selected
locations of five different industrial areas of Chennai. Physico-chemical parameters
such as DO, Salinity, Alkalinity, pH, Calcium, Magnesium, Hardness, Iron Nitrite,
Phosphate, Silica and Chloride were estimated following the procedures of NEERI.
Institution:
Highest nitrate level of (0.15mg/L) was observed at Guindy Industrial Estate, whereas
Unit of Aquaculture and
Aquatic Toxicology, P.G and the lowest level (0.02mg/L) was noticed at Avadi Industrial area. Silica content was
Research Department of found to be highest (66.67mg/L) at Ambatthur Industrial Estate and the lowest
Zoology, The New College, level (12.29mg/L) was observed at Guindy Industrial Estate. Highest iron content
Chennai 6000 14. India. (0.59mg/L) was recorded at Ambatthur Industrial Estate and the lowest level
(0.13mg/L) was noticed at Pallavaram Industrial area. Chloride level was highest
(861mg/L) at Ambatthur Industrial area and the lowest level (223mg/L) was noticed at
Pallavaram Industrial area. The results are discussed in the light of available literature.
Keywords:
Arun kumar MS. Physicochemical parameters, Ground water pollution, Permissible limit,
Industrial area.
Web Address:
http://jresearchbiology.com/ Article Citation:
documents/RA0259.pdf. Abdul kadhar M, Arun Kumar MS, Sivaraj S and Jawahar Ali A.
Studies on ground water pollution in industrial areas of Chennai.
Dates:

474-481 | JRB | 2012 | Vol 2 | No 5

An International
Kadhar et al., 2012
INTRODUCTION physico chemical properties of ground water in the

Water is extremely essential for the survival of industrial areas of Chennai.
all living organisms. Ground water is the ultimate and
most suitable fresh water resource for human MATERIALS AND METHODS
consumption in both urban as well as rural areas. The Ground water samples were collected during
importance of ground water for existence of human March 2008 from 10 selected locations of five different
society cannot be overemphasized. Increasing population industrial areas of Chennai namely, Guindy, Pallavaram,
and its necessities have led to the deterioration of surface Ambatthur, Avadi and Manali. Samples were collected in
and sub-surface water (Dhiviyaa Pranavam et al., 2011). one litre polythene sterilized bottles without any air
There are several states in India where more than 90% bubbles and transported to laboratory immediately for
populations are dependent on groundwater for drinking analysis.
and other purpose (Ramachandraiah, 2004). Ground The following Physico-Chemical parameters,
water is also frequently used as alternative source for pH, Chlorides, Sodium, Calcium, Magnesium,
agricultural and industrial sector. Phosphates, Nitrites, Iron, Silica, Chloride, Hardness,
The ground water pollution is caused by the Dissolved Oxygen, Salinity, Alkalinity and Turbidity
bleaching of industrial waste into the aquifers. Water were estimated by following procedure suggested
being a universal solvent, contains materials such as by NEERI and compared with standards of
bicarbonates, chlorides, sulphate, calcium, potassium and the World Health Organization (WHO) and the
sodium in ionized or dissolved forms, nitrite and iron are Bureau of Indian Standards (BIS) (Table.1).
also present in small amounts. Good quality drinking
water can be consumed in any desired amount without RESULTS
any adverse effect on health and also free from The results showed that the pH varied from
impurities is declared as potable. Improper use of water 6.0 to 7.5 (Fig.1). Magnesium hardness varied between
resources is causing catastrophic effects (Kumar, 2004). 30 and 75mg/L whereas calcium hardness ranges from
The most common problem in ground water is attributed 100 to 246mg/L as showed in Fig.2 & Fig. 3
to hardness, iron, sulphate, sodium chloride and acidity Dissolved oxygen (DO) content ranges from
(Ranjana, 2009). 0.6 to 1.8mg/L (Fig.4).
Ground water has been contaminated by various Salinit y levels were ranged between
ways, for e.g. application of chemical fertilizers in 0.3 and 1.6mg/L (Fig.5). Alkalinity was ranging from
agriculture field (Altman & Parizek, 1995), seepage from 260 to 776mg/L (Fig.6). Phosphate levels were varied
effluent bearing water body (Adekunle, 2009). Most of from 0.10 to 0.14mg/L (Fig.7).
the industries discharge their effluent without proper Nitrite levels were varied from 0.02 to 0.15mg/L.
treatment into nearby open pits or pass them through Highest nitrite level was observed at Guindy Industrial
unlined channels, resulting in the contamination of Estate (0.15mg/L) whereas the lowest level was noticed
ground water (Jinwal and Dixit, 2008). The incidence of at Avadi Industrial area 0.02mg/L (Fig.8). The highest
ground water pollution is highest in urban areas where iron content (0.59mg/L) was recorded at Ambatthur
large volumes of waste are concentrated and discharge Industrial Estate and the lowest level (0.13mg/L) was
into relatively small areas (Rao & Mamatha, 2004). observed at Pallavaram Industrial area (Fig.9).
The present study was conducted to evaluate the
Kadhar et al., 2012

Kadhar et al., 2012
from 10 different locations of five selected industrial

areas in Chennai city. The physical parameters were
analyzed for appearance, odour, and turbidity levels were
within the limits prescribed by WHO and BIS.
Water samples were colorless, clear and odourless
indicating the absence of colloidal substances, suspended
and decomposed vegetation. Chemical properties of
water were analyzed for pH, Dissolved Oxygen, salinity,
alkalinity, magnesium, calcium, phosphate, iron, silica
A-M.K.N. Road, Chennai-32, B-Thiru.Vi.Ka and chloride.
Industrial Estate, Chennai-32, C-Kannapiran Kovil
Street, Chennai-43, D-Narayana Samy Pillai Street, pH is a term used universally to express the
Chennai-43, E-Pillaiyar Kovil Street, Chennai-50, intensity of the acidic or alkaline condition of a solution.
F-M.T.H. Road, Chennai-50, G-Periyar Street,
Chennai-54, H-Jothiraman Street, Chennai-54, In this study the pH levels were found within the limits
I-Thillaipuram First Cross Street, Manali, set for domestic use as prescribed by WHO and Indian
Chennai-68, J-Thillaipuram First Street, Manali,
Chennai-68 standards. Most of the waters are slightly alkaline due to
the presence of carbonates and bicarbonates, the range
Silica levels were ranged bet ween less than or more than the permissible level is hazardous
12.29 and 66.67mg/L highest silica content (66.67mg/L) (Ravisankar & Poongothai, 2008).
was noticed at Ambatthur Industrial Estate and the In the present investigation, the magnesium
lowest level (12.29mg/L) was observed at Guindy hardness values are found within the permissible norms
Industrial Estate (Fig.10). In the present analysis, whereas the calcium hardness values found more than
chloride content ranges from 223 to 861mg/L highest the permissible limit of WHO and Indian Standards.
chloride content was found to be at Ambatthur Industrial Magnesium hardness shows minimum 30mg/L and
Estate (861mg/L) and lowest chloride content (223mg/L) maximum of 75mg/L, while calcium hardness shows
was recorded at Pallavaram industrial area (Fig.11). minimum 100mg/L and a maximum 246mg/L. Hardness
is defined as the sum of the polyvalent cations present in
DISCUSSION the water, notably calcium and magnesium. According to
In the present Study an attempt has been made to Dufor and Beckers, 1964 water with more than 180mg/L
study the Physico-Chemical parameters of ground water hardness is very hard. Excessive hardness may cause
Kadhar et al., 2012
Table1: Drinking Water Standards

WHO STANDARDS INDIAN STANDARDS
PHYSICAL PARAMETERS Permissible Excessive Permissible Excessive
Colour CLEAR CLEAR CLEAR CLEAR
Turbidity (NTU) 5 25 10 25
Odour Unobjectionable Unobjectionable
CHEMICAL PARAMETERS
pH Ranges 7 8.5 6.5 8.5
Dissolved Oxygen (mg/L) 0.3 1.1 - -
Salinity (mg/L) 0 5 0 5
Alkalinity (mg/L) 45 200 Not Perscribed
Ca Hardness (mg/L) 20 75 20 75
Mg Hardness (mg/L) 30 100 30 100
Po4 0.1 - - -
Nitrite (mg/L) 0.5 1 Not Perscribed
Iron (mg/L) 0.3 1 0.05 1.5
Silica (mg/L) 0 60 Not Perscribed
Chloride (mg/L) 200 600 250 1000
hea lt h hazar d s lik e k id ne y st o nes and Alkalinity of samples ranges between

other ailments (Jain et al., 1999). 260 and 776 mg/L which are above the normal limit
Dissolved oxygen (DO), a vital parameter in the prescribed by WHO indicates that water from all
stability of their aquatic eco-system, was found within locations are very hard. Vass et al., (1977) reported that
the prescribed norms of 0.6 to 1.8mg/L. It depends on total alkalinity value of 60mg/L or more indicates hard
physical chemical, biochemical and microbial factors. In water. Alkalinity of water depends on concentration of
general unpolluted water has dissolved oxygen level of hydroxides. Hydroxides are generally absent in natural
3 mg/L. Water depleted in oxygen provides shelter for waters however their concentration may increase in the
anaerobic bacteria which is injurious to human health polluted water. The water for domestic use
(Karnath, 1987). The range of salinity is within the having alkalinity less than 100mg/L is safe
permissible limit of WHO. Highest amount of salinity is (Saravanakumar and Ranjith Kumar, 2011).
recorded in Avadi industrial area whereas lowest amount Important sources of phosphate depend on
of salinity was observed in Guindy Industrial area. geochemical conditions, surface runoff from surrounding
Salinity in general depends on four major cations field and cattle dung. The high concentration of
namely, calcium, magnesium, sodium, potassium and phosphate give rise to an algal bloom and it also brings
major anions like bicarbonates, chlorides, carbonates and eutrophication (Pulle and Khan, 2003). Concentration of
phosphates Wetzel, (1983) reported that the phosphate in the samples ranges from 0.05 to 0.4mg/L.
concentration of sodium and chloride predominate in A fairly high amount of phosphate is found in Ambatthur
concentration which contribute to salinity. industrial Estate and low in Pallavaram industrial area.

Kadhar et al., 2012
Phosphate content may increase when contamination groundwater is almost exclusively and unequivocally a
with sewage occurs. In general high amount of phosphate result of water rock interaction (Hem, 1985, Ali Khan
that occurs in natural waters is usually harmful to and Umar 2010). Concentration of SiO2 in ground water
human health (Ellis et al., 1948). varies from 60 to 100mg/L is very rare. Higher
Concentration of nitrate varies from concentrations of silica are found in alkaline waters as
0.02 to 0.15mg/L and found to be within the permissible also in some acidic waters (Suerdrup et al., 1961).
limit of WHO. Highest level of nitrite was found in Relatively high concentration of chloride content
Manali industrial area and the lowest level was observed is observed in the collected groundwater samples. The
in Avadi industrial area. It may be due to agricultural amount of chloride ranges from 223 to 861mg/L all the
runoff from field (Wagh et al., 2009). High nitrate values were found to be slightly higher than the
concentration in water bodies lead to organic pollution permissible limits of WHO. High chloride concentration
that causes blue baby syndrome or cyanosis and gives an undesirable taste to water and cause many
methemologlobinemia in infants (Young et al., 1976; harmful effects in human beings (Suresh et al., 1992).
Vigil et al., 1965, Karunakaran et al., 2009) and
development of cancer in adults (Gass, 1978, CONCLUSION
Udayalaxmi et al., 2010). The physico-chemical parameters of groundwater
Iron occurs in both soluble and insoluble form in from industrial areas of Chennai showed that the pH, mg
water. It is present as ferric hydroxide in concentration hardness, salinity, phosphate, nitrate, Iron and chloride
renders water turbid and cause light yellowish brown were within the permissible norms while some of the
color to water. Concentration more than 1 to 2mg/L is parameters like calcium hardness; alkalinity and silica
generally held as an indication of the state of pollution. have the values more than the permissible limit as per
In the present analysis iron content varies from WHO and BIS norms for drinking purposes in the
0.13mg/L to 0.59mg/L which was found to be within the studied area. The findings of the present work are also
permissible limit of WHO and Indian standards. In recommended ground water is suitable for irrigation and
general surface water contains less than 0.5mg/L of iron domestic use. Some of locations in the study area are
whereas the concentration perhaps increases in the unfit for drinking as well as other domestic purpose.
ground water due to low cost iron pipes in bore wells laid
some decades ago could have been rusted. The rusted ACKNOWLEDGEMENTS
iron piece peels off and retained in particulate and The authors are thankful to the Management,
colonial form in drinking water. Further holes that Principal and HOD (Zoology), The New College
develop in rusted pipes pave the way for mixing up of Chennai-14 for providing necessary facilities.
the sewage water (Karunakaran et al., 2009).
Silica of water samples lies in the range from REFERENCES
12.29 to 66.67mg/L. Sample has moderately high values Adekunle AS. 2007. Effects of Industrial Effluent on
of silica and exceeds the permissible limit proposed by Quality of Well Water within Asa Dam Industrial Estate,
BIS and WHO. Silica released as a result of chemical Ilorin, Nigeria. Nature and Science 7(1).
breakdown of silicate minerals in rocks and sediments by
Ali Khan MM and Umar R. 2010. Significance of
chemical weathering is acquired by circulating
silica analysis in groundwater in parts of Central Ganga
groundwater and therefore the source of silica (SiO2) in
Kadhar et al., 2012
Plain, Uttar Pradesh, India Current science 98(9). Kumar A. 2004. Water Pollution. Nisha Enterprises
New Delhi. 1-331.
Altman SJ, Parizek RR. 1995. Dilution of nonpoint
source nitrate in ground water. J. Environ. Quality Pulle JS and Khan AM. 2003. Poll. Res., 22:393.
24:707-717.
Ramachandraiah CV. 2004. Right to drinking water in
Dhiviyaa Pranavam TS, Venkatesa Rao T, India, Centre for Economic and Social Studies.56.
Punithavathi L, Karunanithi S and Bhaskaran A
Ranjana Agarwal. 2009. Study of Physico-Chemical
2011. Groundwater pollution in the Palar Riverbed near
parameters of groundwater quality of dudu town in
Vellore, Tamil Nadu, India. Indian J. Sci. Technol. 4 (1),
Rajasthan. Rasayan J.Chem 2(4):969-971.
19-21.Domain site: http://www.indjst.org.
Rao Sudhkar M and Mamatha P. 2004. Water quality
Dufor CN and Becker E. 1964. Public water supplies of
in sustainable water management, Current science 87(7).
the 100 largest cities in the United States. US. Georg.
Sur. Water supply paper. 1812. 364. Ravisankar N and Poongothai S. 2008. A study of
groundwater quality in Tsunami Affected areas of
Ellis MM, Westfal BA and Ellis MD. 1948.
Sirkazhi Taluk, Nagapattinam District, Tamilnadu, India.
Determination of water Quality U.S. Government
Science of Tsunami hazards 27 (49).
office.
Saravanakumar K and Ranjith Kumar R. 2011.
Gass. 1978. Drinking water and your health, Part-II.
Analysis of water quality parameters of groundwater
Water Well J., 702(4):30-31.
near Ambatthur industrial area, Tamil Nadu, India 4(5).
Hem JD. 1985. Study and interpretation of the chemical
Suerdrup H, Martin V and Johnson W. 1961. The
characteristics of natural water.USGS Water Supply
Oceans, Asia Publishing House, New York, 1961, First
Paper, 2254,117-120.
Edition, 165-227.
Jain GKS Bhatia and Kumar SR. 1999. Ground Water
Suresh C, Tripathi BD and Mishra BP. 1992. Com
Contamination in Greater Guwahati, Assam, J.E.P
Physico Eco.,17(3):92-96.
20(1):641-649.
Udayalaxmi G, Himabindu D and Ramadass G. 2010.
Jinwal A and Dixit S. 2008. Pre and post monsoon
Geochemical evaluation of groundwater quality in
variation in physio-chemical characteristic in
selected areas of Hyderabad, A.P., India. Indian J.
groundwater quality in Bhopal, India. Asian j. Exp. Sci.,
Sci.Technol., 3(5):548.
22(3).
Vass KK, Raina HS, Zutshi DP and Khan MA. 1977.
Karnath KR. 1987. Ground Water Assesment and
Geobios, 4, 238.
Management, Tata Mc Graw Hill Publishing House,
1987. Vigil J, Warburton S, Haynes W and Kaiser LR.
1965. Nitrates in municipal water supply causing
Karunakaran KP. Thamilarasu and Sharmila R.
methemoglobinia in infants. Public Health Report.
2009. Statistical Study on Physicochemical
80(12):119-1121.
Characteristics of Groundwater in and around Namakkal,
Tamilnadu, India 6(3):909-914.
Kadhar et al., 2012
Wagh CV, Sudarshan J. Kokate, Haribhau R and

Kuchekar R. 2009. Physico-chemical analysis of ground
water in Pravara area, district Ahmednagar, Maharashtra
2:237.
Wetzel RG. 1983. Limnology II Edition, Saunders

College Publishing, New York,753.
Young CP, Oakes DB and Wilkinson WS. 1976.

Prediction of future nitrate concentration in groundwater.
Groundwater 4(6):426-438.

Advantages
Affordable Charges
Quick processing
Extensive indexing

Original Research
Taxonomic discrimination of Solanum nigrum and S. giganteum by

Fourier transform infrared spectroscopy Data
Authors: ABSTRACT:
Anilkumar VS1,
Dinesh Babu KV2,
Sunukumar SS3 and Fourier transform infrared spectroscopy (FTIR) provides biochemical profiles
Murugan K3. containing overlapping signals from a majority of the compounds that are present
when whole cells are analyzed. Leaf samples of higher plant species and varieties were
subjected to FTIR to determine whether plants can be discriminated phylogenetically
Institution:
on the basis of biochemical profiles. The results showed that the infrared spectra of
1. Department of Botany,
Govt. College for Women, Solanum were fingerprint-like patterns which were highly typical for different taxa.
Thiruvananthapuram, Kerala The principal component analysis of Fourier Transform Infrared (FTIR) data confirmed
695 014. most of morphological classifications of the species proposed in previous works.
The protein absorption bands located between 1800-1500 and the bands between
-1
2 Department of Chemistry, 1500-1000 cm (finger print region) showed variation between the two species
Govt. College for Women, S. nigrum and S. giganteum. Infrared spectra of leaves are of taxonomic value in genus
Thiruvananthapuram, Solanum, and this technique can be widely used for identification and classification of
Kerala 695 014. other taxa when standard spectra are available.
3. Plant Biochemistry and

Molecular Biology
Laboratory, University Keywords:
College, Thiruvananthapuram, Solanum, analysis, infrared spectra, taxonomic significance.
Kerala 695 034.
Corresponding author: Article Citation:

Murugan K. Anilkumar VS, Dinesh Babu KV, Sunukumar SS and Murugan K.
Taxonomic discrimination of Solanum nigrum and S. giganteum by Fourier transform
infrared spectroscopy Data.
Email: Dates:
harimurukan@gmail.com Received: 06 Jun 2012 Accepted: 28 Jun 2012 Published: 09 Jul 2012

Web Address: licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
http://jresearchbiology.com/ reproduction in all medium, provided the original work is properly cited.
482-488 | JRB | 2012 | Vol 2 | No 5

An International
Anilkumar et al., 2012
INTRODUCTION be analyzed by means of multivariate analysis when

The genus Solanum L. consists of over 2000 multiple samples are compared. FTIR has been shown to
species distributed worldwide (Knapp, 1991) is the be a valuable tool for differentiating, classifying and
largest in Solanaceae and is one of the largest among discriminating closely related microbial strains
flowering plants (Olmstead and Palmer, 1997). (Lamprell et al., 2006; Rebuffo et al., 2006). In plant
The sp ecies are a co mmo n so urce of classification, Kim et al ., (2004) have proposed this
vegetables (Omidiji, 1982), medicinal herbs approach is robust in chemotaxonomic classification of
(Caicedo and Schaal, 2004) and contain unique alkaloids flowering plants, and we previously have used this
and other biochemical constituents used for the treatment method to identify the species in Hypericum L. and
of diverse ailments (diabetes, cholera, bronchitis, high Triadenum Raf. (Lu et al., 2004). All these previous
blood pressure) and as laxatives (Lester and Seck, 2004). studies showed that this approach is a valid
In spite of the economic and medicinal value of Solanum representation of phylogenetic relationships between
species, no serious attention has been paid to diversity, plant taxa even closely related.
characterization and taxonomical identification at the In this report, we conducted a comprehensive
biosystematic level. This is a prerequisite to the FTIR analysis of carbohydrates, proteins, lipids, and cell
exploitation of the vast genetic variability available for wall pectin from Solanum nigrum and S. giganteum
the improvement of the quality and quantity of their drug leaves. De-convolution and curve-fitting analysis of IR
contents. Although the species discovered in this genus spectrum could acquire accurate data, thus helping for
have been sorted out, classified and revised many times quantitatively analyzing some functional groups.
during generic revision, there is much disagreement The presence of secondary metabolites and the value of
concerning combination of species depending on the FTIR method in this field were also considerate in this
taxonomic authority (Gracelin et al., 2011). Hence, it is study.
necessary to further investigate the classification of the
species using other technologies. MATERIALS AND METHODS
Chemotaxonomy has strongly influenced the Plant materials
entire field of biology, which is also useful for plant Fresh specimens of two species of Solanum such
systematics. Fourier Transform Infrared (FTIR) as S.nigrum and S.giganteum were collected from
Spectroscopy is a rapid, noninvasive, high-resolution Kerala, India, and were identified by comparison with
analytical tool for identifying types of chemical bonds in the voucher specimen from Kerala Forest Research
a molecule by producing an infrared absorption spectrum institute (KFRI, Kerala). One voucher was deposited at
that is like a molecular fingerprint. This technology the herbarium of the Department of Botany, University
allows detecting the whole range of infrared spectrum College, Kerala.
in measurement s of bio lo gical specimens IR spectroscopy
(Griffiths and de Haseth, 1986). Thus, these The leaves (approximately 3-4cm) were taken
fingerprints are made up of the vibrational features of from different plants and were pooled as one sample.
all the cell components, i.e., DNA, RNA, proteins, and Then the samples were immediately dried in an oven for
membrane and cell-wall components. The biochemical 2days at 60C. Tablets for FTIR spectroscopy were
profiles of FTIR from whole cell samples are extremely prepared in an agate mortars, by mixing leaves powder
high density data sets and, consequently, FTIR data must (2 mg) with KBr (1:100 p/p). The absorbance spectra
Figure 1. IR spectrum of Solanum nigrum.
were measured between 300 and 4500 cm-1.

At least three leaves were collected and at least three
Figure 2. IR spectrum of S. giganteum.
spectra were obtained from each sample.
A FTIR spectrometer (FTIR Shimadzu Prestige 40001500 cm-1 are due to functional groups
21) was used to collect spectra. Spectra were obtained in (e.g., OH, C=O, NH, CH3, etc.), while the region
32 scans co-added, 4000 resolution, and 2.0 gains. 1500675 cm-1 is referred to as the fingerprint region,
The parameters for the Fourier self-deconvolution were a which is highly specific for each taxon (Pan et al., 2000).
-1
smoothing factor of 15.0 and a width factor of 30.0 cm . Similarly, in mid-IR region (2000-1000cm-1) appeared
De-convolved and second-derivative spectra were large numbers of sharp peaks, indicating that the leaves
calculated for Fourier self-deconvolution and the bands have a rich chemical composition, such as carbohydrates,
were selected and normalized to unity with Omnic 7 proteins and lipids. However, this region yielded broad
software. Curve-fitting of the original spectra was and overlapped bands.
performed with Origin 7 software. The band position Knowitall software was used to find the
of functional groups was monitored with functional groups for preliminarily analyzing IR spectra
Knowitall 7.8 software. The spectral region between collected. The bands around 3370 cm-1 represent O-H
3000 and 2800 cm-1 was selected to analyze lipids. The and N-H stretching vibrations that are mainly generated
-1
spectral region between 1800 and 1500 cm was selected by proteins and carbohydrates (Wolkers et al., 1998).
to analyze proteins. The spectral region between The bands between 3000 and 2800 cm-1 represent C-H
1200 and 1000 cm-1 was selected to analyze stretching vibrations that are mainly generated by lipids
carbohydrates. (Wolkers and Hoekstra, 1995). The proteins absorption
bands mainly located between 1800 and 1500 cm-1
RESULTS AND DISCUSSION c o nt a in e d a m id e - I and a m id e - I I band s
FTIR spectra of Solanum species (Stehfest et al., 2005), but overlapped with other
FTIR detected all compounds, including absorption bands within this region. Amide III, the
polymers and low-molecular weight compounds in function group of nucleic acid and carbohydrates
whole cell samples, subsequently providing biochemical contributed to these absorption bands in the leaves.
profiles of extremely high-density data sets. Amide-I and amide-II bands are particularly useful for
Representative baseline-corrected and normalized FTIR determining the protein IR absorption changes. Amide-I
spectra for Solanum nigrum and S. giganteum are shown region (1700-1600 cm-1) mainly represent C=O
in Fig. 1 and 2. Absorption bands in the range of stretching vibrations of polypeptide, which can detect

Table 1. IR bands obtained in S. nigrum and S.giganteum changes of the overall protein conformation and content
Solanum nigrum Solanum giganteum (Surewicz et al., 1993). S.nigrum displayed a band at
409.88 596 1630.84. Meanwhile, S.giganteum showed five bands
438.81 617.22 viz. 1535.34, 1571.99, 1624.06, 1639.49 and 1670.35
473.53 663.51
(Table 1). The protein banding pattern show diversity
517.9 698.23
605.66 779.24 between the two species and this may be used to demark
647.13 829.39 the plants at species level. Further analysis by
830.37 914.26 de-convolution and curve fitting process in amide-I
1024.22 1020.34 region between 1700 and 1600 cm-1 can give additional
1062.8 1066.64
information about the protein structure: the band around
1099.43
1685 cm-1 assigned to the turn structure, the band around
1237.36 1236.37
1263.37 1656 cm-1 are assigned to the -helix structure, and the
1290.38 band around 1621 cm-1 are assigned to the
1328.98 1319.31 -sheet structure.
1394.56 1392.61 The bands around 2850 cm-1 and 2921 cm-1
1450.47
represents C-H asymmetric or symmetric stretching
1535.34
vibration, which belongs to the -CH2 group of lipids. The
1571.99
1630.84 1624.06 results show the total band areas (3000-2800 cm-1) were
1630.84 1639.49 similar. This implies that lipid profiles in the species are
1670.35 similar. The IR spectra between 1200 and 1000 cm-1
1731.14 1743.65 mainly occur from carbohydrates. The band size at
2326.15
1024.22 in S.nigrum more or less matches with that of
2370.51
S.giganteum (1020.34). The band around 1743 cm-1
2853.73 2854.65
2925.1 2926.01 represents -COOR stretching vibration (Fig 1.), which
3010.88 belongs to characteristic group of cell wall pectin.
3032.1 S.giganteum possess the characteristic band width at
3062.96 1743.65 while, S.nigrum showed only a lower band
3122.75
width of 1731.14.
3223.05
The peak 3200- 3300 may represent NH group of
3282.84
3305.99 Solanum alkaloids. Similarly the peak at 1635 forms
3329.14 C=N group containing alkaloids. S.giganteum showed
3360 peaks at 3223.05 and 3282.84 but the peaks in this range
3387 were absent in S.nigrum. Both the species possess peaks
3418.88 3419.79
at 1635 region. The bands at 617.22, 779.24, 914.26,
3441.01
1099.43, 1263.37, 1290.38 and 1450.47 are unique and
3479.58
3500.8 can be used as IR finger prints to identify S.giganteum
3523.95 from S.nigrum. Parallely, bands from 1328-1394 are
3554.81 shared between the species (Fig. 1 and 2).
3579.88 FTIR spectroscopy allows detecting the whole
range of infrared spectrum simultaneously providing S. nigrum and S.giganteum have fused petals
speed and accuracy in measurements of biological strongly adnated to androecium, anthers open by apical
specimens (Griffiths and de Haseth, 1986). With this pores which are the characteristic of genus. Previous
technique, Sheng et al., (2006) reported the effect of systems are based on the macro-morphological
MG132 on the change of FTIR spectra of cell wall taxonomy, while the relationships of these species in our
during pollen germination and pollen tube growth, and study are investigated on IR spectral characteristics.
Wu et al., (2003) studied the chemical characterization Hence, the difference may have some relationship with
of casparian strip in needles of Pinus bungeana. The what the characteristics are studied. However, we still
application of a combination with numerical cannot exclude the possibility that the population size in
methodologies, FTIR is recommended and has many the species may lead to the discrepancy, since chemical
advantages. This technique has been successfully components may be varied a little in different population
exploited for classifying normal and aged soybean seeds sizes.
(Kusama et al., 1997) and distinguishing cell wall In conclusion, the infrared spectra of Solanum
mutants from wild-type Arabidopsis (Chen et al., 1998; are fingerprint-like patterns which are highly typical for
Mouille et al., 2003). These studies, including different taxa. The FTIR data shows relationship
determination ofthe fruit content in processed foods between species that are in agreement with most of the
(Wilson et al., 1993) and discrimination of the previous proposed morphological classifications.
genuineness of Chinese medicines (Hong et al., 2006), Differences in cell compositions of the species by
have also been conducted. In plant taxonomic infrared spectroscopy thereby can provide the basis for
classification related studies, Sene et al., (1994) showed chemotaxonomy of species. Infrared spectra of leaves
differences in the plant cell walls of five angiosperms. appear to have taxonomic value and be more useful for
Further, Kim et al., (2004) proposed that FTIR was an discriminating closely related species or varieties in the
excellent method for determining phylogenetic genus, and this technique can be widely used for
relationships between flowering plants, and identification and classification of other taxa when
Lu et al., (2004) used this method to identify the species standard spectra are available. In our studies, the sections
in Hypericum and Triadenum. and the interspecific relationships we concluded are
The species differ in many morphological based on the chemical bonds of total mixture of leaf
characteristics. S.nigrum plants are unarmed, small erect cells, reflecting the interspecific differences on chemical
herbs with white flowers while, tall tree like armed components. Therefore, additional evidence, such as
plants and purple flowers in S.giganteum. In the past the DNA data, is still needed for interpreting the
taxonomic status of Solanum nigrum remained highly classification of Solanum.
controversial (Jennifer and James, 1997). Clarke, (1885)
did not separated Solanum nigrum, S. americanum and REFERENCES
S. villosum from each other and considered all of them Caicedo AL, Schaal BA. 2004. Heterogeneous
along S. nigrum. Rechinger, (1958) findings were evolutionary processes affect R gene diversity in
contradicted to it. According to him a plant sample with natural populations of Solanum pimpinellifolium. Proc of
white flowers and black berry must be identified as the Nat Aca of Sci., USA 101 (50):17444-17449.
S. nigrum Whereas, Edward (1990) mentioned S. nigrum
with orange colour fruit.

Chen L, Carpita NC, Reiter WD, Wilson RH, Jeffries Kusama T, Abe H, Kawano S, Iwamoto M. 1997.
C, McCann MC. 1998. A rapid method to screen for Classification of normal and aged soybean seeds by
cell-wallmutants using discriminant analysis of Fourier discriminant analysis using principal component scores
transformation infrared spectra. The Plant Jour., of near infrared spectra. Nippon Shokuhin Kogyo
16:385-392. Gakkai-Shi .,44:569-578.
Clarke CB. 1885. Solanaceae. In: Flora of British India, Lamprell H, Mazerolles G, Kodjo A, Chamba JF,
(Eds.): H.D. Hooker and C.B., K.C.S.I. Vol. IV. Bishen Noel Y, Beuvier E. 2006. Discrimination of
Singh Mahendra Pal Singh, New connaught Place, Staphylococcus aureus strains from different species of
London. 229-237. Staphylococcus using Fourier transform infrared (FTIR)
spectroscopy. Int J Food Microbiol., 108:125-129.
Edward ES. 1990. The black nightshades
(Solanum section Solanum) of the Indian subcontinents. Lester RN, Seck A. 2004. Solanum aethiopicum L. In:
Bot J of Linn Soc., 102:253-259. Gruben GJH, Denton OA (eds.) Plant Resources of
Tropical Africa 2. Vegetables. Wageningen: PROTA
Gracelin DHS, DE Britto AJ, Stephan Raj TL,
Foundations/Backhuys Publishers/CTA.
Rathna kumar PBJ. 2011. Assessment of genetic
relationships among five species of Solanum as revealed Lu HF, Cheng CG, Tang X, Hu ZH. 2004. Spectrum
by RAPD markers .Life sci Leaflets., 19:809-814 . of Hypericum and Triadenum with reference to their
identification. Acta Botanica Sinica., 46:401-406.
Griffiths PR, de Haseth JA. 1986. Fourier transforms
infrared spectroscopy. New York: Wiley. Mouille G, Robin S, Lecomte M, Pagant S, Hofte H.
2003. Classification and identification of Arabidopsis
Hong QH, Cheng ZF, Cheng CG. 2006. Application of
cell wall mutants using Fourier-Transform InfraRed
FTIR spectroscopy to the analysis of quality mensuration
(FT-IR) microspectroscopy. The Plant Journal.,
of Paeonia lactiflora Pall. from native habitat. Spectro
35:393-404.
and Spect Anal .,26:1610-1613.
Olmstead RG, Palmer JD. 1997. Implications for the
Jennifer ME, James AC. 1997. Black nightshades,
phylogeny, classification, and biogeography of Solanum
Solanum nigrum L and related species. International
from cpDNA restriction site variation. Syst Bot.,
Plant Genetic and Research institute (IPGRI)., Italy. 113.
22(1):19-29.
Kim SW, Ban SH, Chung H, Cho S, Chung HJ, Choi
Omidiji MO. 1982. Interrelationships of Solanum
PS, Yoo OJ, Liu JR. 2004. Taxonomic discrimination
species in different series of the subgenus
of flowering plants by multivariate analysis of Fourier
Leptostemonum (Dun) Bitt. Crop Research., 22:13-21.
transform infrared spectroscopy data. Plant Cell Reports.,
23:246-250. Pan YB, Zhao Y, Zhang FY. 2000. IR fingerprint
spectrum and Its Analyzing Method. Modern
Knapp S. 1991. A revision of the Solanum sessile
Instruments., 1:1-13.
species group (Section Germinate parte) (Solanaceae).
Bot J Linn Soc., 105:179-210. Rebuffo CA, Schmitt J, Wenning M, von Stetten F,
Scherer S. 2006. Reliable and rapid identification of
Listeria monocytogenes and Listeria species by artificial
neural network-based Fourier transform infrared Surewicz WK, Mantsch HH, Chapman D. 1993.
spectroscopy. App and Environ Microbiol., 72:994-1000. Determination of Protein Secondary Structure by Fourier
Transform Infrared Spectroscopy: A Critical
Re ch i n g e r KH. 1958. S o la n a ce a e. I n:
Assessment. Biochem., 32(2):389-393.
Symbolae Afghanicae. (Eds.): M. Koie and K.H.
Rechinger. Kommission hos Ejnar Munksgaard. 85-88. Wilson RH, Slack PT, Appleton GP, Sun L, Belton
PS. 1993. Determination of the fruit content of jam using
Sene CFB, McCann MC, Wilson RH, Grinter R.
Fourier transform infrared spectroscopy. Food Chem.,
1994. Fourier transform Raman and Fourier-transform
47:303-308.
infrared spectroscopy: an investigation of five higher
plant cell walls and their components. Plant Wolkers WF, Hoekstra AF. 1995. Aging of Dry
Physiol.,106:1623-1631. Desiccation-Tolerant Pollen Does Not Affect Protein
Secondary Structure. Plant Physiol., 109:907-915.
Sheng XY, Hu ZH, L HF, Wang XH, Baluska F,
Samaj J, Lin JX. 2006. Roles of the Wolkers WF, Oldenhof H , Alberda M, Hoekstra FA.
Ubiquitin/Proteasome pathway in pollen tube growth 1998. A Fourier transform infrared micro spectroscopy
with emphasis on mg132-induced alterations in ultra study of sugar glasses: application to anhydrobiotic
structure, cytoskeleton, and cell wall components. Plant higher plant cells. Biochim Biophys Acta.,
Physiol., 141:1578-1590. 1379(1):83-96.
Stehfest K, Toepel J, Wilhelm C. 2005. The application Wu XQ, Lin JX, Zhu JM, Hu YX, Klaus H, Lukas S.
of micro-FTIR spectroscopy to analyze nutrient 2003. Casparian strips in needles of Pinus bungeana:
stress-related changes in biomass composition of isolationand chemical characterization. Physiologia
phytoplankton algae. Plant Physiol and Biochem., Plantarum., 117:421-424.
43:717-726.

Advantages
Affordable Charges
Quick processing
Extensive indexing

Original Research
Calculating Integrated Pollution Indices for Heavy Metals in Ecological

Geochemistry Assessment Near Sugar Mill
Authors: ABSTRACT:
Sarala Thambavani D1, The sugar mill is a good example of a site where human pressures and
Sabitha MA 2. ecological values collide with each other. One of the aims of this work was to select
different types of index to aggregate and assess heavy metal contamination near
sugar mill in an accessible manner. Concentrations of heavy metals (Iron, Manganese,
Zinc and Copper) are studied in the soil near sugar mill to asses metal contamination
due to industrialization. The soil samples were collected from three different depths
Institution: A (0 cm), B (5 cm) and C (10 cm) for a period between October 2010 and March 2011
1. Associate professor, (winter and summer) and the heavy metal contents were analyzed by Atomic
Department of chemistry, Sri Absorption Spectrophotometer. Pollution index is a powerful tool for ecological
Meenakshi Government Arts
geochemistry assessment. Nine integrated indices were divided into two groups.
College for Women
One group is suitable for the normal distribution single indices including the average,
(Autonomous),
vector modulus, and Nemerow pollution indices, and the other for log-normal
Madurai-625 002.
distribution including the product, root of the product, and weighted power product
2. Assistant professor, pollution indices. Using background levels as reference values, five contamination
Department of chemistry, classes were divided, and the terminologies are suggested for the integrated indices to
J.A.College for Women unify the assessment results. The pollution load index (Ecological risk index) indicates
(Autonomous), that soil near sugar mill was highly polluted due to heavy metals
Periyakulam-625 601. (PLIFe = 0.30, PLIMn = 0.58, PLIZn = 0.24 and PLICu = 0.34). The results of contamination
index, index for chemistry and metal pollution were in agreement with pollution load
index. Average and vector modulus of pollution index and Nemerow pollution index
indicated slightly polluted domain. Since the aim of work on contamination evaluation
is to assess the overall contamination of a study area, the indices are highly
appropriate.
Keywords:
Sabitha MA. Atomic Absorption Spectrophotometer, integrated indices, pollution index,
heavy metals, ecological risk index, Nemerow pollution index.

sarala_dr@yahoo.in Sarala Thambavani D, Sabitha MA .
sabiarsh@yahoo.in Calculating Integrated Pollution Indices for Heavy Metals in Ecological Geochemistry
Assessment Near Sugar Mill.
Web Address: Dates:

http://jresearchbiology.com/ Received: 24 May 2012 Accepted: 01 Jun 2012 Published: 11 Jul 2012

489-498 | JRB | 2012 | Vol 2 | No 5

An International
Thambavani and Sabitha, 2012
INTRODUCTION assess soil and sediment quality for decision making and
With increasing population and industrial spatial planning.
expansion, the need for the treatment and disposal of the Pollution index is a powerful tool for processing,
waste has grown (Sarala and Sabitha, 2012a; Sarala and analyzing, and conveying raw environmental information
Sabitha, 2012b). Pollution of the natural environment by to decision makers, managers, technicians, and the public
heavy metals is a worldwide problem because these (Caeiro et al., 2005). This article presents the results
metals are indestructible and most of them have toxic from the study of pollution indices by heavy metals in
effects on living organisms (Sarala and Sabitha, 2009a). ecological geochemistry assessment. The aim of this
There is increased awareness that heavy metals present work is to select different types of indices to aggregate
in the soil may have negative consequences on human and assess the heavy metal contamination near sugar
health and on the environment (Sarala et al., 2009b). mill. The different types of indices are compared and
From the environmental point of view, all heavy discussed.
metals are largely immobile in the soil system, so they
tend to accumulate and persist in agricultural soils for a MATERIALS AND METHODS
long time. The most frequently reported heavy metals In the present study, stratified regular sampling
with potential hazards in soils are Cadmium, method was adopted for soil sample collection as in
Chromium, Lead, zinc and copper (Alloway, 1995). geo-assessment of the variables estimated; the stratified
The concentration of these toxic elements in soils may regular sampling is more suitable because this kind of
increase from various sources including anthropogenic sampling draws homogenous error (Burges and Webster,
pollution, weathering of natural high background rocks 1980; Burges et al., 1981). For this purpose the grid map
and metal deposits (Senesi et al., 1999). These chemicals of the study area has been used to know the distribution
in the terrestrial environment clearly pose a significant of heavy metal concentration in the whole region by
risk to the quality of soils, plants, natural waters and stratifying the region into a regular-sized grid cells, each
human health (Hooda and Naidu., 2004; U.S.E.P.A, grid cell is further divided into many smaller subcells for
2003; W.H.O, 2004; Sarala and Sabitha., 2011). a period between October 2010 and March 2011
With the development of ecological (winter and summer). Five sampling points in a grid of
geochemistry survey and exploration geochemistry 0.51km at each sampling station (4 at the corners and
survey, a great deal of data related to heavy metal one at the centre of the grid) were selected and
concentration in soils and water sediments have been composite samples consisting of three sub-samples were
measured which can be used to assess the quality of collected from the top (0 to 10 cm) layer of the soil using
ecological geochemistry environment. Many calculation plastic spatula after removing the debris, rock pieces and
methods have been presented to assess the environmental physical contaminants. In order to have the background
quality, such as pollution index, principle component concentration values of the heavy metal elements, three
analysis (Cheng et al., 2007), gray correlation, and fuzzy soil samples were collected, each from 100 cm below
decision. Different calculation methods on the basis of ground level, which are least affected by the sugar mill.
different algorithms might lead to discrepancy on The samples were placed in the clean polythene bags,
pollution assessment when they are used to assess the which were brought to the laboratory.
quality of soil and/or sediment ecological geochemistry. Laboratory methodology
So it is of great importance to select a suitable method to The samples were brought to the laboratory
where they were air dried and mixed thoroughly to might be composed by the above single indices
obtain the representative samples. Soon after drying the separately. According to algorithm, eight integrated
debris and other objects were hand picked up and the methods were illustrated as following.
sample was ground in a mortar to break up the Average of Pollution Index
aggregates or lumps, taking care not to break actual soil An average of pollution index (PIAvg) can be
particles. Soil samples were then passed through a 2mm defined as
sieve in order to collect granulometric fraction. Since
trace metals are often found mainly in clay and silt
fractions of soil and hence the size fraction <63m is
most commonly in the recommended size. For this where Pi is the single pollution index of heavy
purpose the granulometric fraction was added with the metal i, and m is the count of the heavy metal species.
dispersing agent and after shaking the sand fraction was This kind of pollution index was used by
separated from the clay and silt with <63m sieve (wet Bhattacharya et al., (2006). A PIAvg value of
sieving) and was used to measure the concentration of >1.0 indicates low quality soil because of contamination.
the heavy metals Fe, Mn, Zn and Cu from all the samples Vector Modulus of Pollution Index
collected. A vector modulus of pollution index (PIvectorM)
For this purpose the clay and silt fraction were can be defined as
digested by acids to get the solution by taking 5g of
sample into a 300ml polypropylene wide-mouthed jar
and distilled water was added to make a total 200ml.
Then it was acidified with 10ml HF, 5ml HClO4, 2.5ml
HCl and 2.5ml HNO3 in order to completely digest the
soil. This jar was shaken on an orbital shaker for 16 where Pi is the single pollution index of heavy
hours at 200-220 rpm before being filtered through metal i and m is the count of the heavy metal species.
whatman filter paper (No. 42) into acid washed bottles. Nemerow Pollution Index
The solution was stored and heavy metal contents were A Nemerow pollution index (PINemerow) was
analyzed by Atomic Absorption Spectrophotometer as applied to assess the quality of soil environment widely
per the method recommended by Committee of Soil (Cheng et al., 2007) and was defined as
Standard Methods for Analyses and Measurement
(1986).
Pollution Indices
Caeiro et al., (2005) analyzed the pollution
indices to assess heavy metal contamination and
classified them into two types: (i) contamination indices
and (ii) ecological risk indices. where Pi is the single pollution index of heavy
Integrated Indices metal i; Pimax is the maximum value of the single
Integrated indices are indicators used to calculate pollution indices of all heavy metals, and m is the count
more than one metal contamination, which were based of the heavy metal species. The quality of soil
on the single indices. Each kind of integrated index environment was classified into five grades from

Nemerow pollution index: PINemerow< 0.7, safety domain; Varies from 10 (unpolluted) to 0 (highly polluted).
0.7PINemerow<1.0, precaution domain; 1.0PINemerow<2.0, Metal Pollution Index
slightly polluted domain; 2.0PINemerow <3.0, moderately Metal pollution index was given by Usero et al.,
polluted domain; and PINemerow >3.0, seriously polluted (1996) which was categorized as contamination index.
domain by Cheng et al., (2007). MPI = (M1, M2, ..Mn)1/n
Pollution Index where Mn is the concentration of metal n
Johansson and Johnsson (1976) and Ott (1978) expressed in mg/kg of dry weight.
developed pollution index (contamination index) which Metal Enrichment Index
was given by the formula Riba et al., (2002a) developed the metal
enrichment index (contamination index) as follows:
SEF = Ci - Co
Co
where Ci is the total concentration of each metal i; C0 the
where Wi is the weight for pollution variable i; Ci heavy metal background level established for the
the highest concentration of pollution variable i reported ecosystem studied.
in a location of interest. For each pollutant i, the weight Potential Ecological Risk Index
was based on the reciprocal of the median of observed Potential ecological risk index (ecological risk
concentrations. index) was framed by Riba et al., (2002a) as given
This index allows the identication of priority below:
contaminations sites for implementation of ERF = Ci - CSQV
decontamination action. It requires several measurements CSQV
in the same sampling location. No threshold where Ci is the total concentration of each metal
classication from unpolluted to high pollution. i measured; CSQV the highest concentration of the heavy
Pollution Load Index metal non-associated with biological effects (chemical
Wilson and Jeffrey (1987) framed the pollution concentration associated with adverse effects); polluted
load index (ecological risk index) as follows: stations have values equal to or greater than 1.
Five classes of terminologies were suitable to
describe the degree of contamination. The five classes of
contamination degrees, and their terminologies for soils
were tabulated in Table 1.
Table 1: Terminologies for contamination
where B is the baseline value not contaminated; classes on integrated indices
T the threshold, minimum concentrations associated with S. No. Values Contamination classes
1 1 Unpolluted
degradation or changes in the quality of the estuarine
2 2 Low polluted
system. Wilson and Jeffrey (1987) dened B and T for 3 3 Moderately polluted
the different contaminants; C the concentration of the 4 4 Strongly polluted
5 5 Extremely polluted
pollutant. For each place the PLI calculation takes into
account all the n contaminants: When the PIAvg and PIvectorM are used with the
1/n
PLI = (PLI1, PLI2, PLIn) wi=1 condition, terminologies can also be used like
single indices. Tr or Tr would be used for the criterion presented above, and a total performance score
integrated indices based on the single index. was summarized for all the indexes used (table 2).
Index Comparison In each management unit the indices were
Co nt aminat io n ind ices measu r e t he calculated using the median values of chemical
contamination or enrichment levels and ecological risk concentration in all the locations belonging to each
indices; evaluate the potential for observing adverse management area. This mode was also used where the
biological effects. For index performance evaluation the index was nominal. These measures of the central
indices were scored on the basis of qualitative expert tendency were used instead of an arithmetic mean as the
knowledge and judgment (the project research team), objective of the analysis is to show the main trend in the
using the following criteria: index values for each management area. Moreover, the
Comparability: the existence of a target level or arithmetic mean should only be used for normal
threshold against which to compare it so that users distributions and should not be used in the present of
are able to assess the signicance of the values outliers (Wheater and Cook, 2002).
associated with it. For MPI and PLI a geometric increment was
Representativity: ability to provide a spatially employed which was divided into four classes. MPI used
representative picture of estuarine environmental a classication from clean to highly contaminated
states and impacts. (as it is only a contamination index); for PLI a
Credibility: a good theoretical basis in technical and classication from unimpacted to highly polluted was
scientic terms; applicability to estuaries. given, according to the index authors classication.
Simplicity: ease of calculation and interpretation. In an overall comparison of the contamination
Sensitivity and robustness: responsiveness to change and ecological risk indices the MPI and PLI indices have
in the environment. vi. Acceptable levels of the highest performance scores, according to the
uncertainty. indicator criteria MPI due to its simplicity and PLI due to
Each index was scored from 1 (lowest its simplicity, representative, comparability, sensitivity
performance) to 3 (highest performance) for every and robustness. It has the lowest performance score since
it needs reference site values. It may give imprecise
Table 2 Score of the metal assessment indices, based on several criteria
Contamination indices
Ecological risk indice (PLI)
MPI I
Simplicity 3 3 3
Representative 1 2 3
Credibility 2 3 2
Comparability 1 1 3
Sensitivity and robustness 1 3 3
Acceptable levels of uncertainty 2 2 2
Total 16 14 16
Table 3: Pollution index calculated at three depths using average,

vector modulus and Nemerow pollution indices
Depth cm
S. No. Indices
0 5 10
1 Average of pollution index 1.08 1.28 1.21
2 Vector modulus of pollution index 1.08 1.29 1.24
3 Nemerow pollution index 1.16 1.37 1.04

Table 4: Pollution index for soil near sugar mill Table 6: Index for chemistry for soil near sugar mill
at three depths (0 cm, 5 cm and 10 cm) at three depths (0 cm, 5 cm and 10 cm)
Metals Depth Wi Ci WiCi Metals Depth Vi (Vi)o RTRi = Vi/Vio
0 cm 0.05 20.10 1.01 0 cm 14.78 7.54 1.96
Fe 5 cm 0.09 15.44 1.39 Fe 5 cm 10.71 2.88 3.72
10 cm 0.10 16.86 1.69 10 cm 10.34 2.84 3.64
0 cm 0.16 7.66 1.23 0 cm 6.42 12.82 0.50
Mn 5 cm 0.20 7.24 1.45 Mn 5 cm 5.24 3.84 1.36
10 cm 0.17 6.60 1.12 10 cm 5.84 2.90 2.01
0 cm 0.18 5.87 1.06 0 cm 5.29 3.14 1.68
Zn 5 cm 0.26 4.10 1.07 Zn 5 cm 3.70 1.80 2.06
10 cm 0.11 9.21 1.01 10 cm 8.19 0.76 10.78
0 cm 0.14 7.31 1.02 0 cm 6.74 1.74 3.87
Cu 5 cm 0.21 5.80 1.22 Cu 5 cm 4.83 2.12 2.28
10 cm 0.10 10.11 1.01 10 cm 8.22 1.44 5.71
values because of the undue inuence of one of the The pollution index calculated was tabulated in
measurements used in the nal composite values table 4 which shows that the surface soil was strongly
(DelValls et al., 1998). It has no threshold for maximum polluted (PI0cm = 30.78) and at depths 5 cm and 10 cm
pollution and does not allow comparison between were moderately polluted (PI5cm = 10.26 and PI10cm = 9.66
ecosystems. respectively).
Pollution load index which indicates the
RESULTS AND DISCUSSION ecological risk index predicted that the surface soil was
Average, vector modulus of pollution index and moderately polluted compared to depths of 5 cm and 10
Nemerow pollution index calculated at three depths were cm which were unpolluted and the result was in
tabulated in table 3. accordance with the findings of Sayadi et al., (2009).
The results of average and vector modulus The PLI values were calculated to be 4.02, 1.80 and 1.70
pollution indices show that the area was unpolluted respectively (table 5).
where as Nemerow pollution index categorizes the Indices for chemistry (I) at three depths were
region as slightly polluted domain. The values of 16.02, 18.84 and 44.28 respectively. According to the
Nemerow were slightly larger than those of average and classes categorized depths 0 cm and 5 cm were strongly
vector modulus and the latter two were almost consistent polluted and depth of 10 cm was extremely polluted
(Gong Qingjie et al., 2008). where as results of metal pollution index predicts that all
Table 5: Pollution load index for soil near sugar mill Table 7: Metal enrichment index for soil near
at three depths (0 cm, 5 cm and 10 cm) sugar mill at three depths (0 cm, 5 cm and 10 cm)
Metals Depth C T B PLI Metals Depth Ci Co SEF
0 cm 14.78 2.96 7.54 0.26 0 cm 14.78 7.54 0.96
Fe 5 cm 10.71 6.00 2.88 0.03 Fe 5 cm 10.71 2.88 2.72
10 cm 10.34 5.26 2.84 0.01 10 cm 10.34 2.84 2.64
0 cm 6.42 5.75 12.82 1.24 0 cm 6.42 12.82 -0.49
Mn 5 cm 5.24 4.25 3.84 0.00 Mn 5 cm 5.24 3.84 0.36
10 cm 5.84 5.15 2.90 0.49 10 cm 5.84 2.90 1.01
0 cm 5.29 3.76 3.14 0.00 0 cm 5.29 3.14 0.68
Zn 5 cm 3.70 3.12 1.80 0.36 Zn 5 cm 3.70 1.80 1.06
10 cm 8.19 5.86 0.76 0.35 10 cm 8.19 0.76 9.78
0 cm 6.74 5.62 1.74 0.51 0 cm 6.74 1.74 2.87
Cu 5 cm 4.83 4.22 2.12 0.51 Cu 5 cm 4.83 2.12 1.28
10 cm 8.22 1.86 1.44 0.00 10 cm 8.22 1.44 4.71
Table 8: Potential ecological risk index for soil near Nemerow pollution index classify the area as slightly
sugar mill at three depths (0 cm, 5 cm and 10 cm)
polluted domain. Most of the integrated indices predicted
Metals Depth Ci CSQV ERF the place as strongly polluted. These results indicate that
0 cm 14.78 20.10 -0.26 the soil quality varied from strongly polluted to
Fe 5 cm 10.71 15.44 -0.31
10 cm 10.34 16.86 -0.39 unpolluted as the depth increases. This lack of consistent
0 cm 6.42 7.66 -0.16 correlation of the anthropogenic metals in the slightly
Mn 5 cm 5.24 7.24 -0.28 deeper soil layers may indicate that there is minimal
10 cm 5.84 6.60 -0.12
bioaccessibility and/or bioavailability of these metals
0 cm 5.29 5.87 -0.09
Zn within the region, an observation not necessarily
5 cm 3.70 4.10 -0.09
10 cm 8.19 9.21 -0.11 consistent with the conjecture of Nriagu et al., (1998)
0 cm 6.74 7.31 -0.08 about long term release of metals to regional waters as a
Cu 5 cm 4.83 5.80 -0.17
result of soil weathering.
10 cm 8.22 10.11 -0.19
Geochemical carriers
the three depths were extremely polluted with Pearson correlation matrix, standard deviation
MPI0, 5 and 10 cm = 7.63, 5.63 and 7.98 respectively and variance for analyzed soil parameters were
(table 6). These findings were in accordance with the calculated to determine the interrelation between the
results of Bakkialakshmi and Vinodhini (2008) as they parameters (table 9 and 10). Examination of the matrix
reported high contamination of the heavy metals in soils also provides clues about the carrier substances and the
near sugar mill. chemical association of trace elements in the studied
The metal enrichment index categorized different areas (Forstner, 1981; Jaquet et al., 1982).
depths as moderately and extremely polluted with Standard deviation measures the absolute
SEF0, 5 and 10 cm = 10.00, 10.84 and 36.28 respectively dispersion of a distribution. A small standard deviation
(table 7) where as potential ecological risk index means a high degree of uniformity in the observations as
categorizes the three depths as unpolluted well as ho mo g e ne it y of the se r ie s
(Bhupander Kumar et al., 2011) (table 8). (P.N.Arora et al, 2007). Variance has a great practical
Soil Quality Near Sugar Mill significance and is the best measure of comparing the
Average and vector modulus predict that the variability of the series. Except iron all the metals show
region near sugar mill was unpolluted where as
Table 9: Statistical data of heavy metals near sugar mill

Metals Depth Mean Minimum Maximum Standard deviation Variance
A (0 cm) 14.78 2.96 20.1 7.41 54.93
Fe B (5 cm) 10.71 6.00 15.44 3.45 11.89
C (10 cm) 10.34 5.26 16.86 4.98 24.79
A (0 cm) 6.42 5.75 7.66 0.79 0.64
Mn B (5 cm) 5.24 4.25 7.24 1.00 1.01
C (10 cm) 5.84 5.15 6.60 1.41 1.98
A (0 cm) 5.29 3.76 5.87 0.86 0.75
Zn
B (5 cm) 3.70 3.12 4.10 3.59 12.91
C (10 cm) 8.19 5.86 9.21 1.32 1.75
A (0 cm) 6.74 5.62 7.31 0.65 0.42
Cu B (5 cm) 4.83 4.55 5.80 2.03 4.13
C (10 cm) 8.22 1.86 10.11 1.28 1.64

Table 10: Correlation coefficient matrix between evaluation is to assess the overall contamination of a
heavy metal concentration in soil near sugar mill study area, the indices are highly appropriate.
Fe Mn Zn Cu Heavy metal assessment indices are not to be
Fe 1 -0.430 0.189 0.282 used as the only evidence of soil quality. In future
Mn 1 0.018 0.020 developments, organic compounds will be integrated into
Zn 1 0.808 the contamination evaluation, which can be correlated
Cu 1 with data on the different sources and spatial distribution

of pollution. Furthermore, the integration of
homogeneity in values with depth. Iron shows high contamination assessment with biota and toxicity
variation with depth. evaluation will be carried out in each management unit to
Iron showed negative correlation (-0.430) with allow a weight of evidence for soil quality assessment
Manganese which indicate that increase in the near sugar mill.
concentration of iron will lead to decrease in the
concentration of manganese. Zinc exhibited significant REFERENCES
positive correlation with Copper which indicate that the Aikpokpodion PE, Ipinmoroti RR and Omotoso SM.
presence of Zinc in the soil will lead to the accumulation 2010. Evaluation of Camellia sinensis (tea) biomass in
of Copper. This result is at variance with the report of nickel contaminated waste water treatment, J. Soil
Aikpokpodion et al., (2010) where existed positive Nature 4(1): 7-16.
correlation between large number of heavy metals.
Alloway JB. 1995. Soil Pollution and Land
Contamination, in Pollution: Causes, Effects and
CONCLUSION
Control, ed. R. M. Harrison. Cambridge: The Royal
Different metal assessment indices were used
Society of Chemistry, 318.
and discussed. Some indices gave equivalent information
but others gave complementary information Arora PN, Sumeet Arora, Arora S. 2007.
(e.g. contamination or background enrichment indices Comprehensive Statistical Methods, S.Chand and
and ecological risk indices) that can be developed for Company Ltd, New Delhi.
different purposes. There should be better methods of
Bakkialakshmi S and Vinodhini R. 2008.
standardization for indices to allow better comparability
Spectro-chemical study of Ambika sugar factory waste
between them (as several assess the same information).
affected soil and water in Pennadam, Cuddalore District,
According to the evaluation of the index criteria
Tamil Nadu, Indian journal of Environmental Protection,
performance, PLI had the highest score particularly in
28(5): 405-414.
the group of ecological risk indices. This index can be
complemented with the MPI contamination index. MPI Bhattacharya A, Routh J, Jacks G. 2006.
does not evaluate the potential for adverse effects and the Environmental Assessment of Abandoned Mine Tailings
results from one ecosystem are more difcult to compare in Adak, Vsterbotten District (Northern Sweden).
with others, but it allows more site-specic and accurate Applied Geochemistry, 21:1760-1780.
information on contamination levels. The results of the
Bhupander Kumar, Sanjay Kumar, Dev Prakash,
indices per management unit are in accordance with the
Singh, SK, Meenu Mishra Jain PK, Lal, RB, Sharma
surface areas of each metal. If the aim of contamination
CS. and Mukherjee, DP. 2011. A study on sugar mill Hooda PS and Naidu R. 2004. Speciation,
pressmud compost for some heavy metal content and bioavailability and toxicity relationships of contaminants
their bio-availability, Asian Journal of Plant Science and in the terrestrial environment, in: Proceedings of
Research, 1 (3): 115-122. International Contaminated Site Remediation
Conference, Adelaide, South Australia, 1518,
Burges TM and Webster R. 1980. Optimal
September.
interpolation and arithmic mapping of soil properties,
Journal of Soil Sciences, 31: 315-331. Jaquet JM, Davaud E, Rapin F and Vernet JP. 1982.
Basic concepts and associated statistical methodology in
Burges TM, Webster R and Mc Bratney AB. 1981.
geochemical study of lake sediments, Hydrobiologia,, 91
Optimal sampling and isarithmic mapping of soil
(1): 139-146.
properties, IV-Sampling strategy. Journal of Soil
Sciences, 32:1028-1032. Johansson SAE, Johansson TB. 1976. Analytical
application of particle induced X-ray emission. Nuclear
Caeiro S, Costa MH, Ramos TB. 2005. Assessing
Instruments and Methods 137(3):473-516.
Heavy Metal Contamination in Sado Estuary Sediment:
An Index Analysis Approach. Ecological Indicators, Nriagu J, Wong HKT, Lawson G and Daniel P. 1998.
5:151-169. Saturation of ecosystems with toxic metals in Sudbury
basin, Ontario, Canada, The Science of the Total
Cheng JL, Shi Z, Zhu YW. 2007. Assessment and
Environment, 223, 99-117.
Mapping of Environmental Quality in Agricultural Soils
of Zhejiang Province, China. Journal of Environmental Ott WR. 1978. Environmental IndicesTheory and
Sciences, 19:50-54. Practice. Ann Arbor Science, Michigan, USA, 371.
Committee of Soil Standard Methods for Analyses Riba I, DelValls TA, Forja JM, Go mez-Parra, A.
and Measurements. 1986. Soil Standard Methods for 2002a. Evaluating the heavy metal contamination in
Analyses and Measurements. Hakuyusha, Tokyo, Japan. sediments from the Guadalquivir estuary after the
Aznalco llar mining spill (SW Spain): a multivariate
DelValls TA, Forja JM, Go mez-Parra A. 1998.
analysis approach. Environ. Monit. Assess. 77:191-207.
Integrated assessment of sediment quality in two littoral
ecosystems from the Gulf of Ca diz, Spain. Environ. Sarala Thambavani D and Sabitha MA. 2012a.
Toxicol. Chem., 17:1073-1084. Multivariate statistical analysis between COD and BOD
of sugar mill effluent, Scholarly Journal of Mathematics
Forstner U. 1981. Metal pollution assessment from
and Computer Science Vol. 1(1):6-12.
different analysis, In: Forstner, U. and Wittmann,
G.T.W., (eds) metal pollution in the aquatic environment Sarala Thambavani D and Sabitha MA. 2011.
Springer, Berlin Heidelberg, New York, p 486. Variation in air pollution tolerance index and anticipated
performance index of plants near a sugar factory:
Gong Qingjie, Deng Jun, Xiang Yunchuan, Wang
implications for landscape-plant species selection for
Qingfei, Yang Liqiang. 2008. Calculating Pollution
industrial areas, Journal of Research in Biology,
Indices by Heavy Metals in Ecological Geochemistry
7:494-502.
Assessment and a case study in Parks of Beijing, Journal
of China University of Geosciences, 19(3): 230-241.

Sarala Thambavani D and Sabitha, MA. 2012b. Soil U.S.E.P.A. 2003. Drinking water contaminants, National
physico-chemical characteristics affected by sugar primary drinking water regulations, EPA 816-F-03-016.
industry effluent at Alanganallur, Tamil Nadu, J.
Usero J, Gonza lez-Regalado E, Gracia I. 1996. Trace
Ecotoxicol. Environ. Monit., 22(3):245-252.
metals in the bivalve mollusc Chamelea gallina from the
Sarala Thambavani D, Sabitha MA and Rajeswari G. Atlantic Coast of Southern Spain. Mar. Pollut. Bull.
2009a. Tolerance of plants to air pollution near leather 32(3):305-310.
tanneries, J. Ecotoxicol. Environ. Monit., 19(6):609-612.
W.H.O. 2004. Guidelines for Drinking Water Quality,
Sarala Thambavani D, Sabitha MA and Selvasundari 3rd ed., ISBN 9241546387, Retrieved from http://
R. 2009b. Air Pollution Tolerance Index of tree species www.who.int/water sanitation health/dwq/guidelines/
growing in traffic area of Madurai, Tamil Nadu, The en/.
Asian Journal of Experimental Chemistry,
Wheater CP, Cook PA. 2002. Using Statistics to
4(1 & 2):126-132.
Understand the Environment. Routledge, London, 254.
Sayadi MH, Sayyed MRG and Shabani N. 2009.
Wilson JG, Jeffrey DW. 1987. Europe-wide indices for
Quantification of heavy metal pollutants in the surface
monitoring estuarine quality. In: Kramer, K.J.M. (Ed.),
soils of Chitgar industrial area Tehran, Iran with spatial
Biological Indicators of Pollution. Royal Irish Academy,
references to their spatial pattern, Pollution Research,
Dublin, Ireland, 225-242.
28: 345-351.
Senesi GS, Baldassarre G, Senesi N, Radina B. 1999.

Trace element inputs by anthropogenic activities and
implications for human health. Chemosphere
39:343- 377.

Advantages
Affordable Charges
Quick processing
Extensive indexing

Original Research
Radiosensitizing effects of 2-deoxy-D-glucose and ferulic acid on mouse

Ehrlichs Ascites Carcinoma in Swiss albino mice
Authors: ABSTRACT:
Bandugula Venkata
Reddy, Nagarajan The objective of the present study is to evaluate the radiosensitizing activity
Rajendra Prasad. of combination of 2-deoxy-D-glucose (2DG) and ferulic acid (FA) against
Ehrlichs Ascites Carcinoma (EAC) in Swiss albino mice. Extensive DNA damage has
been observed in the cells collected from the peritoneal cavity of combined treatment
group (2DG+FA+IR) than the other treatment modalities. Treatment of EAC tumor
bearing mice with 2DG and FA before 8 Gy of hemi-body -radiation has resulted in
Institution: the significant decrease of tumor volume and tumor weight compared with
Department of Biochemistry EAC control group. Further, combination of 2DG and FA along with radiation
and Biotechnology, decreased the viability of tumor cells in the peritoneal fluid of EAC bearing mice. It has
Annamalai University, been also found that the percentage of EAC apoptotic cells in the peritoneal fluid has
Annamalainagar-608 002, been significantly increased in the combined treatment group (2DG+FA+IR) compared
Tamilnadu, India. with other treatment groups. Combined treatment of 2DG and FA activated
caspase-3 and 9, when compared with the treatment given with radiation alone
indicated radiosensitizing effect of this combination (2DG+FA). This has been
further evidenced by the massive decrease of LDH activity in the 2DG+FA+IR group.
Corresponding author: On other hand, 2DG and FA combination protects the hematological changes occurred
Nagarajan Rajendra during radiation treatment. Histopathological examinations showed that the
Prasad. combination of 2DG and FA offers protection to normal tissues during radiation
treatment. Taken together, the results of the study clearly suggested 2DG and FA
combination sensitizes EAC bearing mice to radiation effects at the same time offers
protection to normal tissues from radiation-induced damage.
Keywords:
Email:
drprasadnr@gmail.com Ehrlich ascites carcinoma; radiosensitization; apoptosis; 2-deoxy-D-glucose;
ferulic acid.
Phone No: Article Citation:

+91 9842305384. Bandugula Venkata Reddy, Nagarajan Rajendra Prasad.
Radiosensitizing effects of 2-deoxy-D-glucose and ferulic acid on mouse Ehrlichs
Ascites Carcinoma in Swiss albino mice.
Fax:
+91 4144 239141. Dates:
Received: 27 Jun 2012 Accepted: 12 Jul 2012 Published: 14 Jul 2012
Web Address:
499-512 | JRB | 2012 | Vol 2 | No 5

An International
Reddy and Prasad, 2012
INTRODUCTION glutathione/glutathione peroxidase/glutathione reductase

Radiotherapy is one of the major treatment system, has also been shown to participate in the
options in cancer management. About 52% of cancer metabolic decomposition of hydrogen peroxide and
patients receive radiotherapy at least once during their organic hydroperoxides (Ahmad et al., 2004).
treatment period (Delaney et al., 2005).The goal of Therefore, in addition to its well known role in energy
radiotherapy is to destroy or inactivate cancer cells while production, glucose metabolism appears to be integrally
preserving the integrity of normal tissues within the related to the metabolic detoxication of intracellular
treatment field. Many tumors develop resistance to hydroperoxides formed as byproducts of oxidative
radiotherapy when given alone for prolonged periods. metabolism. It was reported that glucose deprivation
In most cases radiotherapy is given along with surgery or causes cytotoxicity in the MCF-7/ADR human
chemotherapy. Newer approaches in combination multidrug-resistant breast carcinoma cell line (Lee et al.,
therapy are gaining momentum because of the beneficial 1998). 2DG inhibits growth and induces apoptosis in a
effects offered. The most frequently used combination number of cancer cell lines (Lee et al., 1998).
of ch em oth er a p eut i c a g en t s in cl u d es 2DG exhibits a cytotoxic effect in cancer cells, but the
cis-dichlorodiamine-platinum (II), paclitaxel, docetaxel, same dose spares normal cells (Reddy and Prasad, 2011).
gemcitabine and topotecan (Dimitroulis et al., 2006). In addition, the cytotoxic effect of 2DG is greater in
To reduce the toxic effects of combination regimens, cancer cells that exhibit mitochondrial respiratory defects
new class of compounds are required which shall reduce (Gogvadze et al., 2008). Many mechanisms are
the side effects and give optimum therapeutic benefits. postulated to contribute to the antitumor effect of 2DG,
Combination regimens using phytochemicals and including inhibition of glucose transport and hexokinase
metabolic analogs may be suited to alleviate the side II activity, depletion of cellular ATP, blockage of cell
effects of radiotherapy and chemotherapy and increase cycle progression, induction of apoptosis, induction of
the effect of radiation on neoplastic cells, while endoplasmic reticulum stress, and/or induction of
protecting normal cells from radiation damage oxidative stress (Coleman et al., 2008).
(Rao et al., 2008). 2DG significantly sensitizes human osteosarcoma and
2-deoxy-D-glucose (2DG), an analog of glucose, non-small cell lung cancer to adriamycin and paclitaxel
is a glycolytic inhibitor. After the formation of in mouse models (Maschek et al., 2004).
glucose 6-phosphate (via hexokinase) the major Plant phenolics could fit as promising adjuvants
pathways of glucose metabolism include glycolysis and for radiotherapy by enhancing radiosensitivity of cancer
the pentose phosphate cycle (Sharma et al., 2007). cells and by decreasing radiation effects in normal cells
Glycolysis results in the formation of pyruvate and the (Kim et al., 2011). Radiosensitizing effects of many
pentose phosphate pathway results in the formation of phytochemicals are thought to interact with several
NADPH (Sharma et al., 2007). Pyruvate, in addition to intracellular signaling molecules which then mediate
being a substrate for the formation of acetyl-CoA and signaling cascades including cell cycle arrest and cell
energy metabolism via the tricarboxylic acid (TCA) death (Kim et al., 2011). Curcumin, a polyphenol, has
cycle and mitochondrial oxidative phosphorylation, has been reported to confer radiosensitizing effect in
been shown to scavenge hydrogen peroxide and other relatively radio- resistant prostate cancer cell line PC-3
hydro peroxides (Ahmad et al., 2004). NADPH, by (Chendil et al., 2004). Ferulic acid (FA)
virtue of being the source of reducing equivalents for the (4-hydroxy-3-methoxycinnamic acid) (C10H10O4) is a
Figure 1 Effect of 2DG, FA and/or IR on (a) tumor Figure 2 Effect of 2DG, FA and/or IR on (a) viable cell
volume and (b) tumor weight in EAC bearing mice. count and (b) nonviable cell count in EAC bearing mice.
Values are given as means S.D. of six experiments in Values are given as means S.D. of six experiments in
each group. Values not sharing a common marking each group. Values not sharing a common marking
(a,b,cetc) differ significantly at p<0.05 (DMRT). (a,b,cetc) differ significantly at p<0.05 (DMRT).
ubiquitous phenolic compound in plant tissues; therefore, the highest purity and were purchased from Himedia and
it constitutes a bioactive ingredient of many foods. NICE chemicals, Mumbai, India.
Prooxidant activity of hydroxycinnamic acids in different Animal care and handling
experimental models has been previously reported Ten- to twelve-week-old female Swiss albino
(Zheng et al., 2008). It has been reported earlier from our mice weighing 25 to 30 g were selected from an inbred
laboratory that FA sensitizes human cervical carcinoma colony maintained under the controlled conditions of
cells to radiation and this property is attributed to its temperature (23 2C), humidity (50 5%) and light
pro-oxidant nature (Karthikeyan et al., 2011). Further, (14 and 10 h of light and dark, respectively). The animals
FA in combination with 2DG enhanced radiation effects had free access to sterile food and water. The animals
in NCI-H460 cells in vitro (Reddy and Prasad, 2011). were housed in a polypropylene cage containing sterile
There were number of experimental models available to paddy husk (procured locally) as bedding throughout the
study in vivo anticancer effect of test compounds. EAC is experiment. The study was approved by the Institutional
one of the models still best suitable to study Animal Ethical Committee of Annamalai University,
radiosensitizing mechanisms due to its rapid growth rate
and undifferentiated nature (Ozaslan et al., 2011). Hence,
in the present study, an attempt has been made to
evaluate the radiosensitizing potential of 2DG and FA in
EAC bearing experimental animals.
MATERIALS AND METHODS

Chemicals
2 - d eox y- D- gl u cose, ferulic a ci d,
ethidium bromide and acridine orange were purchased
from Sigma Chemicals Co., St. Louis, USA. Figure 3 Effect of 2DG, FA and/or IR on serum LDH
activity in EAC bearing mice. Values are given as
ApoAlert caspase 3 and 9 fluorescent assay kit was
means S.D. of six experiments in each group.
purchased from Clontech Laboratories, Inc., Canada. All Values not sharing a common marking (a,b,cetc)
differ significantly at p<0.05 (DMRT).
other chemicals and solvents used in the study were of

B C
Figure 4 Effect of 2DG, FA and/or IR on (a) DNA damage (b) Percentage tail DNA (c) tail length in
EAC bearing mice. Values are given as means S.D. of six experiments in each group.
Values not sharing a common marking (a,b,cetc) differ significantly at p<0.05 (DMRT).
Chidambaram, India. growth phase, the animals were randomly divided into
Transplantation of tumor and treatment schedule nine groups (each group=6 animals) as follows: 54 Swiss
EAC cells were obtained from Amala Cancer albino mice were divided into nine groups (n=6) and
Research Institute, Thrissur, Kerala, India. The EAC given food and water ad libitum. All the animals in each
cells were maintained in vivo in Swiss albino mice by groups except Group I received EAC cells
6 6
intraperitoneal transplantation of 2 x 10 cells per mouse (2 x 10 cells/mouse i.p.). Group III and GroupVII
every 10 days. Ascitic fluid was drawn out from EAC received 0.5 g/kg body weight of 2DG through i.p.
tumor bearing mouse at the log phase (days 7-8 of tumor injection on every alternate day for 14 days. Group IV
bearing) of the tumor cells. Each animal received 0.1 ml and Group VIII received 50 mg/kg body weight of FA
of tumor cell suspension containing 2 x 106 tumor cells through i.p. injection on every alternate day for 14 days.
intraperitoneally. On the 7th day after EAC tumor Group V and Group IX received 0.5 g/kg body weight of
inoculation, when the tumors were in the exponential 2DG and 50 mg/kg body weight of FA through i.p.
Figure 5 Effect of 2DG, FA and/or IR on (a) apoptotic morphology (b) percentage of apoptosis in
EAC bearing mice. Values are given as means S.D. of six experiments in each group.
Values not sharing a common marking (a,b,cetc) differ significantly at p<0.05 (DMRT).
injection on every alternate day for 14 days. On the LDH activity. Tissues (Liver and spleen) were dissected
15th day, animals from Group VI to IX received to observe the histopathological changes.
8 Gy hemi-body -radiation (2 Gy/min). 24 hours after Tumor volume
the exposure of animals to -radiation and 18 h of The ascitic fluid was collected from the
fasting, the animals were anesthetized and sacrificed by peritoneal cavity of the mouse. The volume was
cervical dislocation. Peritoneal fluid containing EAC measured by taking it in a graduated centrifuge tube
cells was collected and used to estimate DNA damage (Bhattacharya et al., 2011).
and apoptotic morphological changes and tumor cell Tumor weight
count. Blood was collected to analyze the hematological The tumor weight was measured by taking the
changes. Serum was collected to analyse the weight of the animal before and after the collection of

A B LDH was expressed as the change in optical

density/min/mg of protein in serum sample. Total protein
content was determined at 650 nm using freshly prepared
solutions of 2% alkaline sodium carbonate, 0.5% copper
sulphate in 1% sodium potassium tartrate and
FolinCiocalteus phenol reagent (Lowry et al., 1951).
Measurement of DNA damage
DNA damage was estimated by alkaline single
Figure 6 Effect of 2DG, FA and/or IR on (a) caspase 3
and (b) caspase 9 expressions in EAC bearing mice. cell gel electrophoresis (comet assay) according to the
Values are given as means S.D. of six experiments in
method of Singh et al., 1988 (Singh et al., 1988) with
each group. Values not sharing a common marking
(a,b,cetc) differ significantly at p<0.05 (DMRT). slight modification (Reddy and Prasad, 2011). The extent
of DNA damage was estimated by fluorescence
the ascetic fluid from peritoneal cavity microscopy using a digital camera and analyzed by
(Bhattacharya et al., 2011). image analysis software, CASP. For each sample,
Tumor cell count 100 cells were analyzed and classified visually into one
The ascitic fluid was taken in a WBC pipette and of five classes according to the intensity of fluorescence
diluted 100 times. Then a drop of the diluted cell (DNA) in the comet tail. DNA damage was quantified as
suspension was placed on the Neubauers counting % of tail DNA and tail length.
chamber and the numbers of cells in the 64 small squares Apoptotic morphology
were counted (Bhattacharya et al., 2011). EAC cells were collected from the peritoneal
Viable/nonviable tumor cell count cavity and washed with PBS and fixed on microscopic
The viability and nonviability of the cells were slides with 3:1 ratio of methanol:acetic acid. Later, these
checked by trypan blue assay. The cells were stained slides were stained with 1:1 ratio of ethidium bromide
with trypan blue (0.4 % in normal saline) dye. The cells and acridine orange as described elsewhere (Reddy and
that did not take up the dye were viable and those that Prasad, 2011). Stained cells were washed with PBS and
viewed under a fluorescence microscope. The number of
Number of cells X Dilution factor cells showing features of apoptosis was counted as a
Cell count = ----------------------------------------
Area X Thickness of liquid film function of total number of cells present in the field.
During apoptosis DNA becomes condensed and
took the dye were nonviable. These viable and nonviable fragmented. This could be observed when several small
cells were counted (Reddy and Prasad, 2011). spots of DNA are visible in the nucleus stained by
Lactate dehydrogenase (LDH) assay acridine orange. Later, the cell membrane becomes
The serum sample was added to a freshly permeable to dyes like ethidium bromide. The DNA
prepared NADH solution (2.5 mg/ml in 0.1 M phosphate spots turn orange red after intercalation of ethidium
buffer; pH 7.4), and a sodium pyruvate solution bromide with DNA.
(2.5 mg/ml in 0.1 M phosphate buffer, pH 7.4) was Caspase 3 and 9 activity assays
added to it after 20 min. The absorbance of the mixture ApoAlert caspase-3 and 9 assays were performed
was noted at 340 nm for 5 min at regular intervals according to the manufacturers instructions using
(Wroblewski and LaDue, 1955). The unit activity of DEVD-AFC and LEHD-AMC as substrates,
A B
C D
Figure 7 Effect of 2DG, FA and/or IR on (a) RBC count (b) hemoglobin content (c) WBC count (d) Percentage
of lymphocytes in EAC bearing mice. Values are given as means S.D. of six experiments in each group. Values
not sharing a common marking (a,b,cetc) differ significantly at p<0.05 (DMRT).
respectively. Fluorometric detection for caspase 3 is haemocytometer (Mukherjee, 1988).

performed using a 400 nm excitation filter and
505 nm emission filter. Fluorometric detection for Histopathological studies
caspase 9 is performed using 380 nm excitation filter and Liver and spleen of sacrificed mice were fixed in
460 nm emission filter. Emissions of samples were formaldehyde-saline and processed to prepare slides of
compared with uninduced control cells to determine the the tissues stained with hematoxylin-eosin for
increase in caspase activities. microscopic observation of their histopathological status.
Hematological parameters Statistical analysis
At the end of the experimental period, the next Statistical analysis to evaluate the significance of
day after an overnight fasting blood was collected from any differences between the data of two chosen groups
freely flowing tail vein and used for the estimation of was done by using one-way ANOVA followed by
hemoglobin (Hb) content, red blood cell (RBC) count, DMRT (Duncan Multiple Range Test) taking p<0.05 to
white blood cell (WBC) count and lymphocyte count by test the significant difference between groups. Values are
standard procedures using cell dilution fluids and given as means S.D. of experiments in each group.

Reddy and Prasad,2012
Figure 8 Effect of 2DG, FA and/or IR on histopathological changes in the liver of EAC bearing mice. (A)
migration of tumor cells close to the capsule (B) appearance of tumor cells near capsules minimized, nuclear
disintegration observed (C) presence of tumor cells near the lining of the capsule minimized (D) tumor cells
showed necrosis, nuclear disintegration observed (E) tumor cells appeared adherent to the lining of the
capsule (F) necrosis of the tumor cells and blast formation of the liver cells observed (G) tumor cell neurosis
and blast formation of the liver cells observed (H) Viable tumor cells not seen on the lining of the capsule.
Values not sharing a common marking (a,b,c.. etc.) differ Figure 2 shows the effect of 2DG and FA
significantly at p<0.05. pretreatment on tumor cell viability in -radiation treated
RESULTS EAC bearing mice. Compared with control group, the
Tumor volume and tumor weight viable cell count has been decreased in 2DG, FA and IR
Figure 1 shows the changes in tumor volume and treatment groups. Combined treatment of 2DG and FA
tumor weight in untreated and treated EAC bearing mice. has resulted in the significant decrease in viable cell
EAC control animals showed significant tumor volume count compared with 2DG or FA alone treated groups.
and tumor weight (3.9 ml and 4.1 g, respectively). Tumor Combined treatment group (2DG+FA+IR) has showed a
volume and tumor weight has been reduced significantly further decrease in viable cell count compared with
upon the treatment with 2DG (2 ml and 3.4 g, 2DG and FA treatment group.
respectively), FA (2.1 ml and 3.6 g. respectively) and IR Lactate dehydrogenase activity
(1.5 ml and 2.4 g, respectively) alone when compared Figure 3 shows the effect of 2DG and FA
with EAC control mice. Combination of 2DG and FA pretreatment on LDH activity in -radiation treated EAC
(2DG+FA) (1.4 ml and 2.1 g, respectively) significantly bearing mice. Compared with normal group, EAC
decreased the tumor volume and tumor weight compared control mice have shown high LDH activity. LDH
with 2DG or FA alone treated groups. Combined activity has decreased significantly in 2DG, FA and
treatment group (2DG+FA+IR) (0.5 ml and 0.5 g, 2DG+FA treated groups compared to EAC control.
respectively) has showed a further decrease in tumor 2DG+FA+IR treatment group has showed decreased
volume and tumor weight when compared with all other activity of LDH compared to IR alone treated group.
treatment modalities. The LDH activity is near to normal in 2DG+FA+IR
Tumor cell viability treatment group.
Figure 9 Effect of 2DG, FA and/or IR on histopathological changes in the spleen of EAC bearing mice. (A)
tumor cells seen peripheral to the spleenic capsule (B) spleen shows necrosis and fibrosis around the capsules
(C) viable tumor cells were seen around the lining of the spleenic capsule (D) tumor cells seen decreased
around the capsule and fibrosis observed (E) tumor cells present around the spleenic capsule shows necrosis
(F) less number of tumor cells around the capsule observed (G) tumor cells shows necrosis and fibrinolytic
changes observed (H) viable tumor cells not seen on the lining of the spleenic capsule.
DNA damage apoptosis in the different treatment groups. Percentage of

Figure 4a shows photomicrographs of DNA apoptotic cells has increased in the combined treatment
damage (comet assay) in 2DG, FA and IR treatment of 2DG and FA compared with 2DG and FA alone
groups. The control cell showed largely non-fragmented treatment groups. Combined treatment group
DNA. We observed fragmented DNA in 2DG, FA and (2DG+FA+IR) has showed 79% of apoptotic cells.
radiation treated mice which appear as a comet during Caspase-3 and 9 expression
single cell gel electrophoresis. Figure 4b & 4c shows the Figure 6a and b shows the spectrofluorometric
changes in the levels of % DNA in the tail and tail length readings for the analysis of caspase 3 and 9 in 2DG, FA
of different treatment groups in EAC bearing mice. and IR treatment groups. The activity of apoptotic
There were no changes in levels of DNA damage in the enzymes caspase-3 and 9 were significantly increased in
untreated control cells. Treatment with 2DG, FA and IR 2DG, FA and 2DG+FA treatment groups. The group
alone increased DNA damage compared to untreated with the treatment of radiation alone has also showed a
control cells. 2DG+FA+IR treatment group shows significant increase in the activity of caspase-3 and 9
increased % of DNA in the tail and tail length compared compared with EAC control group. Treatment with 2DG
to any other treatment group. and FA before irradiation has resulted in the increased
Apoptotic morphology expression of caspase 3 and 9, compared with the group
Cells with condensed or fragmented chromatin, treated with radiation alone.
indicative of apoptosis, were observed in different Hematological changes
treatment groups as compared to control cells which Figure 7 shows the effect of 2DG, FA and IR on
showed evenly distributed green fluorescent chromatin hematological parameters in EAC bearing mice. RBC
(Figure 5a). Figure 5b shows the quantitative result of count and hemoglobin content have been increased in the

2DG and FA treatment group compared with 2DG and before 8 Gy hemi-body -radiation caused retardation in
FA alone treated groups. WBC count and % of the tumor volume (0.5 ml) and tumor weight (0.5 g). The
lymphocytes have been decreased in the 2DG and FA decreased tumor volume might be because of the
treatment group. In the combined treatment group inhibition of EAC cell proliferation by the combination
(2DG+FA+IR), RBC count and hemoglobin content had of 2DG and FA. Reduction in viable cell count in the
significantly increased and WBC count had decreased peritoneal fluid of tumor host suggests the
compared with IR treatment group alone. radiosensitizing effect of combination of 2DG and FA in
Histopathological changes EAC bearing mice. It has been reported earlier that the
Figure 8 shows the histopathological results of combination of 2DG and FA decreases the cell
the liver of treated and untreated EAC bearing mice. In proliferation of NCI-H460 cells in vitro
EAC control mice, clumps of EAC cells were observed (Reddy and Prasad, 2011).
close to the capsule of liver. In radiation alone treated The induction of apoptotic pathway is crucial for
mice, few cells were seen adherent to the capsule and the anti-proliferative potential of treatment compounds
appeared viable. But there were no tumor cells outside administered during cancer therapy (Essack et al., 2011).
the capsule and the rest of the hepatocytes exhibited In this study, we observed an increase in the percentage
nuclear disintegration. In 2DG and FA treated group, the of EAC apoptotic cells in the radiation treated EAC
tumor cells were not seen both inside the capsule and mice. Administration of 2DG+FA before irradiation has
outside the capsule and the nuclear disintegration of the resulted in the maximal increase of apoptotic cells in the
hepatocytes was also observed. peritoneal fluid. Recently, specific intracellular proteases
The histopathological results of the spleen of belonging to the protease family have emerged as crucial
treated and untreated EAC bearing mice is shown in the effectors of apoptosis (Eriksson et al., 2009). It is well
figure 9. The tumor cells were seen peripheral to established that activated caspase 9 triggers a cascade
spleenic capsule and the anaphylatic cells were observed that culminates in the activation of caspase 3 resulting in
deeper to the capsule, but in the radiation group, the chromosomal DNA fragmentation and cellular
tumor cells were seen peripheral to the capsule with morphologic changes characteristic of apoptosis
necrosis. Animals received 2DG and FA together (Thornberry and Lazebnik, 1998). The present study
showed that, 2DG and FA increased the radiation effects results demonstrated that initiator caspase 9 from
on tumor cells by increasing the tumor cell necrosis. intrinsic pathway and effector caspase 3 participated in
the apoptotic process following radiation; these results
DISCUSSION are well correlated with the earlier findings
In the present study, we observed the (Eriksson et al., 2009). It was demonstrated that glucose
radiosensitizing potential of 2DG and FA in EAC depr i va t i on pr om ot ed the a ct i va t i on of
bearing Swiss albino mice. Tumor volume and tumor mitochondria-dependent pathways leading to apoptosis
weight has been reduced significantly upon the treatment (Munoz-Pinedo et al., 2003). It has been reported earlier
with 2DG (2 ml and 3.4 g, respectively), FA (2.1 ml and that phytochemicals induce reactive oxygen species,
3.6 g. respectively) and IR (1.5 ml and 2.4 g, which mediate apoptosis in human cervical cancer cells
respectively) when compared with EAC control mice (Javvadi et al., 2008). The induction of DNA strand
(3.9 ml and 4.1 g, respectively). In combined treatment breaks is often used to predict the radiosensitivity of
group (2DG+FA+IR), administration of 2DG and FA tumor cells. Increased DNA damage observed during
radiation treatment in the cells isolated from the and FA. It has been also observed that the combination
peritoneal cavity of EAC mice. In the combined of 2DG and FA also offers protection against
treatment group (2DG+FA+IR), the extent of DNA myelotoxicity, the most common side effect of cancer
damage was more, as is evident by the increased tail radiotherapy. It was reported earlier that 2DG offers
length and % of DNA in tail. 2DG inhibits DNA repair protection against perchloroethylene induced
pathways in cancer cells after exposure to ionizing a l t er a t i on s in h em a t ol og i c a l pa r a m e t er s
radiation (Sinthupibulyakit et al., 2009). Damage to (Ebrahim et al., 2001). Earlier reports also highlighted
DNA by ROS and RNS is reported to initiate signaling the protective effect of FA on hematopoietic cell
cascades and results in activation of transcription of recovery in -irradiated mice (Ma et al., 2011).
specific groups of genes which may lead to apoptosis The multitude of pathological changes caused by
(Valko et al., 2007). Phenolic acids are reported to cause the progression of tumor as well as its inhibition through
DNA damage by increasing the ROS production in treatment compounds are expected to be reflected in the
cancer cells (Kim et al., 2011; Chendil et al., 2004). biochemical parameters of the host system, particularly
The inclusion of phytochemicals in radiotherapy pertaining to the liver. In the present study, increased
regimens will improve the therapeutic index by killing LDH activity in EAC bearing mice might be because of
neoplastic cells and reducing radiation toxicity to normal two reasons; 1) progression of tumor 2) high rate of
tissues. The major problem that arises with radiation glycolysis observed in malignant conditions
treatment is anemia due to reduction in RBC. In this (Hazra et al., 2005). Administration of 2DG+FA before
study, elevated WBC count, reduced hemoglobin and irradiation helps in restoring the activity of LDH to near
RBC count were observed in EAC bearing mice. It was normal. Taken together with the decreased viable cell
observed there was a decrease in the RBC count, count in the combined treatment (2DG+FA+IR), the
hemoglobin content and WBC count in the radiation reason for restoration of LDH activity could partly be
alone treated EAC mice. Myelosuppression and anemia explained. In the present study, experimental animals
(reduced hemoglobin) have been frequently observed in exposed to -radiation alone have showed comparatively
ascites carcinoma (Price et al., 1950). Anemia elevated levels of LDH in serum. In alignment with
encountered in ascites carcinoma mainly due to iron earlier reports, it is assumed that the radiosensitive
deficiency, either by haemolytic or myelopathic organs thymus and spleen might be the possible sources
conditions which finally lead to reduced RBC number of increased amounts of serum LDH after -radiation
(Fenninger et al., 1954). Treatment with 2DG and FA exposure (Hori et al., 1970). It was reported earlier that
before irradiation restored the RBC count and the level of LDH release was reduced significantly by a
hemoglobin content to near normal levels, which treatment with 2DG in neural progenitor cells
indicates the protective action of 2DG and FA against the (Park et al., 2009). Microscopic examination of tissues
radiation induced damage. It has been reported earlier from EAC control mice showed marked alterations in the
that ferulic acid offers protection against -radiation structure of liver and spleen. Presence of tumor cells
induced damage in Swiss albino mice (Maurya and Nair, along the lining of the capsules of liver and spleen are
2006). Intraperitoneal administration of 2DG and FA seen in EAC bearing mice. Treatment with 2DG and FA
before irradiation restored the WBC count to near normal decreased the viable tumor cells along the lining of the
levels. The observed change in hematological parameters capsules and showed near to normal pathological status
supports the hematopoietic protecting activity of 2DG despite being challenged by the tumor cells.
Journal of Research in Biology (2012) 2(5):499-512 509

CONCLUSION Coleman MC, Asbury CR, Daniels D, Du J, Aykin-

In conclusion, the present study findings Burns N, Smith BJ. 2008. 2-deoxy-D-glucose causes
indicated the radiosensitizing potential of combination of cytotoxicity, oxidative stress, and radiosensitization in
2DG and FA, by decreasing the tumor weight, tumor pancreatic cancer. Free Radic Biol Med., 44:322-331.
volume, tumor cell count and increasing the expression
Delaney G, Jacob S, Featherstone C, Barton M. 2005.
of caspase family proteases. It has also been understood
The role of radiotherapy in cancer treatment: estimating
that the combination of 2DG and FA restored the
optimal utilization from a review of evidence-based
hematological and histopathological changes to near
clinical guidelines. Cancer 104:1129-1137.
normalcy. Further studies highlighting the molecular
mechanisms involved in the radiosensitizing activity of Dimitroulis J, Toubis M, Antoniou D, Marosis C,
2DG and FA are warranted. Armenaki U, Stathopoulos GP. 2006. Alternate
paclitaxel-gemcitabine and paclitaxel-vinorelbine
ACKNOWLEDGEMENT biweekly administration in non-small cell lung cancer
The financial assistance in the form of Senior patients: a phase II study. Anticancer Res., 26:1397-
Research Fellowship to Mr. Bandugula Venkata Reddy, 1402.
by the Indian Council of Medical Research (ICMR),
Ebrahim AS, Babu E, Thirunavukkarasu C,
Government of India, New Delhi, is gratefully
Sakthisekaran D. 2001. Protective role of vitamin E, 2-
acknowledged. We greatly acknowledge Dr G.
deoxy-D-glucose, and taurine on perchloroethylene
Prabavathi, Radiation safety officer, GVN cancer
induced alterations in ATPases. Drug Chem Toxicol.
hospital, Tiruchirapalli, India, for giving us technical
24:429-437.
assistance in handling irradiation facility.
Eriksson D, Lfroth PO, Johansson L, Riklund K,
REFERENCES Stigbrand T. 2009. Apoptotic signalling in HeLa Hep2
Ahmad IM, Aykin-Burns N, Sim JE, Walsh SA, cells following 5 Gy of cobalt-60 gamma radiation.
Higashikubo R, Buettner GR. 2004. Mitochondrial Anticancer Res. 29:4361-4366.
O2*- and H2O2 mediate glucose deprivation-induced
Essack M, Bajic VB, Archer JA. 2011. Recently
stress in human cancer cells. J Biol Chem., 280:4254-
confirmed apoptosis-inducing lead compounds isolated
4263.
from marine sponge of potential relevance in cancer
Bhattacharya S, Prasanna A, Majumdar P, Kumar treatment. Mar Drugs 9:1580-1606.
RB, Haldar PK. 2011. Antitumor efficacy and
Fenninger LD, Mider GB. 1954. In: Greenstein JP,
amelioration of oxidative stress by Trichosanthes dioica
Haddow A (ed) Advances in cancer research. Academic
root against Ehrlich ascites carcinoma in mice. Pharm
Press, New York. 244.
Biol., 49:927-935.
Gogvadze V, Orrenius S, Zhivotovsky B. 2008.
Chendil D, Ranga RS, Meigooni D, Sathishkumar S,
Mitochondria in cancer cells: what is so special about
Ahmed MM. 2004. Curcumin confers radiosensitizing
them? Trends Cell Biol., 18:165-173.
effect in prostate cancer cell line PC-3. Oncogene
23:1599-1607. Hazra B, Kumar B, Biswas S, Pandey BN, Mishra
KP. 2005. Enhancement of the tumour inhibitory
activity, in vivo, of diospyrin, a plant-derived quinonoid, increases the efficacy of adriamycin and paclitaxel in
through liposomal encapsulation. Toxicol Lett., 157:109- human osteosarcoma and non-small cell lung cancers in
117. vivo. Cancer Res., 64:31-34.
Hori Y, Takamori Y, Nishio K. 1970. The Effects of X- Maurya DK, Nair CKK. 2006. Preferential
Irradiation on Lactate Dehydrogenase Isozymes in radioprotection to DNA of normal tissues by ferulic acid
Plasma and in Various Organs of Mice. Radiat Res. under ex vivo and in vivo conditions in tumor bearing
43:143-151. mice. Mol Cell Biochem. 285:181-190.
Javvadi P, Segan AT, Tuttle SW, Koumenis C. 2008. Mukherjee KL. 1988. Medical laboratory technology. a
The chemopreventive agent curcumin is a potent procedure manual for routine diagnostic tests. Tata
radiosensitizer of human cervical tumor cells via McGraw Hill, New Delhi.
increased reactive oxygen species production and
Munoz-Pinedo C, Ruiz-Ruiz C, Ruiz DA, Palacios C,
overactivation of the mitogen-activated protein kinase
Lopez-Rivas A. 2003. Inhibition of glucose metabolism
pathway. Mol Pharmacol. 73:1491-1501.
sensitizes tumor cells to death receptor-triggered
Karthikeyan S, Kanimozhi G, Prasad NR, apoptosis through enhancement of death-inducing
Mahalakshmi R. 2011. Radiosensitizing effect of ferulic signaling complex formation and apical procaspase-8
acid on human cervical carcinoma cells in vitro. Toxicol processing. J Biol Chem., 278:12759-12768.
In Vitro., 25:1366-1375.
Ozaslan M, Karagoz ID, Kilic IH, Guldur ME. 2011.
Kim W, Seong KM, BuHyun Youn. 2011. Ehrlich ascites carcinoma. Afr J Biotechnol., 10:2375-
Phenylpropanoids in radioregulation: double edged 2378.
sword. Exp Mol Med. 43:323-333.
Park M, Song KS, Kim HK, Park YJ, Kim HS, Bae
Lee YJ, Galoforo SS, Berns CM, Chen JC, Davis BH, MI, Lee J. 2009. 2-Deoxy-d-glucose protects neural
Sim JE. 1998. Glucose deprivation-induced cytotoxicity progenitor cells against oxidative stress through the
and alterations in mitogen-activated protein kinase activation of AMP-activated protein kinase. Neurosci
activation are mediated by oxidative stress in multidrug- Lett., 449:201-206.
resistant human breast carcinoma cells. J Biol Chem.,
Price VE, Greenfield RE. Anemia in cancer. In:
273:5294-5299.
Greenstein JP, Haddow A (ed) 1950. Advances in
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. cancer research. Academic Press, New York. 199-200.
1951. Protein measurement with the Folin phenol
Rao SK, Rao PS, Rao BN. 2008. Preliminary
reagent. J Biol Chem. 193:265-275.
investigation of the radiosensitizing activity of guduchi
Ma ZC, Hong Q, Wang YG, Tan HL, Xiao CR, Liang (Tinospora cordifolia) in tumor-bearing mice. Phytother
QD. 2011. Effects of ferulic acid on hematopoietic cell Res., 22:1482-1489.
recovery in whole-body gamma irradiated mice. Int J
Reddy BV, Prasad NR. 2011. 2-deoxy-D-glucose
Radiat Biol. 87:499-505.
combined with ferulic acid enhances radiation response
Maschek G, Savaraj N, Priebe W, Braunschweiger P,
in non-small cell lung carcinoma cells. Cent Eur J Biol.,
Hamilton K, Tidmarsh GF. 2004. 2-deoxy-D-glucose
6:743-755.

Sharma S, Guthrie PH, Chan SS, Haq S, Taegtmeyer

H. 2007. Glucose phosphorylation is required for insulin-
dependent mTOR signalling in the heart. Cardiovasc
Res., 76:71-80.
Singh NP, McCoy MT, Tice RR, Schneider EL. 1988.

A simple technique for quantification of low levels of
DNA damage in individual cells. Exp Cell Res.,
175:184-191.
Sinthupibulyakit C, Grimes KR, Domann FE, Xu Y,

Fang F, Ittarat W. 2009. p53 is an important factor for
the radiosensitization effect of 2-deoxy-D-glucose. Int
J Oncol. 35:609-615.
Thornberry NA, Lazebnik Y. 1998. Caspases: Enemies

within. Science 281:1312-1316.
Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur

M, Telser J. 2007. Free radicals and antioxidants in
normal physiological functions and human disease. Int
J Biochem Cell Biol., 39:44-84.
Wroblewski F, LaDue JS. 1955. Lactic dehydrogenase

activity in blood. Proc Soc Exp Biol Med., 90:210-213.
Zheng LF, Dai F, Zhou B, Yang L, Liu ZL. 2008.

Prooxidant activity of hydroxycinnamic acids on DNA
damage in the presence of Cu (II) ions: mechanism and
structure-activity relationship. Food Chem Toxicol.,
46:149-156.

Advantages
Affordable Charges
Quick processing
Extensive indexing

Original Research
Adaptation to Education: An Analysis of College Students Health

Authors: ABSTRACT:
Sagaya rani C1,
Anusuya G2, Keerthana R3, Health is a resource of every days life, not the objective of living. Health is a
Navara banu S4. positive concept emphasizing social and personal resources, as well as physical
capacities. Fifty individuals of age group 20, 21, and 22 were considered. Blood
parameters investigated in the present study included haemoglobin concentration,
bleeding time, clotting time, blood grouping, blood pressure, body mass index and
Institution:
urine analysis. The healthy individuals were considered as control and the unhealthy
1. Associate Professor,
Department of Zoology, were considered as an experimental one. The health problems of the college student
J.A.College for Women are found to be high. The highest sixty percentage of college students found to be
(Autonomous), anemic in the age group of twenty years old. The lowest percentage of anemic
Periyakulam-625 601 condition is found in the age group twenty one and twenty two respectively. 86% of
students were found to be having normal weight, 10% were categorized as
2,3,4. Students, Department underweight and the remaining 4% came under overweight. Of the students analyzed
of Zoology, J.A.College for 64% had hypotension. It was a shocking reality that nearly 8% of the students had
Women (Autonomous), diabetes due to stress. The blood grouping was also performed which revealed that
Periyakulam-625 601 majority of the students (40%) had O+ve blood group. Suitable suggestions such as
intake of iron along with protein were given. Diet with necessary protein and iron was
provided.

Sagaya rani C. Body mass index, bleeding time, blood pressure, anemic condition,
hypotension.
Email:
sabiarsh@yahoo.in Article Citation:
Sagaya rani C, Anusuya G, Keerthana R, Navara banu S.
Adaptation to Education: An Analysis of College Students Health.
Phone No: Journal of Research in Biology (2012) 2(5): 513-522
9344620141
Dates:
Received: 24 May 2012 Accepted: 27 Jun 2012 Published: 14 Jul 2012
Web Address:
513-522 | JRB | 2012 | Vol 2 | No 5

An International
Rani et al.,2012
INTRODUCTION 10% being proteins, carbohydrates, lipid, hormones,

Health is achieved through the combination of enzymes, vitamins and salts. The majority of cells
physical, mental, emotional and social well being which suspend in plasma are erythrocytes. The leucocytes are
together is commonly referred to as the health triangle. much fewer in number than the erythrocytes while the
Health is maintained and improved not only through the platelets are associated with blood coagulation.
advancement and application of health science but also The erythrocytes contain a high concentration of
through the efforts and intelligent lifestyle choices of the haemoglobin in a red coloured pigments which acts as
individual and society. The Lalonde report suggests that the oxygen carrier. Leucocytes exist in different forms,
there are four general determinants of health including each having a physiological function in the defence
human biology, environment, lifestyle and health care (Ochei and Kolhathar, 2000).
services (Marc Lalonde, 1974). The present investigation was focused to study
Public health is the science and the art of the blood parameters, BMI and urine analysis. Fifty
preventing disease, prolonging life and promoting health individuals of age group 20, 21, and 22 were considered.
through the organized efforts and informed choices of The suggestions, awareness and preventive measures
society, organizations, public and private, communities were given to the individuals.
and individuals (Winslow, 1920). The focus of public
health intervention is to prevent rather than treating a MATERIALS AND METHODS
disease through surveillance of cases and prevention of Subjects
healthy behavior. The blood and urine samples from the students of
In a clinical set up a haematology laboratory is age group 20, 21 and 22 years of Jayaraj Annapackiam
concerned with the abnormalities of the constituents of College for Women at Periyakulam were collected.
blood, namely the plasma and the blood cells. General information was collected using questionnaire
The laboratory tests performed include enumeration of cum interview method.
different types of cells, relative distribution of various Sample
categories and their chemical functional and structural The venous blood was used for the present
abnormalities. Haematological tests are also required as a hematological investigation. The individual was asked to
part of patients admission report because many disease sit or lie down. The tourniquet was tied just above the
show signs and symptoms of haematological nature. puncture site. Blood was withdrawn from an anticubital
These tests are commonly referred to as complete blood vein or a prominent well supported vein. The syringe
count (CBC) and include estimation of haemoglobin, was taken in the right hand and the needle inserted at an
enumeration of red and white blood cells and differential angle of 30 to the skin to slowly puncture the vein.
count, (Ochei and Kolhathar, 2000). When proper amount of blood has been withdrawn, the
An average healthy adult has blood equivalent to cotton with spirit was applied over the puncture area and
7 - 8% of the body weight. In quality it is about 6 liters, the patient was asked to fold the arm for some time.
blood is mainly composed of plasma, a fluid in which red The blood was transferred slowly in to vial (Maiti, 1995).
blood cells (RBC) or erythrocytes, white blood cells Anticoagulants
(WBC) or Leucocytes and platelets or thrombocytes are A number of different anticoagulants are in use.
suspended. The plasma occupying 55% of the total Sodium and potassium salts of ethylene-di-amino-tetra
volume of blood contains 90%- water, the remaining acetic acid (EDTA) and trisodium citrate. Sodium citrate
Rani et al.,2012
Table 1 Categories of blood pressure Table 2 Colour change and the indication of sugar level
Systolic Diastolic OBSERVATION PRESENCE OF SUGAR
Categoery
mm/Hg mm/Hg Blue Nil
Hypotension <90 Or<60 Pale Green Trace
Normal 90-119 And 60<79 Greenish precipitate +, less than 0.5%
Pre-hypertension 120-139 Or 80-89 Greenish yellow ++, 0.5% to 1%
Stage: 1 hypertension 140-159 Or 90-99 Orange precipitate +++, 1% to 2%
Stage: 2 hypertension 160 or100 Red precipitate ++++, more than 2%
was preferred for the present haematological Clotting Time

investigations (Sona Yengkokpam et al., 2005). The finger is prepared for a capillary blood
Anticoagulant did not alter the size of the cells and other extraction. With the help of a sterile needle a puncture
haematological parameters. Besides, sodium citrate is made. Blood is collected in a capillary tube
removes calcium which is essential for coagulation. (10 cm. long* 0.8 to 1.2 mm. diameter) leaving some
Haemoglobin Concentration (Cyanmett - portion on either tips. Trapping of air bubble should be
haemoglobin method) avoided. The stop clock was started immediately. As the
0.02 ml (20l) of blood was added to 5ml of bleeding time of the person is already recorded, the
Drabkins solution in a test tube. It was mixed well and examination for blood clotting is started by breaking the
allowed to stand for 10 minutes. The absorbance was capillary tubes as bits by every 15 seconds. The clock is
read colorimatrically at 540 nanometers with Drabkins stopped as soon as the fibrin thread bridges the broken
solution as blank. The absorbance of standard was found ends when they have been separated by distance of
in the same way (Ochei and Kolhathar, 2000). 5 mm, or more. Normal value for clotting time was
Bleeding Time 3 7 minutes.
The finger was rubbed firmly to ensure adequate Blood Grouping
blood supply and is cleaned by clod sterilization method. The finger of the patient was punctured and two
With a sterile needle, the tip of the finger was punched. drops of blood were collected in a small test tube
With the help of blotting papers, the overflowing blood containing about 2ml of normal saline, and mixed gently.
was removed every 15 seconds without touching the A glass slide was taken and divided into two with the
skin. As soon as the blood starts flowing, the stop clock help of a wax pencil and marked as A and B. A drop of
is started. The finger is pressed hard periodically until suspension of blood and a drop of Group A sera (Anti B)
the last drop of blood was removed. When no more are put on one side of the slide, a drop of suspension of
blood comes out, stop clock is stopped and the time blood and a drop of Group B sera (Anti A) and mixed
was noted. Normal value for the bleeding time was gently by rocking the slide, it was allowed to stand for
1 3 minutes. few minutes, occasionally with rolling or tilting the slide
Table 3 Cloudiness and the indication of albumin level Table 4 Classification of body mass index
Observation Presence of Albumin Classification BMI
No cloudiness Negative Under weight >18.5
Cloudiness nearly Trace, less than 0.05 Normal >18.5-24.9
Dense cloudiness (+) upto 0.1g% Over weight >25.0-29.9
Granular cloudiness (++) 0.2 0.3% Obese (grade I) >30.0-34.9
Dense opaque cloudiness (+++) 0.5g% Obese (grade II) >35.0-39.9
Solid looking (++++) more than 0.5% Obese (grade III) >40

Rani et al.,2012
Table 5 Population size
AGE GROUP
PARAMETERS 20 21 22
N A N A N A
HB 28% 60%(11.31) 2% 2% (1.00) 4% 4% (1.41)
BT 86% -(30.41) 4% - (1.41) 10% - (3.54)
CT 80% 6% (26.16) 4% - (1.41) 10% - (3.54)
BMI 36% 50% (4.95) - 4% (1.41) 4% 6% (0.71)
BP 32% 54% (7.78) 4% 6% (0.71) - 4% (1.41)
US 6% 2% (1.41) - 4% (1.41) 2% 8% (2.12)
HB-Haemoglobin, BT-Bleeding time, CT-Clotting time, BMI-Body mass index, BP-Blood pressure,
US-Urine sugar, N-Normal, A-Anaemic, values within brackets show SD.
to ensure thorough mixing. observed. Various observations and the indication of
Blood Pressure level of albumin were tabulated in table 3.
The blood pressures of thse individuals were Bile Salt (Hays method)
measured with the help of sphygmomanometer and 5ml of urine was taken in a test tube. A pinch of
stethoscope. The blood pressure was categorized in dry sulphur powder was sprinkled over the surface of
table 1. urine. Sinking of sulphur powder towards the bottom of
Urine Analysis the test tube indicated the presence of excess bile salt in
The physical, chemical and microscope urine.
examination of urine was known as urine analysis. Urine Bile Pigments (Fouchets test method)
is the fluid containing water soluble waste products Equal volume of urine and 10% barium chloride
excreted from the blood via the kidneys. Urine is mainly solution were mixed thoroughly in the test tube and
composed off 95% water and the rest being made up of allowed to stand for two minutes, a white precipitate
urea, uricacid, cretinine, sodium, potassium, chloride, appears. It was filtered and dried. 2-4 drops of fouchets
calcium and phosphates (Ochei and Kolhathar, 2000). reagent was added to the residue on the filter paper.
Urine Sugar (Benedicts method) A greenish colour indicates the presence of bile pigments
5ml of Benedicts solution was taken in a test of bilirubin in the urine (Maiti, 1995).
tube and eight drops of urine sample was added. The test BODY MASS INDEX
tube was heated and cooled in water bath. Colour change Height
was observed. Various colour changes and the For measuring standard height vertical
indications were tabulated in table 2. anthropometrics rod was used. The height was recorded
Albumin (Heat and acetic acid method) to the nearest centimeter using the standardized
Urine sample was taken in a 5ml test tube. Few procedure.
drops of 3% acetic acid was added. The cloudiness was
Table 6 Percentage of haemoglobin concentration

among college students Table 7 Body Mass Index (BMI) of college students
AGE GROUP FEMALE Grade Age group

NORMAL ANAEMIC 20 21 22
20 28% 60% Under weight - 10% -
21 2% 2% Normal 86% - -
22 4% 4% Over weight - - 4%
Rani et al.,2012
Figure 1 Percentage of Heamoglobin Concentration Figure 2 Body Mass Index (Bmi) of

Among College Students College Students
Weight Where,
Weight was measured using standardized ATCO X = Arithmetic mean
digital balance. The body weight was recorded when the X = Sum of value of variables
digits display of the body weight becomes stabilized. The N = Number of observations
value of body mass index (table 4) was calculated for STANDARD DEVIATION
each by following methods, The standard deviation was calculated using
Weight (kg) the following formula.
BMI =
Height (m2)
STATISTICAL ANALYSIS
The raw data were subjected to the following
statistical analysis for interpreting results. Where,
Mean ai Mean value of the variables
Mean was computed using the following formula n- Number of observations
X= X / N
RESULTS AND DISCUSSION
The results of present study were recorded in the
form of tables and graphs and the same were interpreted
Figure 3 Blood Pressure of College Students Figure 4 Distribution of Blood Group

Rani et al.,2012
Table 8 Blood pressure of college students

Normal Blood Low Blood
Age group
Pressure Pressure
20 32% 54%
21 4% 6%
22 - 4%
assumption that nutritional iron deficiency is the major
cause of anaemia (Ricardo J Soares Magalhaes and
Archie CA Clements, 2011). In targeting communities
with the highest prevalence of anaemia, it is useful to
Figure 5 Percentage of Urine Sugar Among identify and geographically position anaemia-control
College Students resources, based on the relative importance of different
anaemia contributors.
appropriately to realize the objectives. Table 5 shows the The evaluation of BMI can be an indirect way of
total population size studied in the age group of twenty, determining the current nutritional status and
twenty one and twenty two years old college students. subsequently the level of social health care in a country.
The health problems of the college student were Therefore, regulatory policies on the basis of normal
found to be high. Sixty percentage of college students BMI in each nation, which may help overcoming weight
were found to be anaemic in the age group of twenty discrepancies, would be of special importance for
years. The lowest percentage of anaemic condition is improving people's life standards (WHO, 1995). Table 7
found in the age group of twenty one and twenty two (figure 2) shows the college students differing
respectively (table 6 and figure 1). These results were in significantly in their overall body mass index. 86% of
accordance with the findings of Junwal Manju and Bhai students were found to be normal weight. The remaining
Ismail (2012). A large variation in standard deviation differed significantly as underweight (10%) and 4% as
was found for the age group of twenty. The prediction overweight. Standard deviation (4.95) indicates that a
proves that education increases awareness about public large mass of students were anaemic. Even though high
health problem such as: anaemia, diabetes, obesity and percentage of students were having normal weight, the
B.P (Bentley and Griffiths, 2003). anaemic percentage was found to be high (50%).
These statistics underline the failure of national Although in this research, a relatively low
programmes and interventions to reduce the burden of frequency of overweightness was observed, results are
anaemia. One reason for the lack of success is that different in other reports (Heshmat, 2004; Mortazavi and
anaemia-control interventions are designed on the Shahrakipour, 2002). BMI depends on several variables,
Table 9 Distribution of blood group

Blood Group
Age group
O+ O- A+ A- A1+ B+ B- AB+ AB-
-
20 40% 4% 20% - 8% 8% - 8%
-
21 - - 2% - - 2% - -
-
22 - - - 2% - 2% - 4%

Rani et al.,2012
Table 10 Urine sugar analysis among college students were found to have O+ve blood group, 20% have A+ve, 4%
Female have O-ve, 8% have A+ve , 12% have B+ve, 12% have
Age Groups
Diabetic Non- Diabetic AB+ve and zero percentage students were found to have
20 6% 2%
21 0% 2% A-, B- and AB-ve.
22 2% 8% This study further confirms that blood group O
including behavior patterns, nutritional habits, was the most common of the ABO blood group system
social ideals and also changes due to aging in the population studied (Odokuma et al., 2007).
(Paknahad et al., 2001). Therefore, the aforementioned AB blood group was quite rare and Rhesus D was more
differences are to be expected, and BMI discrepancies common than Rhesus D negative phenotype.
may vary from one report to the other upon different The prevalence of diabetes is rapidly rising all
conditions and circumstances in which a study is over the globe at an alarming rate (Huizinga and
conducted. However, the 4% level of overweightness Fothman, 2006). Last three decades, the status of
evidenced in this study indicates a significant potential diabetes has changed from being considered as a mild
danger (high blood pressure, diabetes, heart disease, etc) disorder of the elderly to one of the major causes of
for the individual. Possible solutions are physical activity morbidity and mortality affecting the youth and middle
promotion, extracurricular sporting activities (especially aged people (Subramanian and Venkatesan, 2012). India
for females considering the social constraints on female leads the world with the largest number with
physical activities), counseling sessions on nutritional (40.9 million) diabetic patients (Sicree et al., 2006).
habits (recommendations on consuming fruits, and The most disturbing trend is the shift in age of onset of
vegetables) and an increase in social propaganda diabetes to a younger age in recent years, which could
concerning healthy foodstuffs (El Ansari et al., 2010). have long lasting adverse effect on nation's health and
Blood pressure is the pressure of blood against economy (Suresh et al., 2005). Table 10 and figure 5
the walls of the arteries. Blood pressure results from two represents the percentage of urine sugar among college
forces: one is created by the heart as it pumps blood into students. Nearly 8% of the students were found to be
the arteries through the circulatory system and the other diabetic and the reason was partially due to stress.
is the force exerted by the arteries as they resist the blood Anaemia in female adolescents appears to be a
flow (Kannel et al., 1972). Each time the blood pressure significant health problem in the world. The highest
is checked, two figures are recorded: the upper, or incidence of aneamia among teenage girls was very
systolic pressure, and the lower, or diastolic pressure. common. The prevalence of anaemia among college
The normal range varies with age, but a young adult students found much higher than in developed countries
would be expected to have a systolic pressure of around such as the united states and Norway where the
120 mmHg and a diastolic pressure of around 80 mm of prevalence of iron deficiency and anaemia among
mercury (Nichols and Rourke, 1990). Table 8 and figure adolescent girls was estimated to be 8.7% and 4%
3 represents the results with respect to blood pressure. respectively (Halter man et al., 2001; Ostro and
36% were found to be normal whereas 64% percentage Eskeland, 1999).
were found to have hypotension. These results were in In the recent studies from developing countries
contradiction to the findings of Bimenya et al., (2005). like Kenya, India, Indonesia and Bangladesh, the
The distribution of blood groups was presented prevalence of anaemia among adolescent girls has been
in table 9 and figure 4. It indicates that 40% of students found to be 21.1-27%. (Basu, 2005). However, Anaemia

Rani et al.,2012
in this region cannot be explained by iron deficiency egg. Copper is also essential for the utilization
alone and other causes of anaemia may also exist in this of iron in the building of haemoglobin
populat io n, such as malar ia, vit amin A (Fatin Al-Sayes et al., 2011).
deficiency, parasitic infections etc (Foo et al., 2004;
Bushra H. Al-Naemi et al., 2011). REFERENCES
Cook and Lynch (1986) proportional hazards Basu SM. 2005. Anaemia and Pregnancy, In: Gupta S,
models indicated high risk of elevated blood pressure editor. Obstetric Anaesthesia. 1st ed. Delhi: Arya
among over weight and obese subjects compared with Publications; 433-56.
adolescents with normal body mass index.
Bentley ME and Griffiths PL. 2003. European J.
clinical Nutrition, 57, 52-60.
CONCLUSION
Since females experience the monthly period, the Bimenya GS, Byarugaba W, Kalungi, S, Mayito J,
significant higher frequency of anemia in their group was Mugabe K, Makabayi R, Ayebare E, Wanzira H
predictable. Anaemia is much more easily prevented than and Muhame M. 2005. Blood pressure profiles among
corrected. A liberal intake of iron in the formative years Makerere University undergraduate students, Afr Health
can go a long way in preventing iron deficiency anaemia. Sci., 5(2):99-106.
Diet is of the almost importance in the treatment of
Bushra H. Al-Naemi, Zohair IF. Rahemo, Sundus N.
anaemia. Almost every nutrient is needed for the
Al-Kallak. 2011. Relationship between Anemia and
production of red blood cells, haemoglobin and enzymes,
Parasitic Infections in Shekhan District, Iraq, Journal of
required for their synthesis. Refined food like white
Research in Biology, 1(5):319-324.
bread, polished rice, sugar and desserts, rope the body of
the much needed iron (Harris Pastides, 1981). Cook JD, Lynch SR. 1986. The liabilities of iron
Nutritional counseling, iron supplementation, deficiency, American journal of clinical nutrition
and iron-fortification of foods may serve at allevating the 68:803-809.
burden of this condition in youths. Iron should always be
El Ansari, W, El Ashker S, Moseley L. 2010.
taken in its natural organic form as the use of inorganic
Associations between physical activity and health
can prove hazardous, destroying the protective vitamins
parameters in adolescent pupils in Egypt. Int J Environ
and unsaturated fatty acids, causing serious liver damage
Res Public Health., 7(4):1649-69.
and even miscarriage and delayed or premature births
(Jay B. Wish, 2006). The common foods rich in natural Fatin Al-Sayes, Mamdooh Gari, Safaa Qusti, Nadiah
organic iron are wheat and grain cereals, brown rice Bagatian and Adel Abuzenadah. 2011. Prevalence of
polishing, green leafy vegetables, cabbage, carrot, celery, iron deficiency and iron deficiency anaemia among
beets, tomatoes, spinach; fruits like apples, berries, females at university stage, Journal of Medical
cherries, grapes, raisins, figs, dates, peaches and eggs. Laboratory and Diagnosis, 2(1):5-11.
It has been provided that a generous intake of iron alone
Foo LH, Khor GL, Tee ES. 2004. Iron status and
will not help in the regeneration of haemoglobin.
dietary iron intake of adolescents from a rural
The supplies of protein too, should be adequate. The diet
community in Sabah, Malaysia, Asia Pac J Clin Nutr,
should, therefore, be adequate in proteins of high
13(1):48-55.
biological value such as those found in milk, cheese and
Rani et al.,2012
Halterman JS, Aligne CA, Szilagol P. 2001. Iron Nichols WW, Rourke MF. 1990. McDonald's blood
deficiency and cognitive achievement among school flow in arteries. 3rd Edition. Philadelphia Lea & Febiger,
aged children and adolescents in the United states, 216-250.
Pediatrics 107(6):1381-1386.
Ochei J, Kolhathar A. 2000. Introduction, heamoglobin
Harris Pastides. 1981. Iron deficiency anemia among and Medical Lab Technology 2559-256.
three groups of adolescents and young adults, The Yale
Odokuma EI, Okolo AC and Aloamaka PC. 2007.
Journal of Biology and Medicine, 54:265-271.
Distribution of ABO and Rhesus blood groups in
Heshmat R, Fakhrzadeh H, Pour-ebrahim R, Nouri Abraka, Delta State, Nigerian Journal of Physiological
M, Pazhouhi M. 2004. Evaluation of obesity and Sciences, 22(1-2):89-91.
overweight and their changes pattern among 25-64 aged
Ostro BD, Eskeland GS. 1999. Air Pollution and
inhabitants of Tehran University of Medical Sciences
Health Effects: A Study of Medical Visits among
population lab region, Iran J Diab Lipid Disord., 3:63-70.
Children in Santiago, Chile, Environmental Health
Huizinga MM, Fothman RL. 2006. Addressing the Perspectives, 107(1):69-73.
diabetes pandemic; A comprehensive approach", Indian
Paknahadm Z, Omidvar N, Mahboub S, Afiatmilani
Journal of Medicine research, 124: 481-484.
S, Ostadrahimi AR, Ebrahimi M. 2001. Body mass
Jay B. Wish. 2006. Assessing Iron Status: Beyond index of reproductive age group women and it's
Serum Ferritin and Transferrin Saturation, Clinical relationship with iron status. Journal of Tabriz University
journal of the American Society of Nephrology, of Medical Sciences, 35(51):17-23.
1(1):54-58.
Ricardo J Soares Magalhaes and Archie CA
Junwal Manju and Bhai Ismail. 2012. Studies on Clements. 2011. Spatial heterogeneity of haemoglobin
Human Anaemia Haemoglobin Hb assays R.B.Cs. count concentration in preschool-age children in sub-Saharan
in Ujjain, MP, India, ISCA Journal of Biological Africa, Bull World Health Organ., 89:459-468.
Sciences, 1(2):38-42.
Sicree R, Show J, Zimmet P. 2006. Diabetes and
Kannel WB, Castelli WP, McNamara PM, Mckee PA, impaired glucose tolerance, England, editor diabetes
Feinleib M. 1972. Role of blood pressure in the atlas", International diabetes federation, 3rd edition,
development of congestive heart failure: The Belgium 15-103.
Framingham Study. N Engl J Med., 287:781-787.
Sona Yengkokpam, Narottam, P. Sahu, Asim K. Pal,
Maiti CR. 1995. Urine analysis and observation, Subhas C. Mukherjee and Dipesh Debnath. 2005.
Medical Lab Technology 212-215. Haematological and hepatic changes in Catla catla
fingerlings in relation to dietary sources and levels of
Marc Lalonde. 1974. A new perspective on the health of
gelatinized carbohydrate, ACTA ICHTHYOLOGICA
Canadians-a working document, Government of Canada.
ET PISCATORIA, 35(2):87-92.
Mortazavi Z, Shahrakipour M. 2002. Body mass index
Subramanian SS and Venkatesan P. 2012. Stability
in Zahedan University of Medical sciences students,
Ball Exercises In Type 2 Diabetic Patients, Journal of
Tabib-e-Shargh., 4(2):81-6.
Research in Biology, 2(4):403-409.

Rani et al.,2012
Suresh S, Deepa R, Pradeepa R, Rema M, Mohan V.

2005. Large scale diabetes awareness and prevention in
South India", Diabetes Voice 50:11-40.
Winslow, Charles-Edward Amory. 1920. "The

Untilled Fields of Public Health". Science, 51(1306):23-
33.
World Health Organization Expert Committee on

Physical Status. 1995. In: Physical status: the use and
interpretation of anthropometry. Geneva: WHO; World
Health Organization.

Advantages
Affordable Charges
Quick processing
Extensive indexing

Original Research
Effect of extract of Bacillus megaterium 9554 on oral

cancer cell line SCC131
Authors: ABSTRACT:
Vipin Mohan Dan1,Vinitha
Richard2, Asha Nair2,
Palpu Pushpangadan1, A microbe, identified as Bacillus megaterium 9544 was isolated from the soil
Varughese George1, sample collected from Manali, Himachal Pradesh. The bacterial broth extract had the
3
Ajit Varma and presence of compound(s) that induced apoptotic like features on oral cancer cells, like
Radhakrishna Pillai2.
morphological changes, condensation of nucleus and detachment of cancer cells from
the surface of culture vessel. The extract did not cause any kind of changes in
Institution:
1. Amity Institute of Herbal mitochondrial membrane potential as evidenced from flow cytometric analysis and
and Biotech Product did not cause fragmentation of nuclear DNA. This particular compound(s) which was
Development, Trivandrum, present in the bacterial extract seemed to induce only a weak apoptotic signal that did
Kerala. not lead to complete progression of apoptosis.
2. Department of Molecular
Medicine, Rajiv Gandhi
Centre for Biotechnology,
Trivandrum, Kerala.
Keywords:
3. Amity Institute of Apoptosis, Flow cytometry, Bacillus, Cancer.
Microbial Technology,
Amity University, Noida,
Uttar Pradesh.

Vipin Mohan Dan and Vipin Mohan Dan,Vinitha Richard, Asha Nair, Palpu Pushpangadan, Varughese
Ajit Varma. George, Ajit Varma and Radhakrishna Pillai.
Effect of extract of Bacillus megaterium 9554 on oral cancer cell line SCC131.
Phone No: Journal of Research in Biology (2012) 2(5): 523-528
(0471) 2432138.
Fax Dates:
+91 120 2431268 Received: 03 Jul 2012 Accepted: 18 Jul 2012 Published: 25 Jul 2012
Email:
vipindan@yahoo.co.in
Web Address: reproduction in all medium, provided the original work is properly cited.
523-528 | JRB | 2012 | Vol 2 | No 5
An International
Dan et al., 2012
INTRODUCTION MATERIALS AND METHODS

Soil bacteria have been a treasure of many Cancer cell line
valuable natural products ranging from antibiotics, Human oral cancer cell line SCC131 was
anticancer compounds to other secondary metabolites of procured from National Centre for Cell Sciences
industrial and agricultural importance. Microbes have a (NCCS, Pune). The cell line was grown in DMEM
perplexing relation with humans, from mutual beneficial (Dulbeccos Modified Eagle Media) medium
associations in the human body, to the fatal diseases that supplemented with 10 percent FBS (Fetal Bovine Serum)
other members cause and the counteractive compounds in a 5 percent CO2 incubator at 37C. Antibiotics
yet other family members produce to ward-off or cure penicillin (100g/mL) and streptomycin (100g/mL)
such infections. Cancer is one of the diseases where were also added into the medium to avoid any bacterial
intensive research is carried out globally in search of a contamination.
cure. Soil bacteria have contributed immensely in Microorganism
producing many clinically viable anticancer drugs. The bacterial strain used in this study was
Actinomycin D is the oldest microbial metabolite used in Bacillus megaterium 9554, which was previously
cancer therapy. Its relative, actinomycin A, was the first isolated from soil samples collected from Manali,
antibiotic isolated from actinomycetes and was obtained Himachal Pradesh. The bacterial culture was routinely
from Streptomyces antibioticus by Waksman and maintained on Nutrient agar slants. The organism was
Woodruff (1941).Anthracyclines rank among the most subcultured every month. The bacterial isolate was
effective anticancer drugs ever developed (Weiss, 1992). identified based on morphological, physiological
They are effective against most types of cancer than any and biochemical characteristics as described in
other class of chemotherapy agents. They are used to Bergeys manual of determinative bacteriology
treat a wide range of cancers, including leukemias, (Holt et al., 1994).
lymphomas, and breast, uterine, ovarian and lung Bacterial extract
cancers. Anthracyclines act by intercalating DNA The bacterial culture was grown in nutrient broth
strands, which result in a complex formation that inhibits at 150rpm, 37C for 48 h. The broth was centrifuged and
the synthesis of DNA and RNA. It also triggers DNA the supernatant was filtered to remove bacteria. The pH
cleavage by topoisomerase II, resulting in mechanisms of supernatant was adjusted to 2 with Conc. HCl and
that lead to cell death (Weiss 1992). Among the Bacillus then centrifuged at 16,000g, 10 min at 4C. The
species, a class of protein called Parasporins that come pr ecipitate obt a i n e d wa s d i s s ol v e d in
under Cry proteins produced by Bacillus thuringiensis Dimethyl sulphoxide: DMEM (50:50), pH was
had shown cytocidal activity against human cancer cell maintained at 8,filtered through 0.2m filter and kept
lines (Mizuki et al., 2000). In another study at -20C.
Bacillus vallismortis BIT-33 produced a compound of Microscopic analysis and MTT assay
anticancer nature that was effective against various colon Cancer cells were seeded in 96-well plates at a
cancer cell lines (Jeong et al., 2008). The present study density of 5000 cells per well. The attached cells were
involves the investigation of culture extracts of incubated for 24 h and then treated with different
Bacillus megaterium 9554 for presence of compound(s) concentrations (25g, 50g, 100g, 150g, 200g,
with anticancer potential. 300g and 400g per ml of DMEM medium) of
bacterial extract, and were then incubated for 24 and
524 Journal of Research in Biology (2012)2(5) : 523-528
Dan et al., 2012
Figure 1: Effect of extract treatment on oral cancer cell line. Changes in cell morphology
were observed on treatment with extract.
48 h. The cells were visually analyzed with microscope membrane potential of control and treated oral cancer
for morphological changes. The cell viability was cell line by flow cytometry. Cells were cultured in 12
determined with 20l of MTT solution (5 mg/ml). well plates at a density of 5 x 105 cells per well and
The plates were incubated for 4 h at 37C. DMSO was incubated in CO2 incubator for overnight at 37C.
used to dissolve the formazan crystals that formed and The cells were treated with bacterial extract and
soluble formazan product was spectrophotometrically untreated cells were taken as control and incubated for
quantified using an ELISA reader at 575 nm. 48 h. After the incubation period 100 l of JC-1 staining
Nuclear Staining with Hoechst 3342 solution was added per mL of culture medium to each
Trypsinized cells were seeded into 12 well plates well of the plate. The cells were incubated in a CO2
at 10,000 cells per well and kept for 24 h incubation. incubator at 37C for 15-30 min. The cells were
The spent media was aspirated and cells were rinsed with harvested from each well and were analyzed using flow
PBS, different concentrations of the bacterial extract cytometry.
were added to respective wells. Plates were placed in DNA Fragmentation assay
carbon dioxide incubator. After 24 and 48 h each well Oral cancer cells were plated in culture plates at
was incubated with Hoechst 3342 (5 l/mL) for another a density of 1x105 /ml and then treated separately with
15 min at 37C in carbon dioxide incubator. The samples different concentrations of bacterial extract (150g/ml
were analyzed through fluorescence microscope. and 300g/ml) and curcumin (100g/ml) for 48 h. DNA
Mitochondrial membrane potential analysis was extracted by the method described by
MitoProbe JC-1 Assay Kit procured from Life Mori et al., (2001). DNA fragmentation was analyzed on
technologies was used for analyzing mitochondrial agarose gel electrophoresis.
Figure 2 : Nuclear staining with Hoechst 3342 assay. Condensation of nucleus (intense bright
fluorescence) was observed in the two extract treatments as compared to control cancer cells at 48h.

Dan et al., 2012
Figure 3: Mitochondrial membrane potential analysis of control and extract treated SCC131 oral cancer cell
line at 48h.Viable cells are seen in Quarter Q2 while dead cells are seen in Quarter Q4.The percent of viable
cancer cells remained above 99% in two treatments as well as control.
RESULTS AND DISCUSSION Mitochondrial membrane potential assay,

Bacillus megaterium 9554 was identified based revealed that the bacterial extract had no compounds of
on morphological, biochemical and physiological anticancer nature, with the two treatments and control
characteristics (Table 1) as per the Bergeys manual of registering, above 99 percent healthy viable cells with no
determinative bacteriology (Holt et al., 1994). changes in mitochondrial membrane potential (fig. 3).
The treatment with extract caused morphological DNA fragmentation assay confirmed that the apoptotic
changes in cancer cells as evidenced from microscopic features initially induced by bacterial extract did not lead
analysis, the cells lost their striated shape, attained a to fragmentation of cells DNA while treatment with
rounded morphology and detached from surface of curcumin (positive control) gave the fragmented DNA
culture vessel (fig. 1). Initial analysis with MTT assay ladder pattern (fig. 4). The cells even though initially
revealed that morphologically redefined cells were viable showed apoptotic features like morphological changes,
as it utilized MTT thereby signifying the presence of detachment from culture vessel and condensation of
intact mitochondria with mitochondrial dehydrogenase
enzyme responsible for cleaving the yellow MTT to blue
formazan crystals. Two concentrations of the bacterial
extract, 150 g and 300g, that produced effective
morphological change in more than 50% of the cells
were selected for further study. Hoechst staining
revealed that the oral cell line showed condensation of
nucleus on treatment with bacterial extract (fig. 2).
During the early process of apoptosis, cell shrinkage and
pyknosis are visible by light microscopy
(Kerr et al., 1972). Pyknosis is the result of chromatin Figure 4: DNA Fragmentation assay. A: Extract
condensation and this is the most characteristic feature of (150g/ml) treated SCC131 DNA; B: Extract
(300g/ml) treated SCC131 DNA; C: Curcumin
apoptosis (Elmore 2007). (100g/ml) treated, fragmentation of SCC131 DNA
was caused only by curcumin.

Dan et al., 2012
Table1: Morophological, biochemical and cell line SCC131. Further investigation is required to
physiological characteristics of Bacillus megaterium disclose, the scientific reason that causes, such initial
9554;+: positive; -: negative; (+): Weak positive
apoptotic like features in the cancer cells but which does
Tests
Colony morphology not progress to a complete apoptotic pathway.
Grams reaction +ve
Cell shape rods
ACKNOWLEDGEMENT
Size (m) 2-4
Spores +, central, oval The author would like to thank CSIR, India for
Motility - providing Research associate fellowship. The author
Physiological tests
would like to express sincere gratitude to Prof
Growth Temperatures 4C to 50C
pH range 79 Radhakrishna pillai, Director, Rajiv Gandhi Centre for
NaCl (%) 2 - 10 Biotechnology (RGCB), Kerala for providing research
Biochemical tests
facilities. Special thanks to Mrs Indu Ramachandran,
MacConkey agar -
Indole test - Senior Technical Officer, RGCB for helping with flow
Methyl red test + cytometry.
Voges Prokauer test -
Citrate utilization +
H2S production - REFERENCES
Caesin hydrolysis - Elmore S. 2007. Apoptosis: A Review of Programmed
Esculin hyrolysis -
Cell Death. Toxicol Pathol. 35:495-516.
Gelatin hyrolysis +
Starch hyrolysis +
Hoeppner DJ, Hengartner MO and Schnabel R. 2001.
Nitrate reduction (+)
Catalase test + Engulfment genes cooperate with ced-3 to promote cell
Oxidase test + death in Caenorhabditis elegans. Nature 412:202-6.
Arginine dihydrolase +
Holt JG, Krieg NR, Sneath PHA and Staley JT. 1994.
nucleus on extract treatment eventually did not progress Bergeys Manual of Determinative Bacteriology.
to complete apoptosis. This cell survival phenomenon Nineteenth edition, Williams and wilkins company,
may be due to weak apoptotic signals, as per Baltimore, MD, USA 255-273.
Hoeppner et al., (2001), weak pro-apoptotic signals can
Jeong SY, Park SY, Kim YH, Kim M and Lee SJ.
enhance cell survival. It can also be hypothesized that the
2008. Cytotoxicity and apoptosis induction of
bacterial extract may have presence of compound(s) that
Bacillus vallismortis BIT-33 metabolites on colon cancer
can affect the anchoring proteins of cancer cells and also
carcinoma cells. J. of Appl. Microbio., 104:796-807.
create a slightly stressful environment for the cancer
cells. Kerr JF, Wyllie AH and Currie AR. 1972. Apoptosis:
a basic biological phenomenon with wide-ranging
CONCLUSION: implications in tissue kinetics. Br J Cancer 26:239-57.
The culture broth extract of Bacillus megaterium
Mizuki E, Park YS, Saitoh H, Yamashit S, Akao T,
9554 had the presence of compound(s) that produced
Higuchi K and Ohba M. 2000. Parasporin, a human
weak apoptotic signals or created a slightly stressful
leukemic cell-recognizing parasporal protein of
environment that led to morphological changes,
condensation of nucleus and detachment of oral cancer

Dan et al., 2012
Bacillus thuringiensis. Clin Diagn Lab Immunol.

7:625-634.
Mori H, Niwa K and Zheng Q. 2001. Cell proliferation

in cancer prevention; effects of preventive agents on
estrogen-related endometrial carcinogenesis model and
on an in vitro model in human colorectal cells. Mutat
Res, 480-1, 201-7.
Waksman SA and Woodruff HB. 1941. Actinomyces

antibioticus, a new soil organism antagonistic to
pathogenic and non-pathogenic bacteria. J. Bacteriol.
42: 231-249.
Weiss RB. 1992. The anthracyclines: will we ever find a

better doxorubicin? Semin Oncol. 19:670-686.

Advantages
Affordable Charges
Quick processing
Extensive indexing

Original Research
Criteria for optimization of mass rearing of the parasitoid Cotesia flavipes

in the laboratory
Authors: ABSTRACT:
Ramalho DG1, Viel SR2,
Vacari AM1, In this study, we aimed to evaluate criteria that serve to optimize the mass
De Bortoli SA1, rearing of the parasitoid Cotesia flavipes in the laboratory. The following studies were
Lopes MM3, Laurentis VL1 conducted: (1) Optimal density of the third instar larvae of Diatraea saccharalis was
and Veiga ACP1. parasitized using a rearing unit, (2) Parasitism capacity during the lifespan of
C. flavipes was examined, (3) Pupae of C. flavipes were weighed, and (4) Adult
Institution:
1. laboratory of Biology and C. flavipes were fed with different diets. We found that a density of five larvae of
Insect Rearing (LBRI), D. saccharalis parasitized using a rearing unit is most suitable for the mass production
Department of Crop of C. flavipes. A female C. flavipes may parasitize three larvae of D. saccharalis for up
Protection, College of to 4 h after copulation, generating quality individuals for mass rearing. It is impractical
Agricultural and Veterinary to mass market the parasitoid C. flavipes by weight, the correct is to count the number
Sciences, Sao Paulo State of masses to form the release unit. A diet of 2.5% honey and 5% sucrose is most
University, 14884-900 suitable for adults during the mass rearing of C. flavipes.
Jaboticabal, Sao Paulo,
Brazil.
2. Louis Dreyfus
Commodities (Sugarcane Keywords:
Mill), 14870-904 Quality control, larval parasitoid, mass rearing, biological control, sugarcane.
Jaboticabal, Sao Paulo,
Brazil.
3. College of Education Sao
Luis, 14870-37, Jaboticabal,
Sao Paulo, Brazil.

Vacari AM. Ramalho DG, Viel SR, Vacari AM, De Bortoli SA, Lopes MM, Laurentis VL and
Veiga ACP.
Criteria for optimization of mass rearing of the parasitoid Cotesia flavipes in the
laboratory.
Web Address:
http://jresearchbiology.com/ Dates:
documents/RA0215.pdf. Received: 13 Mar 2012 Accepted: 03 Apr 2012 Published: 28 Jul 2012

529-536 | JRB | 2012 | Vol 2 | No 5

An International
Ramalho et al., 2012
INTRODUCTION in demand, discrediting the use of biological control and

Sugarcane culture is performed in several consequently increasing the use of pesticides. These
Brazilian states, and the country ranks first in the world results are reflected in increased environmental impact in
in the production of sugar and ethanol (Mapa, 2011). Brazil, which has become the largest consumer of
Sustainable agricultural production of sugar cane insecticides since 2008, according to Anvisa (2011).
in Brazil is based on the ability to respond to pests, In this study, we have evaluated the criteria used to
diseases, and climatic variations, while ensuring the least optimize the mass rearing of the parasitoid C. flavipes in
possible impact to the environment through the use of the laboratory.
integrated pest management (Macedo, 2000).
Biological control is a promising alternative for MATERIALS AND METHODS
pest management. Regulating the number of individuals The experiments were performed in a
in the pest population through the action of biotic climate-controlled room at 28 1C, with a relative
mortality agents is a natural process. Biological control humidity (RH) of 50 10% and a photophase for 12 h.
of the pest Diatraea saccharalis (Fabricius) Densities of D. saccharalis larvae parasitized by
(Lepidoptera: Cambridae) by using the endoparasitoid C. flavipes per rearing unit
Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) The exper iment was co ndu ct ed at

is the largest program of biological control in Brazil. Ecoquality -Agents of Biological Control in Piacatu, Sao
Today, this parasitoid is released in just over three Paulo, Brazil. The third instar larvae of D. saccharalis
million hectares, and about 80 biofactories produce this were subjected to parasitism by C. flavipes and placed in
natural enemy. Although there are regional differences, petri dishes (6 2 cm) containing an artificial diet,
the main pests are controlled by biological means by where they remained until a mass of pupae formed.
using insects or pathogens (Alves et al., 2008). Thus, Testing was performed at densities of 3, 4, 5, 6, 7, and 8
sugar cane culture is one of the few crop cultures that has larvae per dish (100 replicates per treatment). After the
focused on sustainable agricultural production since pupae were formed, the masses were removed, and the
1970 (Gallo et al., 2002). dishes were placed in transparent 100-mL plastic cups
During laboratory production of the wasp with lids. We evaluated the number of masses formed
C. flavipes, D. saccharalis larvae is the only food used per larvae per dish.
because during development the wasp requires a Parasitism capacity of C. flavipes females
sugarcane borer in order to multiply (Macedo et al., The exper iment was co ndu ct ed at

1983). Ecoquality -Agents of Biological Control in Piacatu, Sao
To meet the demand and applications for Paulo, Brazil. Thirty C. flavipes females were placed in
sugar cane, mass rearing of the wasp is necessary, and individual petri dishes (6 2 cm) for 5 h, which is the
one of the biggest problems faced during the laboratory time required for coupling (30 replicates). Every 2 h, the
production of C. flavipes is the quality of the parasitoid females received third instar larvae of D. saccharalis for
(Prezotti and Parra, 2002). The low quality of the parasitism. Each parasitized larva was identified with the
parasitoid is responsible for its failure in production and number of females that oviposited and then stored in
non-demand. It also contributes to producers resistance petri dishes (diameter, 6 cm) containing the artificial
to using this parasitoid in the field, given that the goal is diet. Parasitism was performed every 2 h until the death
to control the target pest and failure implies a reduction of the parasitoid. The pupae masses obtained from
parasitized larvae were transferred to 10-mL tubes and masses, weight of 30 pupae masses, number of adults
closed with perforated plastic lids. The newly emerged that emerged from mass, and sex ratio of C. flavipes.
parasitoids were frozen and subsequently separated by Data analysis
sex on the basis of the sexual dimorphism of antennas, The data of different densities of parasitized
as described by Wilkinson, (1928). We then counted the larvae per dish were processed using cluster analysis
number of males and females that emerged from with the program Statistica 7.0 (Statsoft Inc., 2004).
each mass. Cluster analysis was used as an exploratory method,
Weight of C. flavipes pupae which does not investigate the assumptions of
The experiment was conducted at the Laboratory independence and normality.
of Entomology of Louis Dreyfus Commodities Mill, Data weighing of the pupae mass of C. flavipes
Jaboticabal, Sao Paulo, Brazil. The experiment was during pupal stage analysis was conducted using
conducted in a room at 28 1C (RH 40 10%), with a PROC REG SAS Institute (2002) by considering
photophase for 12 h. There were 150 pupae masses of the weight of 30 masses and weight loss as the
C. flavipes in petri dishes (6 2 cm). The pupae masses dependent variables (y) and the time in hours as the
were aged 024 h when they were weighed, and after independent variable (x). The curve model was selected
15 days of parasitism, they were weighed every 12 h on basis of the significance of the coefficient (P<0.05)
until the adults emerged. Five pupae masses were and its contribution to better biological representation.
weighed at 0, 12, 24, 36, 48, and 60 h. The experimental The data parasitism capacity and feeding of
design was completely randomized, with six treatments C. flavipes adults with different diets were analyzed
and five replicates. Thirty masses were weighed and using the Kolmogorov and Barttlet tests for normality
weight loss was calculated. and homogeneity of variance, respectively, and
Feeding of C. flavipes adults with different diets necessary changes were made to satisfy the requirements
The experiment was conducted in a of analysis of variance (ANOVA). ANOVA using
climate-controlled room at 28 1C, RH 40 10%, and PROC ANOVA from SAS Institute (2002) was
a photophase for 12 h, at the Laboratory of Entomology performed, and the average values were compared using
of Louis Dreyfus Commodities Mill, Jaboticabal, Sao Tukeys test at 5% probability when significant on the
Paulo, Brazil. basis of ANOVA.
C. flavipes adults were fed different diets before
beginning parasitism of the host D. saccharalis larvae RESULTS
15 days of age were parasitized by C. flavipes adults fed The results for the productivity of C. flavipes of
with diets previously diluted in water to the following different densities of pupae masses showed that when
concentrations: 2.5% sucrose, 2.5% sucrose + 2.5% there were 3, 4, and 5 larvae per petri dish, a higher
yeast, 2.5% honey, honey + 2.5% yeast 2.5, 5% sucrose, number of masses was produced (Fig. 1). The most
5% sucrose + 2.5 yeast, 5% honey, 5% honey + 2.5% suitable density for mass production was five larvae per
yeast, 10% sucrose, 10% sucrose + 2.5% yeast, 10% dish, which was determined on the basis of the quality of
honey, 10% honey + 2.5% yeast, 2.5% yeast, distilled parasitoids produced, productivity (caterpillars
water, and control (without diet). The experimental inoculated/employee/day), and saving of the supplies
design was completely randomized, with fifteen and materials used.
treatments and five replicates. We weighed the pupae When we studied the time of parasitism after

copulation, we observed that the 0-h and 2-h treatments

yielded the highest number of C. flavipes masses and a
greater number of adults emerged. With regard to the
number of male offspring, the 0-h treatment yielded the
highest number. After 4 h, there was no emergence of
females (Table 1).
We observed a reduction in the weight of pupae
masses: the average weight of the mass were 56.8, 54.8,
47.6, 41.0, 34.2, and 28.6 and the average weight of
30 masses (a unit release) were 1704, 1646, 1428, 1232,
1034, and 440 mg for pupae aged 0, 12, 24, 36, 48, and
60 h, respectively. This loss in weight corresponds to Fig 1: Dendrogram showing the similarity in the
Cotesia flavipes parasitoids mass rearing with
3.52, 16.20, 27.82, 39.80, and 48.64% for 12, 24, 36, 48, different densities of hosts per rearing unit
and 60 h, respectively (Fig. 2). diets containing 5% sucrose and diets containing
The weight of a mass, weight of 30 masses, 10% sucrose + 2.5% yeast were higher than that with the
number of adults that emerged from a mass, and the sex other treatments, which yielded similar results, while
ratio of C. flavipes that originated following parasitism adults fed a diet containing 5% honey + 2.5% yeast
by adults fed different diets were examined, and it was showed lower adult emergence. With regard to the sex
found that pupae masses treated with a diet of ratio of adults that emerged from the pupae mass, the
2.5% honey gained more weight. Further, treatment with biggest difference was found for the diet with
5% sucrose led to an increase in the weight of the 5% honey + 2.5% yeast, with a slight difference for diets
parasitoid mass, and in the control, weight gain was containing 10% sucrose + 2.5% yeast and 10% honey.
similar to that with other treatments. Treatments of The other treatments did not differ in this regard
5% sucrose + 2.5% yeast, 5% honey, and (Fig. 3).
5% honey + 2.5% yeast yielded lower weight per unit The results showed that treatment with
mass (Fig. 3). 5% sucrose leads to greater weight and a positive
The number of adults that emerged from the influence on the emergence of C. flavipes adults.
masses derived by the parasitism of adults fed With regard to adult emergence and sex ratio, treatments
Table 1: Number of masses produced and audits that emerged from pupae
masses of cotesia flavipes after the parasitism of females of different ages.
a a ab
ab ab b
b ab a
b ab
b ab
b ab
b b
Means followed by the same letter in the column do not differ, according to the result of Tukeys test (P>0.05).
emerged per mass, the treatment containing 5% sucrose

yielded positive results, while the treatment containing
10% honey yielded positive results with regard to
sex ratio (Fig. 3).
DISCUSSION
Other studies on the parasitism behavior of
C. flavipes found that females discriminate parasitized
hosts for up to 24 h after parasitism. However, analysis
of the sex ratio of offspring that originated from
caterpillar superparasitism showed that females laid
more eggs than males; this suggested the ability of this
species to identify hosts previously parasitized by
females in an intraspecific manner, even a few days after
the first parasitism (Campos-Farinha et al., 2000).
Yamauchi et al., (2002) observed that two successive
ovipositions of C. flavipes in the same caterpillar
D. saccharalis did not alter the sex ratio of offspring but
increased the number of parasitoids and the number of
unfeasible larvae and pupae. Campos-Farinha (1996)
examined parasitism of D. saccharalis larvae by a
C. flavipes female and found that the number of
ovipositions varied from 1 to 4 times, and larvae that
matured into superparasitadas resulted in more males.
Regarding the quality control of the parasitoid
C. flavipes, Carvalho et al., (2008) observed that in a
laboratory with an average production of 6,000 unit
releases (transparent 100-mL plastic cups) with
30 masses per week, counting the number of adults from
a sample of six cups is sufficient to estimate the sex
ratio of the population released and to perform an
Fig 2 Weight of a mass, 30 masses, and weight loss appropriate quality control.
of pupae of C. flavipes that originated from the
parasitism of audit that were not fed In studies on biological control of sugarcane
borers, inundative releases of C. flavipes happen once or
containing 10% sucrose + 2.5% yeast showed better in installments, when the population reaches 800 to 1000
results, because more adults emerged and the sex ratio larvae (greater than 1.5 cm) per hectare or a minimum of
was smaller. However, positive results were obtained for 10 larvae per man hour of collection (Pinto et al., 2006).
individual treatments containing 2.5% honey, with Thus, it is necessary to calculate the number of wasps
regard to the weight of masses. With regard to adults that that should be released in the field in relation to pest

Fig 3 Weight of a mass. 30 masses, the number of adults that emerged from a mass, and the sex ratio of
cotesia flavipes that originated from the parasitism of adults fed different diets
population density in order to achieve successful pest C. flavipes can be programmed in a dynamic manner so
control rather than using C. flavipes pupae masses on the that the number of offspring is optimized by delimiting
basis of their weight, since variations over time present the area and reserves of eggs and reducing the number
challenges in calculating the number of wasps in the of eggs when supplies are scarce (Parke and Courtney,
field. 1984).
According to Godfray, (1994), since the
nineteenth century, entomologists knew that parasitoid CONCLUSION
wasps change their offspring in response to changes in A density of five larvae of D. saccharalis per
the environment and that this adaptation might explain rearing unit was found to be the most suitable condition
the differences in the sex ratio of wasps. The wasp for the mass rearing of C. flavipes.
A C. flavipes female may parasitize three larvae Gallo D, Nakano O, Silveira Neto S, Carvalho RPL,
of D. saccharalis for up to 4 h after copulation, Batista GC, Berti Filho E, Parra JRP, Zuchi 212 RA,
generating quality individuals for mass rearing. Alves SB, Vendramim JD, Marchini LC, Lopes JRS
It is impractical to market pupae masses of the and Omoto C. 2002. Entomologia agrcola. FEALQ,
parasitoid C. flavipes by weight, the correct is to count Piracicaba, 920.
the masses for the formation release unit.
Godfray HCJ. 1994. Parasitoids: behavioral and
Diets of 2.5% honey and 5% sucrose were
evolutionary ecology. Princeton University Press,
determined to be the most suitable for feeding adults
Pinceton, 99-106.
used for the mass rearing of C. flavipes.
Macedo N. 2000. Mtodo de criao do parasitoide
REFERENCES Cotesia flavipes (Cameron, 1981). In: BUENO VHP
Alves SB, Lopes RB, Vieira AS and Tamai MA. 2008. (Ed.). Controle biolgico de pragas: produo massal e
Fungos entomopatognicos usados no controle de pragas controle de qualidade. UFLA, Lavras, 161-166.
na Amrica Latina. In: Alves SB and Lopes RB.
Macedo N, Botelho PSM, Degaspari N, Almeida LC,
Controle microbiano de pragas na Amrica Latina:
Arajo Jr and Magrini EA. 1983. Controle biolgico
avanos e desafios. Piracicaba, FEALQ. p. 91-104.
da broca da cana-de-acar: manual de Instruo.
Anvisa. 2011. Reavaliao de agrotxicos: 10 anos de Piracicaba, IAA/PLANALSUCAR.
proteo a populao. <http://www.anvisa.gov.br/
Mapa. 2011. Cana-de-acar. <http://
alimentos/alimentacao/2009/0309.pdf>.
www.agricult ura.go v.br/vegetal/cult uras/cana-de-
Campos-Farinha AEC. 1996. Biologia reprodutiva de acucar>.
Cotesia flavipes (Hymenoptera: Braconidae). Tese de
Parke GA and Courtney SP. 1984. Models of clutch
Doutorado. Instituto de Biocincias, Universidade
size in insect oviposition. Theor Popul Biol 26:27-48.
Estadual Paulista, Jlio de Mesquita Filho, Rio Claro,
97. Pinto AS, Cano MA and Santos EM. 2006.
A broca-da-cana, Diatraea saccharalis. In: Pinto AS
Campos-Farinha AEC, Chaud-Netto J and Gobbi N.
(Ed.). Controle de pragas da cana-de-acar. Biocontrol,
2000. Biologia reprodutiva de Cotesia flavipes
Sertozinho, 15-20.
(Camero n) (Hymeno pt era: Braco nidae. IV.
Discriminao entre lagartas parasitadas e no- Prezotti L and Parra JP. 2002. Controle de qualidade
parasitadas de Diatraea saccharalis Fabricius em criaes massais de parasitoides e predadores In:
(Lepidoptera: Pyralidae), tempo de desenvolvimento e Parra, JRP, Botelho PSM, Corra-Ferreira BS & Bento
razo sexual dos parasitides. Arq Inst Biol 67: 229-234. JMS (Eds.). Controle biolgico no Brasil: parasitoides e
predadores. Manole, So Paulo, 295-296.
Carvalho JS, Vacari AM, De Bortoli SA and Viel SR.
2008. Armazenamento de pupas de Cotesia flavipes SAS Institute. 2002. SAS/STAT User`s Guide, version
(Cameron, 1891) (Hymenoptera: Braconidae) em baixa 9.00 TS level 2MO. SAS Institute Inc., Cary, NC.
temperatura. Bol Sanidad Veg Plagas 34:21-26.

Statsoft INC. 2004. Statistica: data analysis software

system, version 7. Tulsa. <http://www.statsoft.com/>.
Yamauchi MN, Gobbi N, Chaud-Netto J and

Campos-Farinha AE. 2002. Retation between number
of ovipositions of Cotesia flavipes (Cam.) and number
of d escend ant s e mer g ed fr o m it s ho st
Diatraea saccharalis (Fabr.). An Soc Entomol Bras
26:87-91.
Wilkinson DS. 1928. Eight new species of Braconidae.

Bull Entomol Res18:33-50.

Advantages
Affordable Charges
Quick processing
Extensive indexing

Original Research
Diversity of aquatic fungi in Hebbe water falls of Chikmagalore Taluk,

Chikmagalore District, Karnataka
Authors: ABSTRACT:
Suresha HR, Raju GH,
Krishnappa M1, Enrique
Descals2 and Syed abrar. The aquatic fungi are commonly found in water bodies. The current study
was carried out in Hebbe waterfalls of Chikmagalore district. The foam samples and
Institution: decaying debris were collected from the water falls, and kept for incubation
1.Department of PG. Studies
respectively. The decaying debris was screened using stereo binocular microscope and
and Research in Applied
Botany, Kuvempu recorded the conidia. The contribution in the frequency of occurrence and abundance
University, of aquatic fungi were also enumerated. A total of thirty three species were isolated
Shankaraghatta-577451, belonging to thirteen genera were identified.
Shivamogga dist. Karnataka.
2. Instituto Mediterrneo de
Estudios Avanzados Keywords:
(CSIC/Univ. Illes Balears). Aquatic fungi, decaying debris, waterfalls.
Calle Maestro Miquel
Marqus, 21, 07190
Esporles, Mallorca, Balears,
Spain.

Krishnappa M. Suresha HR, Raju GH, Krishnappa M, Enrique Descals and Syed abrar.
Diversity of aquatic fungi in Hebbe water falls of Chikmagalore Taluk, Chikmagalore
District, Karnataka.
Email: Journal of Research in Biology (2012) 2(5): 537-543
krishnappam4281@yahoo.com
Dates:
Web Address: Received: 07 Jun 2012 Accepted: 27 Jun 2012 Published: 28 Jul 2012

537-543 | JRB | 2012 | Vol 2 | No 5

An International
Suresha et al.,2012
INTRODUCTION (Barlocher 1992, Suberkropp 1997). Temperate

Aquatic fungi play an important role in aquatic woodland streams, in contrast to alpine streams, receive
ecosystem. These fungi have the capacity to degrade large amounts of litter during autumn leaf fall.
aquatic dead plants and animals. Hebbe falls comes Consequently, leaves from riparian plants have been
under the chikmagalure district. This stream falls down recognized as an essential component of lotic organic
from a height of 168 meters in two stages to form matter dynamics and leaf breakdown considered to be a
Dodda Hebbe (Big Falls) and Chikka Hebbe process in the metabolism of streams. The streambed of
(Small Falls) this falls is surrounded area contains many low order tropical streams is composed mainly by dead
different kinds of trees, and medicinal plants water is leaves and twig pieces originated from this type of forest.
running in this thick vegetation. Aquatic fungi rich This substratum is decomposed by physical, chemical
diversity is reported in this area compared to other study and biological factors, and can colonize by an aquatic
sites decaying leaves using substrata. Aquatic hyphomycetes.
hyphomycetes are decaying the leaves and animals as in The estimated fungal diversity is usually linked
an aquatic ecosystem. The selected study site contains with evolution patterns. The knowledge of general
rich fungal diversity in this location. This aquatic diversity of the native mycota is most important when
hyphomycetes are using leaf substrata, aquatic considering that each fungal species as its own niche in
hyphomycetes were found to be colonizing among the habitat. They can be found participating in the food
themselves. The organism produces an enzyme which webs as organic matter decomposers and contributors to
can degrade it selves. These organisms can rapidly nutrients cycling or symbionts with producers. The group
colonize the available substrata within a few days of aquatic fungi produces conidia exclusively in the
releasing prodigious number of conidia for further aquatic environment or in the terrestrial water among soil
dispersal (Barlocher, 1992). This substratum is particles. The main habitats are streams, reservoirs,
decomposed by physical, chemical, and biological lakes, falls typically, substrates like leaves, twigs and of
factors. Lignocelluloses the major components of residues. Submerged macrophytes and eventually
biomass are degraded by that enzyme itself. healthy roots. The conidia of aquatic hypomycetes may
Natural disturbance, from seasonal changes rainfall and be found trapped in foam, dispersed in the water, floating
tree fall, to hurricane damage, cause population shifts on the surface or associated with decomposing organic
and ch an ges to com m un i t i es of fun gi matter (Ingold, 1975). Leaf breakdown in streams is
(Lodge and Cantrell, 1995). The release of fungal caused by the joint action of physical factors, the activity
propagules follows a similar dynamic, but after peaks of microorganisms, such as aquatic hypomyctes
earlier and declines more rapidly. Biomass estimates (Barlocher, 1992). Together with chemical reactions, the
have generally been based on extracting and measuring activities of these organisms result in changes in litter
ergosterol, a fungal specific indicator that does not allow quality (eg. increases in N and P concentrations), and
differentiating between species (Gessner et al., 2003). cause litter mass loss. A number of factors affect these
Fungal communities have generally have been processes, including pH of the stream water,
characterized by counting and identifying conidia concentration of nutrients, and temperature
released in agitated water. This favours sporulating (Webster and Benfield, 1986, suberkropp, 1998). The
aquatic hypomycetes, widely considered to be dominant role of aquatic hypomycetes as intermediaries between
fungal group involved in leaf decomposition in streams imported plant detritus (leaves, needles, wood) and
Table.1. Occurrence and distribution of aquatic fungi in Hebby falls
Number of Number of Number of

Sl no Species Name Density Frequency Abundance
study site species occurrence individual
1 Alatospora accuminata 8 1 2 0.25 0.13 2.00 0.75 1.15
2 Alatospora pulchella 8 4 5 0.63 0.50 1.25 1.89 4.60
Suresha et al.,2012
3 Anguillospora gigantea 8 1 8 1.00 0.13 8.00 3.02 1.15

4 Anguillospora crassa 8 3 3 0.38 0.38 1.00 1.13 3.45
5 Anguillospora longissima 8 2 6 0.75 0.25 3.00 2.26 2.30
6 Anguillospora pseudo longissima 8 2 4 0.50 0.25 2.00 1.51 2.30
7 Anoptodera indica 8 1 3 0.38 0.13 3.00 1.13 1.15
8 Anoptodera mangrovei 8 2 6 0.75 0.25 3.00 2.26 2.30
9 Centrospora acerina 8 2 6 0.75 0.25 3.00 2.26 2.30
10 Centrospora angulata 8 2 10 1.25 0.25 5.00 3.77 2.30
11 Centrospora filiformis 8 4 11 1.38 0.50 2.75 4.15 4.60
12 Clucidospora aquatica 8 5 7 0.88 0.63 1.40 2.64 5.75

13 Culicidospora gravida 8 1 6 0.75 0.13 6.00 2.26 1.15
14 Dactylella aquatica 8 5 2 0.25 0.63 0.40 0.75 5.75
15 Dactylella submersa 8 3 8 1.00 0.38 2.67 3.02 3.45
16 Lemonniera aquatica 8 3 7 0.88 0.38 2.33 2.64 3.45
17 Lemonniera pseudofloscula 8 3 3 0.38 0.38 1.00 1.13 3.45
18 Lemonniera terrestris 8 2 5 0.63 0.25 2.50 1.89 2.30
19 Lunulospors submersa 8 2 14 1.75 0.25 7.00 5.28 2.30
20 Lunulosporus aquatica 8 3 8 1.00 0.38 2.67 3.02 3.45
21 Lunulospora curvula 8 3 20 2.50 0.38 6.67 7.55 3.45
22 Tetracladium apiense 8 1 10 1.25 0.13 10.00 3.77 1.15
23 Tetracladium furcatum 8 3 10 1.25 0.38 3.33 3.77 3.45
24 Tetracladium marchalianum 8 2 7 0.88 0.25 3.50 2.64 2.30
25 Tetracladium maxilliforme 8 1 8 1.00 0.13 8.00 3.02 1.15
26 Tricladium angulatum 8 4 9 1.13 0.50 2.25 3.40 4.60
27 Tricladium attenuatum 8 2 8 1.00 0.25 4.00 3.02 2.30
28 Tricladium chietocladium 8 3 12 1.50 0.38 4.00 4.53 3.45
29 Tricladium curvisporum 8 2 4 0.50 0.25 2.00 0.75 2.30
30 Tricladium splendens 8 1 10 1.25 0.13 10.00 2.64 1.15
31 Triscelosphorus accuminatus 8 4 12 1.50 0.50 3.00 4.53 4.60
32 Triscelosphorus monosporus 8 8 16 2.00 1.00 2.00 6.04 9.20
33 Triscelosphorus patulum 8 2 15 1.88 0.25 7.50 5.66 2.30
539
Suresha et al.,2012
3.00
2.50
2.00
Density
1.50
1.00
0.50
0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
No Of Species
Fig-1 Graphical representation of the Density against no of species
1.20
1.00
0.80
Frequency
0.60
0.40
0.20
0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
No Of Species
Fig-2 Graphical representation of the Frequency against no of species
12.00
10.00
8.00
Abundance
6.00
4.00
2.00
0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
No Of Species
Fig-3 Graphical representation of the Abundance against no of species
higher tropic levels has been well documented autumnal leaf fall (Barlocher, 1992; Suberkrpopp, 1997).
(Barlocher, 1992; Suberkropp, 1997). In the first and The physical factors like light, temperature and
thus far only published study of annual production of pH are the important environmental factors in aquatic
these fungi, it fell within the same range as that of ecosystem. These environmental factors are very
bacteria and invertebrates, two other major heterotropic essential for productivity of aquatic biotic and abiotic
groups of organisms in wood land streams (suberkropp, components. In the present study, work was carried out
1997). Because of their strong dependence on deciduous on the colonization of aquatic fungi on the specific
leaves in many temperate streams, biomass, growth, and composition of leaf debris and sporulation.
reproduction of aquatic hypomycetes are strongly Study area
seasonal and generally peak a few weeks after the Chikmagalur taluk comes under district of
Suresha et al.,2012
Chikmagalur the south Western parts of Karnataka state and sterile polythene bags, which were used to collect
in India. The hebbe falls situated 13 22' and 13 47 N debris. The fungi were isolated by plating technique.
latitude to 75 29 and 75 46 E longitude. The altitude The substratum of plant leaves and wood pieces were
of hebbe falls is 20975h. The temperature varies from also kept for incubation. The aquatic fungi were
15C to 25C and the annual rain fall is 1904 mm. monocultures and were further studied for their colony
The heavy rain fall has resulted in many water tanks, characters. The diversity studies include chytrids in lotic
streams and reservoirs. A lot of foam is developed in all ecosystem and lentic ecosystems, which revealed
the seasons. zoospores and mitosporic fungi. The incubation study
showed five aquatic fungi. These fungi were further mass
multiplied by the help of baiting technique and were
stored in sterile polythene bags.
Isolation of Spores by using England finder.
Most Ingoldian fungi were isolated by this
technique as it offers several important advantages over
the spore suspension technique. The spores were
identified under compound microscope with great
accuracy that relatively small spores down to 5mm in
spam or less can also be isolated.
The slide finder is a standard sized microscopic
slide bearing a photographically reduced grid pattern
each compartment being identified with letter and /or
METHODOLOGY number coordinates arranged sequentially along the
Foam analysis: horizontal and vertical axes. A piece of agar medium
The foam was scooped with the help of spoon from the foam plates. This foam plates were placed on
and transferred into a jam jar or similar receptacle. the finder, the spores relocated individually under the
Equal parts of FAA (Formal-acetic-alcohol) was added compound microscope, the coordinates directly
and fixed. The germined conidia were examined under underneath each spore were noted and the spores
microscope. relocated under the dissecting microscope for lifting.
Leaf packs baiting experiment: Isolation of suspended spores
The leaves were collected and cut in to small Hair technique
pieces and were kept in nylon mesh. The same set was This technique was used to capture the single
kept in a direction of moving water and observed after spore isolation using hair under the dissection
fifteen days. Later aquatic hyphomycetes were isolated. microscope. The incubated plates were placed under the
Incubation studies: dissection microscope then spore along with leaf bits.
During investigation, water samples were The conidial expression was observed, after confirming
collected from the study sites (from lentic and lotic water the conidia with the help of mounted hair, single conidia
bodies) of Chikmagalur district and the water samples were transferred to malt extract plats. The plates were
were also collected in forest and agriculture fields. incubation at 18C for one week.
The water samples were collected in small plastic bottles

Suresha et al.,2012
Micropipette technique from the phylloplane of the riparian trees

This technique was suitable to capture floating (Barlocher, 1992). The aquatic fungi play important role
spores in incubated plates. The incubated plates were in degradation of organic complex to simpler form.
placed under the dissection microscope and the The species of hyphomycetes occurs abundantly in
spore /conidia were observed. The selected spores were submerged leaves. The study proved that different
sucked using micropipette and same was directly transfer aquatic hyphomycetes species vary in their susceptible to
to the malt extracts plate. The plates were kept for soluble substance present in plant materials. In this study
incubation at 18C for week. area contains rich vegetation of different types tree
Sedimentation Experiment species is commonly found in this study area. The tree
This experiment is a standard technique used in species produces variety of dead leaves and are collected
zooplanktons by limnologists. Its main handicap is that in aquatic ecosystem. The rain fall is very high in
the sedimented samples have to be observed with an compare to other taluks rain fall create aquatic
inverted microscope where resolution is not the best. But ecosystems. The diversity of aquatic fungi rich in this
this could possibly be solved if sedimentation is carried study area compares the other study sites. Producers,
out in a burette and the bottom potion collected such as plants and algae, acquire nutrients from inorganic
and observed. The Spore suspension collected from a sources that are supplied primarily by decomposers
burette could be placed on a disc of cellophane attached where as decomposers, mostly fungi and bacteria acquire
by its margins onto a peteriplate and air dried in front of carbon from organic sources that are supplied primarily
a fan or heater. The spores adhered to the cellophane by producers. This producerdecomposer co-dependency
could then be mounted under cover slips placed directly is important in governing ecosystem processes which
in it and observed under microscope and the cellophane implies that the impacts of declining biodiversity on
disc could be lifted from the petridish and pieces of it cut ecosystem functioning should be strongly influenced by
up and mounted on slides. this process. Present study show, by simultaneously
manipulating producer (green algal) and decomposer
RESULTS AND DISCUSSION (heterotrophic bacterial) diversity in freshwater
During the current study, conidia belonging to microcosms that algal biomass production varies
thirty three species were encountered in hebbe falls, considerably among microcosms. In the present study
aquatic fungi belonging to thirteen genera. The table-1 accounts thirty three species (Table-1) of aquatic fungi
shows total thirty three species were recorded in the belonging to thirteen genera were isolated from the
study site. Tricelosporas monosporus, are the common to study site. In Hebbe falls maximum numbers of aquatic
in all the study site. To describe fungal communities in fungi were recorded out of eight study sites the
streams, biologists have identified sporulating structures domnent species is Tricelosporas monosporus, and
on substrata (collecting a random, or introduced and Alatospora accuminata and Dactylella aquatica are very
studied after variable periods of stream) or characterized rare in species recorded out of two study sites. Density,
numbers and types of aquatic hypomycetes. The second frequency and abundance against number of species,
approach is more direct, and relies on a well-defined which can be shown (Fig-1), (fig-2) and (fig-3) clearly in
stage in the fungal life cycle. One potential drawback is graphical representation. Shannon and shimson test will
that some conidia may have been washed in form the be applied, the past software is used, and version is 2004.
terrestrial surroundings or even dripped in to the stream These results indicate that some species of aquatic
Suresha et al.,2012
hyphomycetes can tolerate adverse environmental hypomycetes in streams. Fungal diversity Research
conditions as also stressed by (Barlocher, 1987). series 10;127-157.1x .
Ingold CT. 1975. Conidia in the foam of two English

CONCLUSION
streams. Transaction of the British mycological society
The role of aquatic fungi found to be more
65:522-527.Text-figs.1-4.1x.
important in the degradation of organic complex to
simpler form. The hyphomycetes occur commonly and Lodge D and Cantrell S. 1995. Fungal communities in
abundantly in submerged leaves. The study proved that wet tropical variation in time and space. Canadian
different aquatic hyphomycetes species vary in their Journal of Botany73:1391-1398.
susceptible to soluble substance present in plant
Suberkropp K. 1997. Annual production of leaf-
materials. We explored the significance of fungal
decaying fungi in a woodland stream. Fresh water
diversity on aquatic ecosystem processes by testing
Biology 38:169-178.
whether microfungal in different tropical leaf species
increases the rate of decomposition. This plant leaves Suberkropp K. 1998. Effect of dissolved nutrients on
revealed aquatic fungal diversity will changes according two aquatic hyphomycetes growing on leaf litter.
to particular species. The physical factors like light, Mycological Research102:998-1002.1x.
temperature and pH are the important environmental
Webster JR and Benfield EF. 1986. Vascular plant
factors in aquatic ecosystem. These environmental
breakdown in fresh water ecosystems. Annual review of
factors are very essential for productivity of aquatic
ecology and systematics17:567-594.
biotic and abiotic components. In the present study, work
was carried out on the colonization of aquatic fungi on
the specific composition of leaf debris and sporulation.
The associated aquatic hypomycetes in plant leaves are
very useful in self purification of the aquatic ecosystem.
ACKNOWLEDGEMENT
I am very thankful to department of Applied
Botany Kuvempu University for providing the laboratory
facilities.
REFERENCES Submit your articles online at jresearchbiology.com
Barlocher F. 1987. Aquatic hyphomycetes spora in Advantages

10 streams of New Brunswick and Nova scotia.
Canadian journal of Botany 65:76-79. Affordable Charges
Quick processing
Barlocher F. 1992. The ecology of Aquatic Extensive indexing
hyphomycetes. Springer-Verlag, Berlin.
Gessner MO, Barlocher F. and Chauvet E. 2003. www.jresearchbiology.com/Submit.php.
Qualitative and quantitative analysis of aquatic

Journal of Research in Biology - Volume 2 Issue 5

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Journal of Research in Biology - Volume 2 Issue 5

Uploaded by

Copyright:

Available Formats

Journal of Research in Biology An International Scientific Research Journal

Studies on some biochemical effects of aqueous leaf

Corresponding author: Keywords:

Email: Article Citation:

410-417 | JRB | 2012 | Vol 2 | No 5

INTRODUCTION (Hwang et al., 2002).

Journal of Research in Biology (2012) 2(5): 410-417 412

ALT 19.20.26 16.20.44 19.50.35 16.40.11 19.60.01 16.50.04 19.60.52 16.50.32

AST 16.30.05 13.30.21 16.40.25 13.50.13 16.50.06 13.50.71 16.50.04 13.60.46

ALP 23.40.06 20.50.29 23.60.02 20.60.16 23.60.24 20.60.93 23.50.16 20.70.46

*Values are mean SD of triplicate determinations M =Male, F=Female

413 Journal of Research in Biology (2012) 2(5): 410-417

Table 2: Total Protein conc. (mg/dl)

Table 4: Lipid peroxidation level [TBARS] (mg/ml)

Journal of Research in Biology (2012) 2(5): 410-417 416

Person JGM, Dilst FJH, Kuijpers RP, Leenwerberg

Agricultural University Paper No.92. 323. Advantages

Effect of starvation period on growth performances and

Corresponding author: Keywords:

Email: Article Citation:

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

418-423 | JRB | 2012 | Vol 2 | No 5

INTRODUCTION invertebrates, investigations on larval starvation or food

Journal of Research in Biology (2012) 2(5): 418-423 420

421 Journal of Research in Biology (2012) 2(5): 418-423

Journal of Research in Biology (2012) 2(5): 418-423 422

Pechenik JA and Cerulii T. 1991. Influence of delayed

423 Journal of Research in Biology (2012) 2(5): 418-423

Physical and chemical characteristics of Paraliyar river in

Corresponding author: Keywords:

Email: Article Citation:

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

424-0438 | JRB | 2012 | Vol 2 | No 5

INTRODUCTION present study an attempt has been made to analyse the

Fig2.Surface water temperature (oC) of the water

Journal of Research in Biology (2012) 2(5): 424-438 426

samples collected from station IV, showed a maximum Dissolved Oxygen

427 Journal of Research in Biology (2012) 2(5): 424-438

Fig 5. Dissolved Oxygen (mg/l) content of the water

Journal of Research in Biology (2012) 2(5): 424-438 428

Journal of Research in Biology (2012) 2(5): 424-438 430

431 Journal of Research in Biology (2012) 2(5): 424-438

Journal of Research in Biology (2012) 2(5): 424-438 432

and low in September. At station V, the potassium was COD

Journal of Research in Biology (2012) 2(5): 424-438 434

435 Journal of Research in Biology (2012) 2(5): 424-438

Journal of Research in Biology (2012) 2(5): 424-438 436

CONCLUSION: Husbandry and Medicine. 242-5. Pergamon Press

revealed that most of the physical and chemical

437 Journal of Research in Biology (2012) 2(5): 424-438

Nema P, Rajgopalan S and Mehta CG. 1984. Quality Science, 12:240-253.

Singh R and Mahajan I. 1987. Phytoplankton and

Sreenivasan A. 1976. Limnological studies and primary

Sudhira HS and Kumar VS. 2000. Monitoring of lake

Journal of Research in Biology (2012) 2(5): 424-438 438

Radio-protective and anti-clastogenic effects of Barleria lupulina Lindl. extract against

Email: Article Citation:

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

439-447 | JRB | 2012 | Vol 2 | No 5

INTRODUCTION identified from Central National Herbarium, Botanical

SET I 300 12000 35 30 18 15 82 16 18 36 36 286 2.38 %

Journal of Research in Biology (2012) 2(5): 439-447

Journal of Research in Biology (2012) 2(5): 439-447

Fig 2: Photomicrographs (PM) showing various types