Pakistan Journal of Scientific and Industrial...

ISSN 0030-9885 Coden: PSIRAA 48 (4) 223-296 (2005)

Vol. 48, No. 4, July-August, 2005

Pakistan Journal of Scientificand Industrial Research

Published Bimonthly byScientific Information Centre

Pakistan Council of Scientific and Industrial ResearchKarachi, Pakistan

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PROF. W. LINERTVienna University of Technology,Vienna, AustriaPROF. B. HIRALAL MEHTAUniversity of Mumbai, Mumbai, IndiaPROF. E. MIRALDIUniversity of Siena, Siena, ItalyDR. J. OZGAUniversity of Alberta, Edmonton, CanadaDR. J. R. OGRENEditor, Journal of Materials Engineeringand Performance, Los Angeles, USAPROF. H. M. ORTNERTechnical University of Darmstadt,Darmstadt, Germany

DR. H. AKHTARAgriculture and Agri-Food Canada,Ontario, CanadaPROF. M. AKHTAR, FRSUniversity of Southampton,Southampton, United KingdomDR. A. G. ATTKINSUniversity of Reading,Reading, United KingdomPROF. G. BOUETUniversity of Angers, Angers,FranceDR. M. A. KHANKing Abdulaziz City for Science andTechnology, Riyadh, Saudi Arabia

DR. M. J. QURESHINuclear Institute for Food andAgriculture, Peshawar, PakistanDR. ZAFAR SAIED SAIFYUniversity of Karachi, Karachi,PakistanDR. F. M. SLATERCardiff University, Powys,United KingdomPROF. M. A. WAQARInternational Centre for ChemicalSciences, University of Karachi,Karachi-75270, Pakistan

EditorsGhulam Qadir Shaikh Gulzar Hussain JhatialShagufta Y. Iqbal Shahida BegumSardar Ahmad Nazish Sajid Ali

ProductionRiazuddin Qureshi Irshad Hussain

Chairman Editorial BoardDr. Anwar ul HaqS.I., Pride of Performance,

Tamgha-e-Baqa, FPAS, FTWAS

Chairman, PCSIRDr. Saeed Iqbal Zafar Dr. Kaniz Fizza AzharEditor-in-Chief Executive Editor

Editorial Board

Pakistan Journal of Scientific and Industrial Research

Pakistan Journal of Scientific and Industrial Research

Vol. 48, No. 4 Contents July - August 2005

Physical Sciences

Some Common Fixed Point Theorems in Fuzzy 2-Metric Spaces

Sushil Sharma and Jayesh Kumar Tiwari 223

The Fe-Gd Phase Diagram

S. Atiq, R. D. Rawlings and D. R. F. West 231

Effect of Sodium Chloride on Dissolution of Galena in Aqueous Acid Solution

Abdul Khalique, Adnan Akram, A. S. Ahmed and Nusrat Hamid 236

Separation of Close Boiling Acidic Isomers by Dissociation Extraction

A. S. Ahmed, M. B. Bhatty, M. T. Saeed and M. P. I. Qazi 240

Determination of the Level of PCBs in Small Fishes from Three Different

Coastal Areas of Karachi, Pakistan

A. B. Munshi, A. S. Hina and T. H. Usmani 247

Biological Sciences

Laboratory Evaluation of Plant Extracts as Antifeedant Against the Lesser Mealworm,

Alphitobius diaperinus and Rice Weevil, Sitophilus oryzae

M. Kamruzzaman, M. Shahjahan and M. L. R. Mollah 252

Quantification of Bactericidal Action of the Ethanolic Extract of Garcinia kola Seeds

Alone, and in Combination with the Branded Antibiotic Septrin, on the Culture

Isolates from Throat Irritation Patients by Bacterial Growth Kinetics

V. A. Ajibade, I. G. Adanlawo, T. T. Akande 257

Assessment of the Safety of Wild Strains of Lactobacillus as Probiotics Orogastrically

Administered to Rats

V. O. Oyetayo 263

Restriction of Root and Shoot Growth Limits Their Growth Rates and Changes the

Morphology of Cotton Seedlings During Emergence

Ghulam Nabi and C. E. Mullins 268

Evaluation of Nutritive Properties of the Large African Cricket (Gryllidae sp)

Yemisi A. Adebowale, Kayode O. Adebowale and Michael O. Oguntokun 274

Prevalence of Intestinal Helminth Parasites of Dogs in Lagos, Nigeria

Comfort Adejoke Ibidapo 279

Correlation and Path Analysis in Candidate Bread Wheat (Triticum aestivum) Lines

Evaluated in Micro-Plot Test Trial

Tila Mohammad, Sajjad Haidar, M. Jamil Qureshi, Abdul Jabbar Khan and Roshan Zamir 284

Short Communication

Studies on the Utilization of Oscillatoria thiebautii as Food Source for Artemia sp Culture

S. G. Abbas Shah, Razia Sultana and Abid Askari 289

Technology

Isolation, Determination and Characterization of Taro (Colocasia esculenta) Starch

Feroz A. Jaffery, Abid Hasnain, Khalid Jamil and Tahir Abbas 292

Introduction

The concept of fuzzy sets was initially introduced by Zadeh

(1965). Since then, for the purpose of using this concept in

topology and analysis, many authors have expansively deve-

loped the theory of fuzzy sets and applications. Kaleva and

Seikkala (1984), Deng (1982), Kramosil and Michalek (1975),

and Erceg (1970) have particularly introduced the concept of

fuzzy metric spaces in different ways. Recently, many authors

have also studied the fixed point theory in these fuzzy metric

spaces (Sharma and Deshpande, 2003; 2002; Chugh and Kumar,

2001; Jung et al., 1996; 1994; Fang, 1994; Hadzic, 1989; Grabiec,

1988; Badard, 1984; Singh and Ram, 1982). Mishra et al. (1994)

proved common fixed point theorems on complete fuzzy met-

ric spaces, which generalized, extended and fuzzified several

known fixed point theorems for contractive type maps on met-

ric and other spaces. They assumed continuity of one map in

each of the two pairs of compatible maps and also the commu-

tativity of continuous maps. Cho (1997) and Jung et al. (1994)

extended and generalized several fixed point theorems on

metric spaces, Menger probabilistic metric spaces and uni-

form spaces, and proved common fixed point theorems on

complete fuzzy metric spaces. The results of Cho (1997) were

extended by Sharma and Deshpande (2002), and Sharma (2001).

Cho (1997), Hadzic (1989), Jungck et al. (1983), and Singh and

Kasahara (1983) have proved the common fixed point theorems

for mappings under the condition of continuity and compati-

bility of type (α) in complete fuzzy metric, probabilistic metric,

and metric spaces. In this paper, the assumption of continuity

was removed, relaxing compatibility or compatibility of type

(α) or compatibility of type (β) to weak compatibility, and the

completeness of space was replaced with a set of alternative

conditions. The assumption of commutativity of continuous

maps was also removed in the case of two pairs of maps. The

results of Sharma and Deshpande (2003) have been extended.

Definitions

Given below are definitions used in the paper.

Definition 1.1. A binary operation *: [0,1] × [0,1] × [0,1] →[0,1] is called a continuous t-norm, if ([0,1], *) is an abelian

topological monoid with unit 1 such that a1 * b

1 * c

1 ≤ a

2 * b

2

* c2, whenever a

1 ≤ a

2 and b

1 ≤ b

2 and c

1 ≤ c

2 for all a

1 a

2, b

1 b

2,

c1 c

2 are in [0, 1] (Sharma, 2002).

Definition 1.2. The 3-tuple (X, M,*) is called a fuzzy 2-metric

space if X is an arbitrary set, * is a continuous t-norm and M

is a fuzzy set in X3 x (0, ∞), satisfying the following conditions:

for all x, y, z, u ∈ X, and t1, t

2, t

3 > 0 (Sharma, 2002).

(FM-1) M(x, y, z, 0) = 0,

(FM-2) M(x, y, z, t) = 1, t > 0 and when at least two of the

three points are equal,

(FM-3) M(x, y, z, t) = M(x, z, y, t) = M(y, z, x, t) = ....

(symmetry about three variables),

(FM-4) M(x, y, z, t1 + t

2 + t

3) ≥ M(x, y, u, t

1)* M(x, u, z, t

2)*

M(u, y, z, t3)

(this corresponds to tetrahedron inequality in 2-met-

ric space ),

(FM-5) M(x, y, z,.) : [0, ∞) à [0, 1] is left continuous.

Example 1.1. Let (X, d) metric space define a *b = ab or a * b

= min a, b and for all x, y ∈ X and t > 0

Some Common Fixed Point Theorems in Fuzzy 2-Metric Spaces

Sushil Sharma*a and Jayesh Kumar Tiwarib

aDepartment of Mathematics, Madhav Science College, Vikram University, Ujjain-456 010,

Madhya Pradesh, IndiabDepartment of Mathematics, Shri Vaishnav Institute of Management, Indore-452 001, Madhya Pradesh, India

(received July 22, 2004; revised January 1, 2005; accepted January 6, 2005)

Pak. J. Sci. Ind. Res. 2005 48(4) 223-230

Abstract. The aim of this study was to prove some common fixed point theorems in fuzzy 2-metric spaces by removing

the assumption of continuity, relaxing the compatibility or compatibility of type (α) or compatibility of type (β) to weak

compatibility, and replacing the completeness of the space with a set of alternative conditions.

Keywords: fuzzy metric spaces, coincidence point, common fixed point, compatible maps, weakly compatible maps

*Author for correspondence; E-mail: [email protected]

M(x, y, t) = t

t + d(x, y)

223

then:

(X, M,*) is a fuzzy metric space

This is called fuzzy metric M induced by metric d, the stan-

dard fuzzy metric.

Definition 1.3. Let (X, M,*) be a fuzzy 2-metric space:

then:

• a sequence xn in fuzzy 2-metric space X is said to be

convergent to a point x ∈ X, if

limn → ∞ M(x

n, x, a, t) = 1

for all a ∈ X and t > 0

• a sequence xn in fuzzy 2-metric space X is called a Cauchy

sequence, if

limn → ∞ M(x

n+p, x

n, a, t) = 1

for all a ∈ X and t > 0, p > 0

• a fuzzy 2-metric space in which every Cauchy sequence is

convergent is said to be complete

Remark 1.1. Since * is continuous, it follows from (FM-4)

that the limit of the sequence in fuzzy 2-metric space is uniquely

determined (Sharma, 2002).

Let (X, M, *) be a fuzzy 2-metric space with the following

condition:

(FM – 6) limn → ∞ M(x, y, z, t) = 1

for all x, y, z ∈ X

Definition 1.4. A pair of mappings A and S is called a weakly

compatible pair if they commute at coincidence points (Jungck

and Rhoades, 1998).

Example 1.2. Define A, S : [0, 3] → [0, 3] by:

then:

for any x ∈ [1, 3], ASx = SAx, showing that A, S are weakly

compatible maps on [0, 3].

Example 1.3. Let X = [0, 2] with the metric d defined by:

d(x, y) = x – y , then for each t ∈ (0, ∞) define

M(x, y, t) = M(x, y, 0) = 0, x, y ∈ X

Clearly, M(X, M,*) is a fuzzy metric space on X, where * is

defined by a*b = ab or a*b = mina, b.

Define A, B: X → X by Ax = x, if x ∈ [0, 1/3), Ax = 1/3, if x

≥ 1/3 and Bx = x/(1 + x ), x ∈ [0, 2].

Consider the sequence xn = ½ + 1/n; n ≥ 1 in X

then:

limn → ∞ Ax

n = 1/3 and lim

n → ∞ Bx

n = 1/3

but

limn →

∞ M(ABxn , BAx

n , t) = ≠ 1

thus:

A and B are non-compatible, but A and B are commuting at

their coincidence point x = 0, that is, weakly compatible at

x = 0

also

limn → ∞ M(ABx

n , BBx

n , t) = ≠ 1

and

limn → ∞ M(BAx

n , AAx

n , t) = ≠ 1

further

limn → ∞ M(AAx

n , BBx

n , t) = ≠ 1

thus:

A and B are not compatible of type (β)

In view of this example, it is observed that:

• weakly compatible maps need not be compatible

• weakly compatible maps need not be compatible of

type (α)

• weakly compatible maps need not be compatible of

type (β)

Lemma 1.1. For all x, y, z ∈ X, M(x, y, z, ·) is non-decreasing

(Sharma, 2002).

Lemma 1.2. Let yn be a sequence in a fuzzy 2-metric space

(X, M, *) with the condition (FM-6), if there exists a number

k ∈ (0, 1), such that:

M(yn+2

, yn+1

, a, kt) ≥ M(yn+1

, yn, a, t)

for all t > 0 and a ∈ X and n = 1, 2,…, then yn is a Cauchy

sequence in X (Sharma, 2002).

Lemma 1.3. If for all x, y, a ∈ X, t > 0 and for a number k ∈(0, 1)

M(x, y, a, kt) ≥ M(x, y, a, t)

then, x = y (Sharma, 2002).

Results and Discussion

Sharma and Deshpande (2003) proved the following:

Theorem A. Let (X, M, *) be a fuzzy metric space with t * t ≥ t

for all t ∈ [0, 1] and the condition (FM- 6). Let A, B, S, T be

mappings from X into itself, such that:

x, if x ∈ [0, 1] 3 - x , if x ∈ [0, 1]A(x) = and S(x) =

3, if x ∈ [1, 3] 3, if x ∈ [1, 3]

t

t + 1/3 - 1/4

t

t + 1/3 - 1/4

t

t + 1/4 - 1/3

t

t + 1/3 - 1/4

t ,t + d(x, y)

S. Sharma and J. K. Tiwari224

(1) A(X) ⊂ T(X), B(X) ⊂ S(X)

(2) there exists a constant k ∈ (0, 1), such that:

M(Ax, By, kt) ≥ M(Ty, By, t) * M(Sx, Ax, t) * M(Sx, By, αt)

* M[Ty, Ax, (2 – α)t] * M(Ty, Sx, t)

for all x, y ∈ X, α ∈ (0, 2) and t > 0

(3) one of A(X), B(X), S(X), or T(X) is a complete subspace of

X,

then:

(i) A and S have a coincidence point

(ii) B and T have a coincidence point

further, if

(4) the pairs A, S and B, T are weakly compatible

then:

(iii) A, B, S and T have a unique fixed point in X

Theorem A for fuzzy 2-metric space, has been proved. The

following are proved as:

Theorem 2.1. Let (X, M, *) be a fuzzy 2-metric space with

t * t ≥ t for all t ∈ [0, 1] and the condition (FM-6). Let A, B, S

and T be mappings from X into itself, such that:

(2.1) A(X) ⊂ T(X) , B(X) ⊂ S(X)

(2.2) there exists a constant k ∈ (0, 1)

such that:

M(Ax, By, a, kt) ≥ M(Ty, By, a, t) * M(Sx, Ax, a, t) * M(Sx, By, a,

αt)

* M[Ty, Ax, a, (2 – α)t] * M(Ty, Sx, a, t)

for all x, y, a ∈ X , α ∈ (0, 2) and t > 0

(2.3) one of A(X), B(X), S(X) or T(X) is a complete subspace

of X

then:

(i) A and S have a coincident point

(ii) B and T have a coincident point

further, if

(2.4) the pairs A, S and B, T are weakly compatible

then:

(iii) A, B, S and T have a unique fixed point in X

Proof. By (2.1), since A(X) ⊂ T(X), so for any arbitrary x0 ∈

X, there exists a point x1 ∈ X, such that Ax

0 = Tx

1. Since B(X)

⊂ S(X), for this point x1, a point x

2 ∈ X can be chosen, such

that Bx1 = Sx

2, and so on. Inductively, a sequence y

n in X

can be defined as:

y2n

= Tx2n+1

= Ax2n

, and

y2n+1

= Sx2n+2

= Bx2n+1

for n = 0, 1, 2, …

By (2.2) for all t > 0 and α = 1 - q, with q ∈ (0,1), it would be:

M(Ax2n+2

, Bx2n+1

, a, kt) ≥ M(Tx2n+1

, Bx2n+1

, a, t)* M(Sx2n+2

, Ax2n+2

,

a, t) * M(Sx2n+2

, Bx2n+1

, a, α t) * M[Tx2n+1

, Ax2n+2

, a, (2 – α) t] *

M(Tx2n+1

, Sx2n+2

, a, t)

M(y2n+2

, y2n+1

, a, kt) ≥ M(y2n

, y2n+1

, a, t) * M(y2n+1

, y2n+2

, a, t) *

M(y2n+1

, y2n+1

, a, α t) * M[y2n

, y2n+2

, a, (2 – α) t] * M(y2n

, y2n+1

,

a, t) ≥ M(y2n

, y2n+1

, a, t) * M(y2n+1

, y2n+2

, a, t) * 1 * M[y2n

, y2n+2

,

a, (1 + q) t] * M(y2n

, y2n+1

, a, t)

On the lines of Sharma (2002) it is:

≥ M(y2n

, y2n+1

, a, t) * M(y2n+1

, y2n+2

, a, t) * M(y2n

, y2n+2

a, tq +

t/2 + t/2)

≥ M(y2n

, y2n+1

, a, t) * M(y2n+1

, y2n+2

, a, t) * M(y2n

, y2n+2

, y2n+1

, qt)

* M(y2n

, y2n+1

, a, t/2) * M(y2n+1

, y2n+2

, a, t/2)

≥ M(y2n

, y2n+1

, a, t) * M(y2n+1

, y2n+2

, a, t) * M(y0, y

2, y

1, t/3q2n ) * M(y

0, y

1,

y1, t/3q2n) * M(y

1, y

2, y

1, t/3q2n) * M(y

2n, y

2n+1, a, t/2) * M(y

2n+1,

y2n+2

, a, t/2)

thus:

since M(y0, y

2, y

1, t/3q2n) → 1 as n → ∞

it is:

(2.5) M(y2n+1

, y2n+2

, a, kt) ≥ M(y2n

, y2n+1

, a, t)* M(y2n+1

, y2n+2

, a, t)

similarly, it is:

(2.6) M(y2n+2

, y2n+3

, a, kt) ≥ M(y2n+1

, y2n+2

, a, t) * M(y2n+2

, y2n+3

, a, t)

from (2.5) and (2.6), it follows that:

M(yn+1

, yn+2

, a, kt) ≥ M(yn, y

n+1, a, t) * M(y

n+1, y

n+2, a, t)

for n = 1, 2,…, and also for positive integer n, p

M(yn+1

, yn+2

, a, kt) ≥ M(yn, y

n+1, a, t) * M(y

n+1, y

n+2, a, t/kP)

thus:

since M(yn+1

, yn+2

, a, t/kP) → 1 as p → ∞

it is:

M(yn+1

, yn+2

, a, kt) ≥ M(yn, y

n+1, a, t)

so, by Lemma 1.2, yn is a Cauchy sequence in X.

Now, suppose S(X) is complete, note that S(X) contained the

sub-sequence y2n+1

in S(X) and has a limit in S(X), it is

called z.

Let u = S-1z, thus, Su = z. The fact will be used that the sub-

sequence y2n

also converges to z

by (2.2) with α = 1, it is:

Theorems in Fuzzy 2-Metric Spaces 225

M(Au, y2n+1

, a, kt) = M(Au, Bx2n+1

, a, kt)

≥ M(Tx2n+1

, Bx2n+1

, a, t) *M(Su, Au, a, t) *M(Su, Bx2n+1

, a, t) *

M(Tx2n+1

, Au, a, t) * M(Tx2n+1

, Su, a, t)

= M(y2n

, y2n+1

, a, t) * M(Su, Au, a, t) * M(Su, y2n+1

, a, t) * M(y2n

, Au,

a, t) * M(y2n

, Su, a, t)

which implies that as n → ∞

M(Au, z, a, kt) ≥ 1 * M(z, Au, a, t) * 1 * M(z, Au, a, t) * 1

≥ M(Au, z, a, t)

Therefore, by Lemma 1.3, Au = z

thus:

Au = z = Su, i.e., u is a coincidence point of A and S. This

proves (i).

Since A(X) ⊂ T(X), Au = z implies that z ∈ T(X). Let v =

T-1z , then Tv = z . It can be easily verified by using similar

arguments of the previous part of the proof that Bv = z

thus:

Bv = z = Tv, i.e., v is a coincidence point of B and T. This

proves (ii).

If it is assumed that T(X) is complete, then argument analo-

gous to the previous completeness argument establishes (i)

and (ii). The remaining two cases pertain essentially to the

previous cases. Indeed, if B(X) is complete, then by (2.1), z ∈B(X) ⊂ S(X). Similarly, if A(X) is complete, then z ∈ A(X) ⊂T(X). Thus (i) and (ii) are completely established.

Since the pair A, S is weakly compatible, therefore, A and S

commute at their coincidence point, i.e., ASu = SAu, or Az = Sz

similarly,

BTv = TBv or Bz = Tz.

Now, to prove Az = z by (2.2), with α = 1

M(Az, y2n+1

, a, kt) = M(Az, Bx2n+1

, a, kt)

≥ M(Tx2n+1

, Bx2n+1

, a, t) * M(Sz, Az, a, t) * M(Sz, Bx2n+1

, a, t)

* M(Tx2n+1

, Az, a, t) * M(Tx2n+1

, Sz, a, t)

= M(y2n

, y2n+1

, a, t) * M(Az, Az, a, t) * M(Az, y2n+1

, a, t) *

M(y2n

, Az, a, t) * M(y2n

, Az, a, t)

Taking limit as n → ∞

M(Az, z, a, kt) ≥ 1 * 1 *M(Az, z, a, t) * M(z, Az, a, t) * M(z,

Az, a, t)

≥ M(Az, z, a, t)

therefore:

by Lemma 1.3, Az = z

thus:

Az = z = Sz

similarly,

Bz = z = Tz.

This means that z is a common fixed point of A, B, S and T.

For uniqueness of common fixed point let w ≠ z be another com-

mon fixed point of A, B, S and T. Then by (2.2) with α = 1

M(z, w, a, kt) = M(Az, Bw, a, kt)

≥ M(Tw, Bw, a, t) * M(Sz, Az, a, t) * M(Sz, Bw, a, t) * M(Tw,

Az, a, t) * M(Tw, Sz, a, t)

≥ 1 * 1 * M(z, w, a, t) * M(w, z, a, t) * M(w, z, a, t)

≥ M(z, w, a, t)

therefore:

by Lemma 1.3, z = w. This completes the proof.

Theorem 2.2. Let (X, M, *) be a fuzzy 2-metric space with

t* t ≥ t for all t ∈ [0, 1] and the condition (FM-6). Let A, B,

S, T and P be mappings from X into itself, such that:

(2.7) P(X) ⊂ AB(X) , P(X) ⊂ ST(X)

(2.8) there exists a constant k ∈ (0, 1), such that:

M(Px, Py, a, kt) ≥ M(ABy, Py, a, t) * M(STx, Px, a, t) * M(STx,

Py, a,α t) * M[ABy, Px, a, (2 – α) t] * M(ABy, STx, a, t)

for all x, y, a ∈ X , α ∈ (0, 2) and t > 0

(2.9) If one of P(X) , AB(X) or ST(X), is a complete sub-space

of X

then:

(i) P and AB have a coincident point

(ii) P and ST have a coincident point

further, if

(2.10) PB = BP ; AB = BA ; PT = TP and ST = TS

(2.11) the pairs P, AB and P, ST are weakly compatible

then:

(iii) A, B, S, T and P have a unique common fixed point in X

Proof. By (2.7), since P(X) ⊂ AB(X) , for any point x0 ∈ X,

there exists a point x1 ∈ X, such that, Px

0 = ABx

1. Since P(X) ⊂

ST(X), for this point x1, a point x

2 ∈ X can be chosen, such

that, Px1 = Sx

2, and so on. Inductively, a sequence y

n in X

can be defined as:

y2n

= Px2n

= ABx2n+1

and y2n+1

= Px2n+1

= STx2n+2

where:

n = 0, 1, 2, …

by (2.8), for all t > 0 and α = 1 - q with q ∈ (0,1), it would be:


M(Px2n+2

, Px2n+1

, a, kt) ≥ M(ABx2n+1

, Px2n+1

, a, t) * M(STx2n+2

,

Px2n+2

, a, t) * M( STx2n+2

, Px2n+1

, a, α t) * M[ABx2n+1

, Px2n+2

, a,

(2 – α)t]* M(ABx2n+1

, STx2n+2

, a, t)

M(y2n+2

,y2n+1

, a, kt) ≥ M(y2n

, y2n+1

, a, t) * M(y2n+1

, y2n+2

, a, t)

* M(y2n+1

, y2n+1

, a, α t) * M[y2n

, y2n+2

, a, (1 + q) t] * M(y2n

, y2n+1

,

a, t)

≥ M (y2n

,y2n+1

, a,t) *M(y2n+1

, y2n+2

, a, t) * 1 * M [y2n, y2n+2

, a,

(1 + q) t] *M (y2n

, y2n+1

, a, t)

≥ M(y2n

, y2n+1

, a, t) *M(y2n+1

, y2n+2

, a, t) * M[y2n

, y2n+2

, a, (1+q) t]

On the lines of Sharma (2002), it is:

≥ M (y2n

, y2n+1

, a, t) * M (y2n+1

, y2n+2

, a, t) * M(y2n

, y2n+2

, a, tq +

t/2 + t/2)

≥ M(y2n

, y2n+1

, a, t) * M(y2n+1

, y2n+2

, a, t) * M(y2n

, y2n+2

, y2n+1

, qt)

* M(y2n

, y2n+1

, a, t/2) * M(y2n+1

, y2n+2

, a, t/2)

≥ M(y2n

, y2n+1

, a, t) * M(y2n+1

, y2n+2

, a, t) * M(y0, y

2, y

1, t/3q2n )

* M(y0, y

1, y

1, t/3q2n) * M(y

1, y

2, y

1, t/3q2n)* M(y

2n, y

2n+1, a, t/2)

* M(y2n+1

, y2n+2

, a, t/2)

thus:

since M(y0, y

2, y

1, t/3q2n) → 1 as n → ∞

it is:

(2.12) M(y2n+1

, y2n+2

, a, kt) ≥ M(y2n

, y2n+1

, a, t) * M(y2n+1

, y2n+2

, a, t)

similarly,

(2.13) M(y2n+2

, y2n+3

, a, kt) ≥ M(y2n+1

, y2n+2

, a, t) * M(y2n+2

, y2n+3

, a, t)

from (2.12) and (2.13) if follows that:

M(yn+1

, yn+2

, a, kt) ≥ M(yn, y

n+1, a, t) * M(y

n+1, y

n+2, a, t)

for n = 1, 2,…, and also for positive integer n, p

M(yn+1

, yn+2

, a, kt) ≥ M(yn, y

n+1, a, t) * M(y

n+1, y

n+2, a, t/kP)

thus:

since M(yn+1

, yn+2

, a, t/kP) → 1 as p → ∞

M(yn+1

, yn+2

, a, kt) ≥ M(yn, y

n+1, a, t)

therefore:

by Lemma 1.2, yn is a Cauchy sequence in X.

Now, suppose ST(X) is complete

note that the subsequence y2n+1

is contained in ST(X) and

has a limit in ST(X) called z

Let u = (ST)-1z, then STu = z.

Applying the fact that the sub-sequence y2n

also converges

to z

by (2.8) with α = 1, it would be:

M(Pu, y2n+1

, a, kt) = M(Pu, Px2n+1

, a, kt)

≥ M(ABx2n+1

, Px2n+1

, a, t) * M(STu, Pu, a, t) * M(STu, Px2n+1

,

a, t) * M(ABx2n+1

, Pu, a, t)* M(ABx2n+1

, STu, a, t)

= M(y2n

, y2n+1

, a, t) * M(z, Pu, a, t) * M(z, y2n+1

, a, t) * M(y2n

, Pu,

a, t) * M(y2n

, z, a, t)


M(Pu, z, a, kt) ≥ 1 * M(z, Pu, a, t) * 1 *M(z, Pu, a, t) * 1

≥ M(Pu, z, a, t)

therefore:

by Lemma 1.3, Pu = z. Since STu = z, Pu = z = STu, i.e., u is

coincidence point of P and ST. This proves (i).

Since P(X) ⊂ AB(X), Pu = z implies that z ∈ AB(X).

Let v = (AB)-1z, then ABv = z. It can easily be verified by using

similar argument of the previous part of the proof that Pv = z.

If it is assumed that AB(X) is complete, then argument analo-

gous to the previous completeness argument establishes (i)

and (ii).

The remaining one case pertains, essentially, to the previous

cases indeed. If P(X) is complete, then by (2.7), z ∈ P(X) ⊂ST(X), or z ∈ P(X) ⊂ AB(X). Thus (i) and (ii) are completely

established. Since, the pair P, ST is weakly compatible, there-

fore, P and ST commute at their coincident point, i.e., P(STu) =

(ST)Pu, or Pz = STz. Similarly, P(ABv) = (AB)Pv or Pz = ABz.

Now, to prove that Pz = z, by (2.8) with α = 1, it would be:

M(Pz, y2n+1

, a, kt) = M(Pz, Px2n+1

, a, kt)

≥ M(ABx2n+1

, Px2n+1

, a, t) * M(STz, Pz, a, t) * M(STz, Px2n+1

, a,

t) * M(ABx2n+1

, Pz, a, t) * M(ABx2n+1

, STz, a, t)

= M(y2n

, y2n+1

, a, t) * M(Pz, Pz, a, t) * M(Pz, y2n+1

, a, t) *M(y2n

,

Pz, a, t) * M(y2n

, Pz, a, t)

Taking the limit n → ∞, it is:

M(Pz, z, a, kt) ≥ 1 * 1 * M(Pz, z, a, t) * M(z, Pz, a, t) * M(z, Pz, a, t)

≥ M(Pz, z, a, t)

therefore:

by Lemma 1.3, Pz = z, thus ABz = z = Pz = STz.

Now, it will be shown that Bz = z. In fact by (2.8) with α = 1 and

(2.10) it is:

M(z, Bz, a, kt), = M(Pz, BPz, a, kt) = M(Pz, PBz, a, kt)

≥ M[AB(Bz), PBz, a, t] * M[STz, Pz, a, t] * M[STz, PBz, a, t]

* M[AB(Bz), Pz, a, t] * M[AB(Bz), STz, a, t]

= 1 * 1 * M(z, Bz, a, t) * M(Bz, z, a, t) * M(Bz, z, a, t)

≥ M(z, Bz, a, t)

which implies, by Lemma 1.3, that Bz = z. Since ABz = z,

therefore, Az = z. Finally, Tz = z. Indeed, by (2.8) with α = 1 and

(2.10)

M(Tz, z, a, kt) = M(TPz, Pz, a, kt) = M(PTz, Pz, a, kt)


≥ M[ABz, Pz, a, t] * M[ST(Tz), PTz, a,t] * M[ST(Tz), Pz, a,t]

* M[ABz, P(Tz), a, t] * M[ABz, ST(Tz), a, t]

= 1 * 1 * M(Tz, z, a, t) * M(z, Tz, a, t) * M(z, Tz, a, t)

≥ M(Tz, z, a, t)

which implies, by Lemma 1.3, that Tz = z. Since STz = z, z =

STz = Sz. Therefore, by combining the above results, Az = Bz

= Sz = Tz = Pz = z, i. e., z is a common fixed point of A, B, S, T

and P.

For uniqueness of common fixed point, let w ≠ z be another

common fixed point of A, B, S, T and P.

then:


M(z, w, a, kt) ≥ M(ABw, Pw, a, t) * M(STz, Pz, a, t) * M(STz,

Pw, a, α t) * M(ABw, Pz, a, t) * M(ABw, STz, a, t)

≥ 1 * 1 * M(z, w, a, t) * M(w, z, a, t) * M(w, z, a, t)

≥ M(z, w, a, t)

therefore:

by Lemma 1.3, we have z = w. This completes the proof.

Theorem 2.3. Let (X, M, *) be a fuzzy-2 metric space with t*

t ≥ t for all t ∈ [0, 1] and the condition (FM-6). Let A, B, S, T,

P and Q be mappings from X into itself, such that:

(2.14) P(X) ⊂ AB(X) , Q(X) ⊂ ST(X)


M(Px, Qy, a, kt) ≥ M(ABy, Qy, a, t) * M(STx, Px, a, t) * M(STx,

Qy, a, α t) * M[ABy, Px, a, (2 – α)t] * M(ABy, STx, a, t)

for all x, y, a ∈ X, α ∈ (0, 2) and t > 0

(2.16) If one P(X), Q(X), AB(X) or ST(X) is complete sub-

space of X

then:

(i) P and ST have a coincident point

(ii) Q and AB have a coincident point

further, if

(2.17) AB = BA, QB = BQ , PT = TP and ST = TS

(2.18) the pairs Q, AB and P, ST are weakly compatible

then:

A, B, S, T, P and Q have a unique common fixed point in X.

Proof. By (2.14), since P(X) ⊂ AB(X), for any point x0 ∈ X,

there exists a point x1 ∈ X, such that Px

0 = ABx

1. Since Q(X) ⊂

ST(X), for this point x1, a point x

2 ∈ x can be chosen, such that:

Qx1 = STx

2, and so on

inductively, a sequence yn in X can be defined as:

y2n

= Px2n

= ABx2n+1

, and

y2n+1

= Qx2n+1

= STx2n+2

where:

n = 0,1, 2,…

For all t > 0 and α = 1 – q, with q ∈ (0,1). As proved in Theo-

rems (2.1) and 2.2, it can be proved that yn is a Cauchy

sequence in X. Now, suppose ST(X) is complete; note that

ST(X) contains the sub-sequence y2n+1

and has a limit in

ST(X), called z. Let u = (ST)-1z, then STu = z. Applying the fact

that the sub-sequence (y2n

also converges to z


M(Pu, Qx2n+1

, a, kt) ≥ M(ABx2n+1

, Qx2n+1

, a, t) * M(STu, Pu, a, t) *

M(STu, Qx2n+1

, a, t) * M(ABx2n+1

, Pu, a, t) * M(ABx2n+1

, STu, a, t)

= M(y2n

, y2n+1

, a, t) * M(STu, Pu, a, t) * M(STu, y2n+1

, a, t) * M(y2n

, Pu,

a, t) * M(y2n

, STu, a, t)


M(Pu, z, a, kt) ≥ M(Pu, z, a, t)

therefore:

by Lemma 1.3, Pu = z. Since STu = z, thus Pu = z = STu, i.e., u

is a coincidence point of P and ST. This proves (i). Since P(X)

⊂ AB(X) and Pu = z implies that z ∈ AB(X).

Let v = (AB)-1 z, then ABv = z


M(Pz, y2n+1

, a, kt) = M(Pz, Qx2n+1

, a, kt)

≥ M(ABx2n+1

, Qx2n+1

, a, t) * M(STz, Pz, a, t) * M(STz, Qx2n+1

,

a, t) * M(ABx2n+1

, Pz, a, t) * M(ABx2n+1

, STz, a, t)

≥ M(y2n

, y2n+1

, a, t) * M(STz, Pz, a, t) * M(STz, y2n+1

, a, t)

* M(y2n

, Pz, a, t) * M(y2n

, STz, a, t)

Taking the limit as n → ∞, it is:

M(Pz, z, a, kt) ≥ M(Pz, z, a, t)

therefore:

by Lemma 1.3, we have Pz = z = STz.

Now, it shows that Qz = z. In fact by (2.15) with α = 1 and

(2.17), it would be:

M(y2n

, Qz , a, kt) = M(Px2n

, Qz, a, kt)

≥ M(ABz, Qz, , a, t) * M(STx2n

, Px2n

, a, t) * M(STx2n

, Qz, a, t) *

M(ABz, Px2n

, a, t) * M(ABz, STx2n

, a, t)

Taking the limit n → ∞

M(z, Qz, a, t) ≥ M(Qz, z, a, t)


therefore:

by Lemma 1.3, Qz = z = ABz. Thus, Pz = Qz = ABz = STz = z.

By putting x = z and y = Bz, with α = 1 in (2.15), using (2.17)

and Lemma 1.3, it is easy to see that Bz = z. Since ABz = z,

therefore, Az = z

similarly,

by putting x = Tz and y = z, with α = 1 in (2.15), using (2.17)

and Lemma 1.3, it is easy to prove that Tz = z. Since STz = z,

Sz = z. Therefore, by combining the above results, it would be:

Az = Bz = Sz = Tz = Qz = z

which mean that z is the common fixed point of A, B, S, T, P

and Q. Thus, it is easy to prove uniqueness.

Theorem 2.4. Let (X, M, *) be a fuzzy metric 2-space with t *

t ≥ t for all t ∈ [0, 1] and the condition (FM-6). Let A, B, S, T

and Pi

i ∈ I be mappings from x into itself:

(2.19) ∪i ∈ I

PI(X) ⊂ AB(X), ∪

i ∈ I P

i(X) ⊂ ST(X), where I is an

index set


M(Pix, P

iy, a, kt) ≥ M(ABy,P

iy, a, t) * M(STx, P

ix, a, t) * M(STx,

Piy, a,α t) * M[ABy, P

ix, a, (2 – α) t] * M(ABy, STx, a, t)

for all x, y, a ∈ X, α ∈ (0, 2), i ∈ Ι and t > 0

(2.21) If one of AB(X), or ST(X), or Pi(X) (i ∈ Ι) is a complete

subspace of X, then:

(i) for all i ∈ Ι, Pi and AB have a coincidence point

(ii) for all i ∈ Ι, Pi and ST have a coincidence point

further, if

(2.22) for all i ∈ Ι, PiB = BP

i AB = BA ; P

iT = TP

i and ST = TS

(2.23) for all i ∈ Ι, the pairs Pi, AB and P

i, ST are weakly

compatible, then

(iii) A, B, S, T and Pi

i ∈ I have a unique common fixed point in X

if we put B = T = Ix (the identity map on X) in Theorem 2.2, the

following result:

Corollary 2.1. Let (X, M, *) be a fuzzy 2-metric space with t *

t ≥ t for all t ∈ [0, 1] and the condition (FM-6). Let A, S and P

be mappings from X into itself, such that:

(2.24) P(X) ⊂ A(X), P(X) ⊂ S(X)


M(Px, Py, a, kt) ≥ M(Ay, Py, a, t) * M(Sx, Py, a, α t) * M[Ay, Py,

a, (2 - α) t]* M(Ay, Sx, a, t)


(2.26) If one of P(X), or S(X) is a complete sub-space of X,

then:

(i) P and A have a coincident point

(ii) P and S have a coincident point

further, if

(2.27) The pair P, A and P, S are weakly compatible, then:

(iii) A, S and P have a unique common fixed point in X

if, A = B = S = T = Ix (the identity mapping on X) in Theorem

2.2, the following results are acquired:

Corollary 2.2. Let (X, M, *) be a fuzzy metric 2-space with t *

t ≥ t for all t ∈ [0, 1] and the condition (FM-6). Let P be

mappings from X itself, such that:


M(Px, Py, a, kt) ≥ M(y, Py, a, t) * M(x, Px, a, t) * M(x, Py, a, αt)

* M[y, Px, a, (2 – α) t] * M(y, x, a, t)


if P(X) is a complete sub-space of X, then P has a unique

common fixed point in X

by using Theorem 2.1, the following results are acquired:

Theorem 2.5. Let (X, M, *) be a fuzzy 2-metric space with t *

t ≥ t for all t ∈ [0, 1] and the condition (FM-6). Let A, B and T

be mappings from X into itself, such that:

(2.29) A(X) ∪ B(X) ⊂ T(X)


M(Ax, By, a, kt) ≥ M(Ty, By, a, t) * M(Tx, Ax, a, t) *

M(Tx, By, a, α t) * M[Ty, Ax, a, (2 – α) t] * M(Ty, Tx, a, t)

for all x, y, a ∈ x , α ∈ (0, 2) and t > 0

(2.31) One of A(X), B(X) or T(X) is a complete sub-space of X

then:

A, B, T have a coincidence point

thus:

Theorem 2.1, for sequence of mappings, is established in the

following manner:

Theorem 2.6. let (X, M, *) be a fuzzy metric 2-space with t * t

≥ t for all t ∈ [0, 1] and the condition (FM- 6). Let S, T, Ai : X →

X, i = 0, 1, 2, …, such that:

(2.32) A0(X) ⊂ T(X) , A

i(X) ⊂ S(X), i ∈ N


M(A0x, A

iy, a, kt) ≥ M(Ty, A

iy, a, t) * M(Sx, A

0x, a, t) * M(Sx,

Aiy, a, α t) * M[Ty, A

0x, a, (2-α) t] * M(Ty, Sx, a, t)



(2.34) the pairs A0, S and A

i, T (i ∈ N) are weakly

compatible

(2.35) if one of S(X), T(X) or A0(X) is a complete sub-space

of X, or alternatively Ai, i ∈ N are complete sub-space of X

then:

S, T and Ai, i = 0, 1, 2, … have a unique common fixed point.

Acknowledgement

Authors extend thanks to Professor P. Veeramani for guidance.

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230 S. Sharma and J. K. Tiwari

The Fe-Gd Phase DiagramS. Atiq*a, R. D. Rawlingsb and D. R. F. Westb

aPCSIR Laboratories, PO Box 387, Quetta, PakistanbDepartment of Materials, Imperial College, London, UK

(received June 26, 2002; revised May 5, 2005; accepted May 12, 2005)

Pak. J. Sci. Ind. Res. 2005 48(4) 231-235

Abstract. The results of an experimental investigation of the iron-rich portion of the Fe-Gd system have confirmedprevious studies showing that four incongruently melting intermetallic compounds exist, namely, Fe17Gd2, Fe23Gd6, Fe3Gdand Fe2Gd. The investigation also provided information about the crystal structures and ranges of stoichiometry of theseintermetallic compounds.

Keywords: Fe-Gd phase diagram, Fe-Gd alloys, intermetallic compounds


IntroductionTransition metal-rare earth (TM-RE) alloy systems, both bi-nary and higher orders, are of considerable interest in relationto the development of materials with attractive magnetic pro-perties, such as large magnetostriction and high energy pro-ducts. A characteristic feature of these TM-RE systems is theoccurrence of a series of intermetallic compounds. The workreported here was part of a wider programme of research oncertain binary and ternary transition metal-rare earth alloysystems (rare earths (RE) = Gd, Tb; transition metals (TM) =Fe, Co). The results on binary Fe-Gd alloys are specially re-ported here, which aim at resolving certain differences inpreviously reported studies.

Early work by Novy et al. (1961) reported seven intermetalliccompounds with the Fe : Gd ratios of 17 : 2, 5 : 1, 4 : 1, 7 : 2, 3 : 1,2 : 1, and 3 : 2. Another study (Savitskii et al., 1961) showed thepresence of only four compounds, namely, 17 : 2, 23 : 6, 3 : 1,and 2 : 1. Other papers have been published on various as-pects of this system including constitutional data, whichhave been the subject of several recent assessments (Zhangand Han, 1998; Okamoto, 1996; Savitskii et al., 1970). A reas-sessment has been also presented very recently (Zinkevichet al., 2000). This report incorporates a comprehensive list ofreferences and also presents a thermodynamically calculatedversion of the phase diagram, which confirms the general fea-tures of the system and the four compounds reported bySavitskii et al. (1961).

The experimental results reported here are in general agree-ment with the assessment of Zinkevich et al. (2000). Thestoichiometery ranges of the intermetallic compounds arealso reported.

Materials and MethodsAlloys of nominal compositions, as listed in Table 1, wereprepared as 5-10 g buttons by arc melting 99.99% purity ironand gadolinium under titanium gettered argon. Melting wascarried out several times to ensure homogeneity. Samples ofeach alloy were subjected to homogenization heat treatmentat 1050 °C for 2 weeks, followed by quenching in iced brine.Samples were sealed in evacuated silica tubes with partialpressure of argon prior to heat treatment.

Metallographic examination of both the as-solidified and ho-mogenized samples, was carried out by optical and scanningelectron microscopy. The composition of the phases appea-ring in various alloy samples was determined by energy dis-persive X-ray (EDX) analysis with ZAF corrections. The com-positions reported in Table 2 are the average of 5 measure-ments, the scatter was < ~0.5% and the error was ~±1% of thevalues reported. The X-ray diffractometry was carried out onpowdered alloy samples employing Cu-Ká radiation so as todetermine crystal structure and lattice parameter(s) of thecompounds by applying the Nelson-Riley function.

231

Table 1. Nominal compositions of the Fe-Gd alloys investigated

Alloy designation Composition (atomic, %)Fe Gd

GF-5 95.0 5.0GF-11 88.5 11.5GF-13 86.5 13.5GF-16 83.4 16.6GF-22 78.0 22.0GF-25 74.5 25.5GF-33 66.5 33.5

Phase transformation temperatures were determined by diffe-rential thermal analysis (DTA) using homogenized samples,weighing 50 mg. An alumina crucible was used with platinumas the reference material and titanium gettered argon as theinert atmosphere. Data were recorded for heating cycle (withrates of 10 °C/min) and the extrapolated onset temperatureswere measured to represent the phase transformation tem-peratures. The peritectoid temperatures were estimated tohave an accuracy of ~±2 °C, while for other parts of the soli-dus the range was ~±5 °C.

A diffusion couple was prepared by clamping a 4 mm thicksample of gadolinium between two strips of iron (Rawlingsand Neway, 1968). The couple was wrapped in tantalum foiland encapsulated in an evacuated silica tube with partial pre-ssure of argon. The couple was annealed for 720 h at 1050 °Cand subsequently quenched in iced brine. This was followedby EDX analysis of the phases appearing in the section ofthe couple.

Results and DiscussionPhase diagram. On the basis of the evidence, as discussedbelow and taking account of the results of all the experimentaltechniques used, the binary phase diagram is shown in Fig. 1.The phase diagram incorporates four intermetallic compou-nds corresponding, respectively, to Fe : Gd atomic ratios of17 : 2, 23 : 6, 3 : 1, and 2 : 1. The basic features of the diagramagree closely with those reported in the recent assessmentby Zinkevich et al. (2000). Some details of the experimentalresults, on which the diagram was based, are presented anddiscussed below.

Alloy phases and microstructure. The EDX data for the fourintermetallic compounds found in the homogenized andas-solidified alloys are shown in Table 2. Some aspects of thecompositional and microstructural features of the individualalloys investigated, in relation to the phase diagram (Fig. 1),are of interest to note. Previously reported versions of thephase diagram have shown the intermetallic as “line com-pounds” with no range of compositions. However, the EDXdata reported here (Table 2) indicate the possible existence ofsmall ranges of stoichiometry (see dashed lines in Fig. 1) forthe 17 : 2 and 2 : 1 compounds (up to ~0.5 atomic %) and for3 : 1 compound (up to ~1 atomic %) but the evidence for the23 : 6 compound is lacking. Also, there was a general trendfor the Fe-Gd atomic ratios (Table 2) to extend slightly in ex-cess (i.e., Gd-rich) of the ideal stoichiometries. However, thisfeature was not seen in every case, particularly for the non-equilibrium structures of the as-solidified alloys. The exis-tence of deviation from ideal stoichiometry has been discus-sed earlier (Atiq et al., 1997) in the context of the results of

X-ray and density measurements of Fe-Gd and Fe, Co-Gdalloys and has been interpreted in terms of vacant lattice siteson the trace metal (TM) lattice.

Investigation of alloys GF-13 and GF-16 confirmed the ab-sence of the compound Fe5Gd, reported by Novy et al. (1961)and supported the results of Savitskii (1970), which have beenincorporated in a later assessment (Novy et al., 1971). How-ever, bearing in mind the report that in some rare earth sys-tems a compound Co5 RE exists, but only at temperaturesabove 1150 °C, alloy GF-16 was heated at 1250 °C for four daysand quenched to retain any high temperature phase thatmight be present. The microstructure showed no difference,apart from coarsening, from the alloy homogenized at 1050 °C,indicating the absence of a different phase existing at hightemperatures.

The homogenized GF-22 alloy provided information on theequilibria between 23 : 6 and 3 : 1 compounds, while the homo-genized alloys GF-25 and GF-33 were very close to the singlephase 3 : 1 and 2 : 1 structures, respectively. Some features ofthe as-solidified structures are of interest to note for the alloys

Fig. 1. The Fe-Gd phase diagram (intermetallic com-pounds have been shown as the ratios of Fe :Gd); Rhom. to Hex. = rhombohedral to hexagonaltype.

232 S. Atiq et al.

1600

1400

1200

1000

800

600

13891325

1215

1282

1171

1057

930

~1 at. % Gd

Fe 10 20 30 40 50 60 70 80 90 GdGd (atomic, %)

Rhom. to Hex. 17:2

Thermal arrests

Fig. 2. Microstructures of alloy GF-22 (Sem/Bei): (a) as-solidified, showing Fe17Gd2 (dark) formed as primary phase, Fe23Gd6 (grey)and Fe3Gd (bright); (b) homogenized, showing Fe17Gd2 (dark) formed as primary phase, Fe23Gd6 (grey) and Fe3Gd (bright).

Fig. 3. Microstructures of alloy GF-25 (Sem/Bei): (a) as-solidified, showing Fe17Gd2 (dark) formed as primary phase, Fe3Gd (grey)and Fe2Gd (bright); (b) homogenized, showing a single phase microstructure comprising of Fe3Gd (bright).

Fig. 4. Microstructures of alloy GF-33 (Sem/Bei): (a) as-solidified, showing Fe17Gd2 (dark) formed as primary phase, Fe3Gd (grey)and Fe2Gd (bright); (b) homogenized, showing a nearly single phase microstructure, Fe2Gd matrix with some Fe3Gd (dark).

GF-22 (Fig. 2a), GF-25 (Fig. 3a) and GF-33 (Fig. 4a), in which theabsence of equilibrium is shown by the presence of threephases associated with the incomplete progress of the rele-vant peritectic reactions. In these three alloys, the as-solidi-fied material contained some 17 : 2 compounds. The homoge-nization treatment did not remove the 17 : 2 compound in alloyGF-22 (Fig. 2b). However, in alloy GF-25 and GF-33 the 17 : 2compound was removed by homogenization (Fig. 3a, Fig. 4b).Concerning alloy GF-25 (Fig. 3a), reference to Fig. 1, suggests

that some Fe23Gd6 should form as the primary phase duringsolidification, but instead some Fe17Gd2 was found in theas-solidified state. Similarly, in alloy GF-33 (Fig. 4a), where theprimary phase should be the 23 : 6 compound, the 17 : 2 com-pound was present, instead, in the as-solidified state. Thus, itappears that nucleation of Fe23Gd6 during the solidificationwas relatively difficult. A similar observation on the nuclea-tion of Fe23Tb6 has been reported in Fe-Tb alloys by Darielet al. (1976).

233Fe - Gd Phase Diagram

Crystal structure. X-ray diffraction data identified intermeta-llic compounds with Fe-Gd ratios of 17 : 2, 23 : 6, 3 : 1 and 2 : 1(Table 3) and the lattice parameters were consistent with thosepreviously reported (Zinkevich et al., 2000).

Phase transformation temperatures. The onset temperaturesof thermal arrests during DTA heating, with correspondingtemperatures as assessed by Zinkevich et al. (2000), are shownin Table 4. The temperatures of the incongruent melting pointsof the compounds are in reasonable agreement with thosereported by Savitskii et al. (1961) and Zinkevich et al. (2000),except for Fe2Gd, which showed a significant difference. Inthe thermodynamically calculated diagram by Zinkevich et al.(2000), there was a good agreement with the experimental datafor the peritectic reaction temperature. However, the experi-mentally determined data (from the current work; Novy et al.,1961; Savitskii, 1970) for the onset of primary solidification inalloys with Gd between ~5 and 20%, showed considerablescatter and were significantly higher than the liquidus valuesshown in the thermodynamically calculated diagram. The solidstate transformation at ~1215 °C in the alloys containingFe17Gd2 corresponds to the allotropic transformation from the

Table 2. Microanalysis of the compounds present in theas-cast and homogenized binary Fe-Gd alloysAlloy designation Compound Fe % Gd % Fe : Gd

GF-5* Fe17Gd2 89.7 10.3 17:1.95Iron S.S. 99.7 0.3

GF-5** Fe17Gd2 89.5 10.5 17:1.99Iron S.S. 99.1 0.9

GF-11* Fe17Gd2 89.2 10.8 17:2.05Fe23Gd6 79.5 20.5 23:5.93

GF-11** Fe17Gd2 89.3 10.7 17:2.03

GF-13* Fe17Gd2 89.5 10.5 17:1.99Fe23Gd6 79.5 20.5 23:5.93

GF-13** Fe17Gd2 89.1 10.9 17:2.07Fe23Gd6 79.3 20.7 23:6.0

GF-16* Fe17Gd2 89.5 10.5 17:1.99Fe17Gd2 79.6 20.4 23:5.89

GF-16** Fe17Gd2 89.2 10.8 17:2.05Fe23Gd6 79.3 20.7 23:6.0

GF-22* Fe17Gd2 89.4 10.4 17:1.97Fe23Gd6 79.7 20.3 23:5.85Fe3Gd 75.1 24.9 3:0.99

GF-22** Fe17Gd2 89.4 10.6 17:2.01Fe23Gd6 79.2 20.8 23:6.04Fe3Gd 74.7 25.3 3:1.01

GF-25* Fe17Gd2 88.4 11.6 17:2.23Fe3Gd 73.5 26.5 3:1.08Fe2Gd 65.6 34.4 2:1.05

GF-25** Fe3Gd 73.9 26.1 3:1.06

GF-33* Fe17Gd2 89.2 10.8 17:2.05Fe3Gd 74.2 25.8 3:1.04Fe2Gd 66.3 33.7 2:1.01

GF-33** Fe3Gd 74.6 25.4 3:1.02Fe2Gd 66.1 33.9 2:1.03

*as-cast; **homogenized

Table 3. Lattice parameters for various Fe-Gd intermetalliccompounds

Compounds Structure Lattice parameters

“a” (Å) “c” (Å)

Fe17Gd2 Rhombohedral 8.501 12.492Fe23Gd6 Cubic 12.141 -Fe3Gd Rhombohedral 5.167 24.692Fe2Gd Cubic 7.3895 -

Table 4. Thermal arrests (°C) on heating corresponding to phase transformations in Fe-Gd system

Phase transformationsAlloy Peritectoid Fe3Gd* Fe2Gd* Fe23Gd6* Fe17Gd2* Fe17Gd2 .ä- iron to Melting

ã-iron + Fe17Gd2 rhom. L+ã- ironto á-iron to hex.

GF-5 930 - - - 1325 1215 1389 1465GF-11 931 - - - 1324 1216 1389 1429GF-13 - - - 1283 1325 1215 - 1418GF-16 - - - 1283 1324 1214 - 1387GF-22 - - 1171 1282 - - - 1331GF-25 - - 1173 1281 - - - 1311GF-33 - 1057 1171 - - - - 1289

Reported 932 ± 5 1080 ± 10 1160 ± 10 1280 ± 10 1335 ± 10 1215 ± 2 1380 ± 10data**

*Incongruent melting temperatures of compounds; **Zinkevich et al., 2000; rhom. to hex. = rhombohedral to hexagonal type

234 S. Atiq et al.

low temperature allotrope (rhombohedral, Zn17Th2 type) to thehigh temperature form (hexagonal, Ni17Th2), as previouslyrepor-ted (Atiq et al., 1990a; 1990b).

ConclusionThe results reported here showing the equilibria involvingfour intermetallic compounds, namely, Fe17Gd2, Fe23Gd6,Fe3Gd, and Fe2Gd are in agreement with the recent assess-ment of the Fe-Gd system (Zinkevich et al., 2000). The datareported here indicate the possibility of small compositionranges of stoichiometry and of a trend for the ratios to beslightly rich in gadolinium as compared with the ideal val-ues. The temperatures of the peritectic and solid state trans-formations generally agree reasonably with the data asassessed by Zinkevich et al. (2000), but often the experi-mental melting temperatures were higher. The crystal struc-ture and lattice parameters of the compounds were consis-tent with previous works.

ReferencesAtiq, S., Rawlings, R.D., West, D.R.F. 1997. Defects in lattices

of (Fe1-x Cox)17 RE2 compounds (RE=Gd and Tb). Mater.Sci. Technol. 13: 375-378.

Atiq, S., Rawlings, R.D., West, D.R.F. 1990a. Phase relation-

ships in the iron-cobalt-gadolinium system. J. Mater. Sci.Lett. 9: 518-519.

Atiq, S., Rawlings, R.D., West, D.R.F. 1990b. Crystal structureof compounds iron-gadolinium (Fe17 Gd2) and iron-ter-bium (Fe17 Tb2). Mater. Sci. Technol. 6: 778-780.

Dariel, M.P., Holthuis, J.T., Pickus, M.R. 1976. The terbium-iron phase diagram. J. Less-Common Met. 45: 91-101.

Novy, V.F., Vickery, R.C., Kleber, E.V. 1961. The gadolinium-iron system. Trans. AIME 221: 580-585.

Okamoto, H. 1996. The Fe-Gd (iron-gadolinium) system. J.Phase Equilibria 17: 552-561.

Rawlings, R.D., Newey, C.W.A. 1968. Study of the iron-molyb-denum system by means of diffusion couples. J. IronSteel Inst. 206(Pt.7): 723.

Savitskii, E.M., Terekhova, V.F., Burov, I.V., Chistyakov, O.D.1961. Equilibrium diagram for alloys of the gaddinum-ironsystem. Russ. J. Inorg. Chem. 6: 883-885.

Savitskii, E.M. 1970. Etudes des properties physiques etchimiques of alliages de torres rares. In: Proc. Conf. LesElements des Terres Rares, pp. 47-60, CNRS, Paris, France.

Zhang, W., Han, K. 1998. An updated evaluation of the Fe-Gd(iron-gadolinium) system. J. Phase Equilibria 19: 56-63.

Zinkevich, M., Mattern, N., Seifert, H.J. 2000. Reassessment ofthe Fe-Gd (iron-gadolinium) system. J. Phase Equilibria21: 385-394.

235Fe - Gd Phase Diagram

Introduction

Non-oxidative dissolution of the base metal sulfide in acidic

solution has been studied in order to understand the leaching

kinetics and to develop suitable conditions for a non-oxidative

leaching method for the treatment of base metal sulfide ores.

The kinetics of sphalerite in sulfuric acid solutions, with and

without the addition of sodium chloride, have been reported

(Eguchi et al., 1982). These authors noted that addition of so-

dium chloride to the sulfuric acid solution containing zinc ions

greatly enhanced the dissolution rate which depended on the

concentration of zinc ions. Tarabaev and Milyutina (1956) stud-

ied the kinetics of the dissolution of synthetic and natural sul-

fides of lead, copper, zinc and iron in sulfuric acid solutions

containing chloride ions. They reported that sulfides of base

metals dissolved most readily in these solutions. Scott and Nicol

(1976) also studied base metal dissolution in concentrated

chloric acid solutions and proposed a general model for the

diffusion-controlled dissolution. On the contrary, Awakura

et al. (1980) proposed that base metal dissolution in concen-

trated acid solution is controlled by the first order chemical

reaction. Nunez et al. (1990; 1988) reported that the order of

reaction for ionic activity of HClO4 is of the first order, whereas

for HCl the order of reaction over a wide range of concentra-

tion is 3/2 with respect to mean ionic activity of hydrochloric

acid. They also reported that activation energy is independent

of the chloride ion used to increase the activity of HCl.

Mackovich and Ptitsyn (1999) studied the leaching kinetics of

galena with a mixture of nitrous acid and sulfuric acid. They

observed a dependence on the content of H2SO

4 in the presence

of the leaching agent HNO2, which had a catalytical effect, thus

increasing the oxidation rate of galena to two times even at a

very low concentration (0.01 mole per litre). These kinetic stud-

ies are, however, insufficient to explain the mechanism of

dissolution of galena in acidic solution. It was, therefore, con-

sidered appropriate to reinvestigate the kinetics in order to

better understand the dissolution of galena in acidic solution.

This paper thus presents the results of a kinetic study on non-

oxidative dissolution of galena in hydrochloric acid with the

addition of sodium chloride.

Materials and Methods

Materials. A natural galena sample, having a composition of

85.54% Pb and 14.46% S, was used in this study. The main

impurities identified by spectrographic analysis were silica

and silver. The sample was mounted in such a way that only a

cleavage face of galena was introduced to a dissolution me-

dium. The galena surface was ground with corundum number

1500 and then polished with fine alumina. Distilled and

deiodized water with the average specific conductivity of 107

Ω/cm was used in all the experiments. Hydrochloric acid so-

lution of the desired concentration was prepared by diluting

the standard IM HCl solution. Nitrogen gas was used for

deoxygenation of the solution and purging of the reactor. All

chemical reagents used were of Analytical grade.

Experimental procedure. A 500 ml separable glass flask,

with lid having four necks was used as the reaction vessel for

Pak. J. Sci. Ind. Res. 2005 48(4) 236-239

Effect of Sodium Chloride on Dissolution of Galena in Aqueous Acid

Solution

Abdul Khalique*, Adnan Akram, A. S. Ahmed and Nusrat HamidMaterials Science Research Centre, PCSIR Laboratories Complex, Lahore-54600, Pakistan

(received June 3, 2004; revised March 22, 2005; accepted March 25, 2005)

Abstract. This paper presents the results obtained on the kinetic study of non-oxidative dissolution of natural galena

in aqueous hydrochloric acid with the addition of sodium chloride. A chemical reaction on the surface of galena

controlled the dissolution rates under the experimental conditions of investigation. The galena dissolution rate was of

the first order with respect to hydrochloric ion activity in hydrochloric acid solution. The addition of sodium chloride

to the acid solution greatly enhanced the dissolution rate. The effect of sodium chloride has two possible interpreta-

tions: firstly, it may be the result of an increase in the hydrogen ion activity; secondly, the enhancement of the dissolu-

tion rate, observable at the high sodium chloride concentration, may be due to the specific absorption of chloride ions

or the surface complexing of chloride ions on galena surface.

Keywords: dissolution mechanism, kinetics of galena, effect of sodium chloride, non-oxidative dissolution, galena

dissolution rate

*Author for correspondence

236

all dissolution experiments (Fig. 1). The central hole of the

lid was used for the accommodation of condenser. The other

three necks were used for the introduction of nitrogen gas

into the dissolution medium, thermometer and the fixation of

galena specimen at the predetermined place, respectively. The

evolved hydrogen sulfide gas was collected, through the other

end of condenser, in the absorbing flask. A waterbath pro-

vided with a temperature controller was used as the thermo-

stat. Each dissolution medium was deoxygenated with nitro-

gen gas for 30 min prior to each experiment. A galena sample

was placed, facing downwards, in a dissolution medium (500

ml) stirred with a magnetic stirrer. Nitrogen gas was bubbled

into the dissolution medium throughout each run. Dissolution

rate was followed by the analysis of hydrogen sulfide gas ab-

sorbed in the absorbing solution (KOH + H2O

2 + MoO

3). The

Pb ion concentration in the pipetted solution, drawn at appro-

priate time intervals, was quantitatively analysed for dissolved

Pb by atomic absorption spectrophotometry (Sidney, 1984).


Linear rate determination. The dissolution of Pb in 1 M

HCl solution was determined by maintaing constant the stir-

ring speed at 1100 rpm and temperature of 328 K for 20 h

(Table 1). The results show that galena dissolved at a constant

rate except at the initial stage of dissolution. The reaction rate

was, therefore, determined from the data given in Table 1. It

appears that the rate was 0.4 x 10-5 moles/cm2 per h for the 0-6 h,

but then it attained its constant value of 0.6 x 10-5 moles/cm2 per

h for the next 15 h. Therefore, this reaction rate was used for the

subsequent examination of reaction kinetics.

Effect of temperature. The effect of different temperatures

on the dissolution rates was examined over a range of

313-343 K at the constant stirring speed of 1100 rpm in so-

lutions containing 1M HCl, and 1M HCl-1M NaCl (Fig. 2).

The dissolution rates so obtained, as shown in Table 2, were

plotted against 1/T, which showed a linear relationship over

the entire temperature range studied (Fig. 2). The activation

energies were found to be 13.6 Kcal/mole for 1M HCl, 18.4

Fig. 1. Experimental apparatus: A, reaction flask (500 ml, inner dia

85 mm); B, sample feed and sampling hole; C, thermom-

eter; D, bubbling tube; E, stirrer; F, condenser; G, waterbath;

H, heater; I, magnetic stirrer; J, bath agitator; K, absorption

bottle.

Table 2. Effect of different temperatures on the dissolution

rate of Pb in I M HCl and 1 M HCl-I M NaCl at 100 rpm

stirring speed

Temperature 1 Dissolution rate, R (x10-5 moles/cm2/h)

(K)

T(K-1) 1M HCl 1 M HCl - 1 M Nacl

313 3.2x10-3 0.32 1.6

323 3.1x10-3 0.45 2.5

333 3.0x10-3 0.93 5

343 2.9x10-3 1.81 12

237

R (

x10

-5 m

ole

s/c

m2/h

)

20

109876

5

4

3

2

Dissolution of Glena

Table 1. The dissolution of Pb in 1 M HCl as a function of

time at 1100 rpm stirring speed at 328 K

Time (h) 0 2 4 6 8 10 12 14 16 18 20

Pb dissolved

(x 10-5 moles/cm2) 0.0 0.6 1.3 2.4 3.6 4.8 6.0 7.2 8.4 9.6 10.8

Fig. 2. The effect of temperature on the dissolution rate of galena;

R = dissolution rate, = reciprocal of absolute temperature.1T

curve 2

18.4 Kcal/mole

∆

∆

∆

∆

curve 1

13.6 Kcal/mole

(x10-3 K-1)

2.9 3.0 3.1 3.2 3.3

∆ = 1mole/1 HCl .1mole/1 NaCl

= 1mole/1 HCl

ω = 1100 rpm

1

0.90.80.7

0.6

0.5

0.4

0.3

0.2

0.1

1T

Kcal/mole for 1M HCl-1M NaCl. These high activation ener-gies found for non-oxidative dissolution of galena showedthat dissolution in this system was controlled by the diffu-sion of either the reactants at the surface, or the reactionproducts from the surface of galena instead of diffusion inthe solution. The rate would not be, therefore, dependent onthe stirring speed of the solution in this case. Similar ten-dency was also reported by Awakura et al. (1980) who alsoobserved the independence of dissolution rate from stirringspeed.

Effect of stirring speed. Different stirring speeds wereused to determine their effects on the dissolution of ga-lena (Table 3). Linear dissolution rates at the stirring speedsof 900-1100 rpm were independent of stirring speed, whichwere slightly higher than that at 800 rpm. This independentbehaviour of the dissolution rate from stirring speed seems tosuggest that diffusion was not a rate controlling step. It isreasonable to consider that the rates of non-oxidative disso-lution of galena in HCl, with or without the addition of sodiumchloride, were controlled by a chemical reaction on the sur-face of galena. Hereafter, all experiments were run at 1100 rpmthroughout the later studies.

Effect of hydrochloric acid concentration. Dissolution ratesof galena were measured at different acid concentrations,ranging from 0.1 M to 4.0 M HCl, with or without the addi-tion of NaCl, keeping constant the stirring speed of 1100rpm at the temperature of 328 K. Dissolution rates wereplotted against hydrogen ion concentration of hydrochlo-ric acid (Fig. 3), which show that dissolution rates increasedwith increase in acid concentration in all the solutions ex-amined. It seems that the reaction rate was of the first orderwith respect to the hydrogen ion concentration over theconcentration range from 0.1 M to 2.0 M hydrochloric acidsolutions (Fig. 3; curve 1) and its value at 2 M HCl was 10-

5 moles/cm2/h. The dissolution rate of galena exhibited ahigher dependence, than first order, upon hydrogen ionconcentration in HCl solutions having concentration be-yond 2 M. Nunez et al. (1988) also observed similar ten-dency during base metal dissolution in HCl and HClO4.Therefore, with the analogy to their studies, it may be con-sidered that a special role of chloride ions of HCl, such as

specific absorption and/or complexing ability, would affectthe dissolution rate.

Since acid concentration appears to be the most appropri-ate parameter to determine dissolution kinetics, it can bespeculated that a rate determining process is the release ofHS − from the surface of galena as follows:

PbS + H+ PbS H+ad

PbSH+ad Pb2+ + HS−

The hydrogen sulfide ion, HS−, thus formed may react rapidlywith a hydrogen ion to form hydrogen sulfide.

The dissolution rate for HCl solution containing 1 M NaCl wasalso of the first order with respect to HCl concentration whentotal chloride ion concentration was less than 2 M and its valuewas 5 x 10-5 moles/cm2/h at the acid concentration of 2 M (Fig.3; curve 2). These findings suggest that HCl concentrationdetermined the dissolution kinetics, which was of first order forHCl solution in the presence of NaCl when total chloride ionconcentration did not exceed 2 M. It is also concluded that theaddition of sodium chloride upto the total chloride ion concen-tration of 2 M to acid solutions resulted in the enhancement ofdissolution rate, which may be due to the increase in hydrogenion concentration of the leachant. Similar behaviour was alsoobserved by Awakura et al. (1980) during their kinetic studieson galena dissolution.

→

238

Table 3. Effect of stirring speed on linear dissolution rates (R = x10-5 moles/cm2/h)

Stirring speed (rpm)

400 500 600 700 800 900 1000 1100 1200 1300Linear dissolution rate(R moles/cm2/h) 2.00 3.00 4.75 6.10 7.00 7.10 7.15 7.18 7.20 7.21

A. Khalique et al.

Fig. 3. Effect of acid concentration with and without NaCl onthe dissolution rates of galena; R = dissolution rate.

R (x

10-5 m

oles

/cm

2 /h)

Acid concentration (mole/l)

Temp: 328 Kω = 1100 rpm

curve 2

curve 1HCl

HCl - 1M NaCl

0.0 0.1 0.5 1 5

00

50

10

5

1

0.5

00

Dissolution of Glena

Temp: 32% K u, = 1100 rprn - 100 - HCI = 1 mold c

;s E $ 5 0 - 2 E

'P 10 - z 25

NaCl (molen) Fig. 4. Effect of the addition of sodium chloride to hydrochlo-

ric acid on dissolution rates of galena.

Effect of sodium chloride. The effects of the addition of sodium chloride to l M HCl solution were examined at 328 K and 1 100 rpm (Fig. 4). From the data shown in Fig. 4, it appears that dissolution rate was slow between the concentration of NaCl from 0-1 M. It was, however, greatly enhanced at the concentration of more than 1 M NaCl. From this observation it may be speculated that increase in the chloride ion concentration itself played a role in the increase of the dissolution rate. This effect of chloride ions was revealed at higher concentrations of NaCl. Such an effect was also studied by a number of researchers (Awakura et al., 1980; Majima et al., 1980; Tarabev and Milyutina, 1956). It is concluded, therefore, that it may be due to specific absorption of chloride ions on the protonated galena surface, or due to the surface complexing of chloride ions on the galena surface playing an important role in the enhancement of dissolution rate.

Conclusion The non-oxidative dissolution of galena was studied kineti- cally in hydrochloric acid solution, with and without the addition of sodium chloride. Under the experimental conditions employed during this study, the dissolution rates were controlled by a chemical reaction on the surface of the galena sample. The galena dissolution rate was of the first order with respect to hydrogen ion activity in hydrochloric acid solution. The addition of sodium chloride to the acid solutions

greatly enhanced the dissolution rate. This effect of sodium chloride has two possible interpretations: fustly, it may be the result of an increase in the hydrogen ion activity; secondly, the enhancement of the dissolution rates observable at high sodium chloride concentration may be due to the specific absorption of chloride ions or the surface complexing of chloride ions on galena surface. A combination of these effects is supported by the observations made.

References

Awakura, Y., Kamei, S., Majima, H. 1980. A kinetic study of nonoxidative dissolution of galena in aqueous acid solution. Metall. Tram. 11B: 377-38 1.

Eguchi, M., Khalique, A., Nakamurah, H. 1982. A kinetics study on the sulphuric acid leaching of sphalerite with hydrogen sulphide formation. J. Japan Inst. Metals 47: 1061-1067.

Mackovich, T.I., Ptitsyn, A.B. 1999. Specifics of processing of galena concentrate with sulphuric acid in the presence of nitrous acid. Fiz. Tekh. Probi. Razrab. Polezn. ISKOP 4: 56-64.

Majima, H., Awakura, Y., Yazaki, T., Chikamori, Y. 1980. Acid dissolution of cupric oxide. Metall. Trans. 11B: 209-2 14.

Nunez, C., Espien, F., Garcia-Zayas, J. 1988. Kinetics of nonoxidative leaching of galena in perchloric and hydrochloric acid solutions. Metall. Trans. 19B: 541-516.

Nunez, C., Espien, F,, Garcia-Zayas, J. 1990. Kinetics ofgalena leaching in hydrochloric acid-chlorides solutions. Metall. Trans. 21B: 11-17.

Scott, P.D., Nicol, M.J. 1976. Dissolution ofbase metal sulphides in concentrated chloric acid solutions. Trans. Inst. Min. Metall. 1: C40-C44.

Sidney, W. 1984. Oflcial Methods ofAnalysis, Association of Official Analytical Chemists 25: pp. 1-464, 14h edition, Washington, USA.

Tarabev, S.I., Milyutina, N.A. 1956. Kinetic studies of synthetic and natural sulphides in sulphuric acid solutions. Isvest. Akad Nauk Kazakh SSR Ser Gom. Dela Stroi- rnaterialov. Met. 6: 76-88.

Separation of Close Boiling Acidic Isomers by Dissociation Extraction

A. S. Ahmed*, M. B. Bhatty, M. T. Saeed and M. P. I. QaziGlass and Ceramics Research Centre, PCSIR Laboratories Complex, Lahore-54600, Pakistan

(recived March 18, 2004; revised January 27, 2005; accepted January 30, 2005)

Abstract. The separation of m-cresol and p-cresol was studied using dissociation extraction technique. The separation

factors obtained for cresols-carbon tetrachloride-caustic soda system were in the range of 1.40 to 1.52, whereas for cresols-

carbon tetrachloride-monoethanolamine system were in the range of 1.297 to 1.417. Isomer ratios, concentration of cresols

and the strength of aqueous extractant have a significant influence on the separation of cresols. The organic acid (m-cresol)

was successfully back-extracted from aqueous caustic soda with a non-polar organic solvent, octanol. pH of the aqueous

phase and the choice of organic solvent have an important role in the back-extraction of m-cresol.

Keywords: dissociation extraction, cresols, monoethanolamine, isomer separation, close-boiling isomers


Introduction

The coal-tar produced in steel industries on carbonization of

coal contains industrially important phenolic compounds

(Micheal and Muder, 1974). These are used as intermediates,

or starting compounds, for the production of dyes, herbi-

cides, pesticides, medicines, and several other useful chemi-

cals (Hutton et al., 2000; Lo and Baird, 1996; Anwar et al.,

1995). These phenolic compounds cannot be separated by

the well established unit operations, as they exhibit normal

boiling points, differing by only a fraction of a degree and

having similar solubilities in most solvents. However, the dif-

ference in molecular arrangements of the isomers does result

in an appreciable difference in their strength as acids or bases,

as shown by their relative dissociation constants, which are

9.8x 10-11 for m-cresol, 6.7x10-11 for p-cresol, 4.54x10-9 for 3-

picoline and 10.62x10-9 for 4-picoline. This difference is being

exploited as the basis of practicable separation by the less

known process of dissociation extraction. The basic principles

of dissociation extraction were reported by Warnes (1924),

long before the term “dissociation extraction” was suggested

(Twigg, 1949). The commercial applications of dissociation

extraction technique, based on the earlier studies (Pratt, 1967;

Wise and Williams, 1964), were impeded by the continuous

consumption of strong bases or strong acids for the recovery

of phenolic compounds. This was due to the difficulty in re-

versing the reaction in order to free the purified isomer, as well

as to regenerate the extractant. Ideally, there should only be a

weak interaction between the extractant and the desired iso-

mer, yet sufficiently strong to achieve the separation but weak

enough to be broken-down without using excessive chemi-

cals, or thermal energy. Considering this, weak acids and bases

(organic and inorganic) in water were successfully used as

extractants (Anwar et al., 1979; 1971). These studies also pro-

vided the theorctical basis for such applications. The findings

reported in these studies prompted Sharma and coworkers

(Pahari and Sharma, 1992; Gaikar and Sharma, 1987; Wadekar

and Sharma, 1981) to study other weak acids and bases for the

separation of close boiling substances using the dissociation

extraction technique.

In the dissociation extraction process, the weakly acidic or

basic organic or inorganic compounds may be used as

extractants as long as they are preferentially soluble in the

aqueous phase (Ahmed et al., 2002; Anwar et al., 1979), and

insoluble in the organic solvent. Earlier work (Ahmed et al.,

2003) established that aqueous sodium dihydrogen phosphate,

a weak acid, could be used successfully to separate the mix-

ture of 4-picoline and 3-picoline. Although experimental re-

sults confirmed the viability of the process, the low separa-

tion factors and aqueous phase loadings of picoline implied

that a liquid-liquid extraction contactor, with a large number of

stages, is required for the desired results.

To illustrate the principles of dissociation extraction, let us

consider a mixture of weak organic acids, m-cresol and p-

cresol obtained from the coal-tar distillate. These, in the

organic diluent, are partially neutralized by an aqueous weak

organic base, such as monoethanolamine, C2H

4(NH

2)OH.

There will thus be a competition between the two organic

acids to react preferentially with the aqueous organic base.

The organic acid (m-cresol), having higher dissociation

constant, will react preferentially with the base to form an

ionized salt in the aqueous phase, insoluble in the organic

phase. The other organic acids (p-cresol), having a lower

dissociation constant, will remain predominantly in its un-

Pak. J. Sci. Ind. Res. 2005 48(4) 240-246

240

dissociated form, soluble in the organic phase. Due to the

weakly basic nature and stoichiometric deficiency of

monoethanolamine (MEA) in the aqueous phase, as com-

pared with total cresols, the reaction does not proceed to

completion in the aqueous phase. Therefore, the isomers

are partially separated and high degree of separation and

purification may be achieved by contacting aqueous MEA

containing cresols with a fresh feed of cresols in a number

of similar contacts. The loaded aqueous phase is contacted

with a suitable organic solvent to extract-back m-cresol from

the aqueous MEA and consequently regenerate MEA to

recycle in the process. The cresol is recovered from the

organic solvent by distillation.

The theory described above was applied to separate m-

cresol from the mixture of m-cresol and p-cresol present in

carbon tetrachloride. The effect of various parameters, such

as isomer ratios, concentration of feed, and the organic

and aqueous phase loadings on the separation factors was

studied. It was shown that separation of cresols could be

achieved by MEA, and consequently m-cresol can easily

be back-extracted from aqueous MEA by a secondary sol-

vent, toluene.

The present work also combined with the earlier work (Anwar

et al., 1979; 1971; Pratt, 1967; Wise and Williams, 1964) to

describe another approach to the dissociation extraction

process. This process employs a strong aqueous base,

aqueous caustic soda, for the extraction of m-cresol from

the mixture of cresols according to the dissociation extrac-

tion theory. Then, the reaction was reversed by using an

organic reagent, having a higher affinity for the dissoci-

ated m-cresol, to extract-back m-cresol from the aqueous

caustic soda. This process produced higher aqueous phase

loadings of cresols and as a result higher separation fac-

tors were realized. The use of organic solvent to extract-

back m-cresol from the aqueous caustic soda gave the ad-

vantage of separation, without continuous consumption,

of bases or acids for the recovery of the desired product.

Hence, the reagents were regenerated and recycled to

economise the process.

The influencing factors on the back-extraction of cresols

were found to be the organic solvent, as discussed earlier

(Ahmed et al., 2003; 2002), the aqueous phase pH, and the

concentration of m-cresol in the aqueous phase. Therefore,

the variation of pH of the aqueous phase with different con-

centrations of m-cresol was studied to find its effect on the

back-extraction by the chosen secondary solvent. Although

the aqueous phase remained alkaline, the back-extraction of

m-cresol from the aqueous caustic soda was successfully

achieved by using octan-1-ol as the solvent.


The equilibrium concentrations of m-cresol and p-cresol were

determined for (a) aqueous MEA and carbon tetrachloride

system, and (b) aqueous caustic soda and carbon tetrachlo-

ride system.

Aqueous MEA-carbon tetrachloride system. For the MEA-

carbon tetrachloride system, a 20 ml mixture of known con-

centration of m-cresol and p-cresol, dissolved in carbon tetra-

chloride, was equilibrated with 20 ml of aqueous MEA. The

two phases were shaken vigorously for 5 min and then kept in

a thermostat at 25+1°C for 20 min. These conditions were

shown to be fully adequate for the equilibrium distribution of

cresols in the conjugate phases. After equilibration, the two

phases were separated and the isomer ratios of cresols in the

organic phase were found by gas liquid chromatography (GLC).

The concentration of cresol isomers in the aqueous phase

could then be calculated by mass balance, since the total quan-

tity of each isomer in the feed was known. The concentration

of isomer ratios of cresols could be verified by direct injection

of aqueous phase into the GLC column. The GLC column was

a coil-shaped glass column having 3 mm dia and 1.5 m length.

The column was filled with chromosorb GAW DMCS of 80-

100 mesh as support material and 8% w/w matexil as the

stationary phase. A high resolution of m-cresol and p-cresol,

with 30 min retention time, was achieved at the column tem-

perature of 195 °C. The GLC was calibrated with standard

solutions of m-cresol and p-cresol in carbon tetrachloride.

The reproducibility of analytical results was +0.1%.

Aqueous NaOH-carbon tetrachloride system. For the caustic

soda-carbon tetrachloride system, equilibrium distribution of

m-cresol and p-cresol between carbon tetrachloride and aque-

ous caustic soda was determined using different cresol ratios

and different aqueous caustic soda concentrations. 10 ml of

each solution was equilibrated according to the experimental

procedure described for the aqueous MEA-carbon tetrachlo-

ride system. After equilibration, phases were separated and

samples of the organic phase were analysed by GLC as de-

scribed previously. The aqueous phase samples were neutral-

ized with dilute hydrochloric acid and made homogeneous by

the addition of methanol before injecting into the GLC glass

column, since direct injections of aqueous caustic soda seemed

to deteriorate the column, resulting in merged peaks of both

cresols on the chromolog integrator. All the chemicals used

were of Analar grade (Merck).


Separation of m-cresol and p-cresol by aqueous MEA. The

equilibrium distribution of an equimolar mixture of m-cresol

and p-cresol between 20 ml carbon tetrachloride and 20 ml

Separation of Close Boiling Acidic Isomers 241

aqueous MEA of concentrations ranging from 2-10% (w/w) is

given in Table 1. The total weight of cresol added to the sys-

tem, equilibrium weights of both the isomers in each phase,

and the separation factors have been listed (Table 1). The

expression for the separation factors is defined as:

[m-cresol]a [p-cresol]

o / [m-cresol]

o [p-cresol]

a

where:

terms in square brackets = concentrations

subscripts a and o = the aqueous and organic phases,

respectively

For 100 g/l MEA, the cresol feed concentration was 50 to 150

g/l. The separation factors varied only between 1.28 to 1.37.

These results showed that although separation factor in-

creased with the increase in cresol feed concentration, it was

virtually insensitive to MEA concentration. The GLC analysis

indicated slight solubility of cresol-MEA salt in the organic

phase at 100 g/l MEA feed concentration. Therefore, higher

MEA concentrations were not used for the cresols-MEA- car-

bon tetrachloride system.

The concentration of total cresols in the aqueous phase (dis-

sociated plus undissociated) increased with the increase in

cresol loading in the system, but reached an almost con-

stant level for a given MEA concentration: 24, 31, 38, 45 and

53 g/l, respectively, for the MEA strengths of 20, 40, 60, 80

Table 1. Distribution of the cresol mixture (equimolar) between 20 ml carbon tetrachloride (organic phase) and 20 ml aqueous

monoethanolamine (MEA) (aqueous phase) system

Aqueous MEA Total Equilibrium distribution of cresols (g)

concentration cresols Organic phase Aqueous phase Separation

(w/w, %) (g) m-cresol p-cresol m-cresol p-cresol factor

2 1.0 0.244 0.280 0.256 0.220 1.34

2.0 0.733 0.788 0.267 0.212 1.35

3.0 1.229 1.292 0.271 0.208 1.37

4 1.0 0.184 0.218 0.316 0.282 1.33

2.0 0.661 0.725 0.339 0.275 1.35

3.0 1.153 1.230 0.347 0.270 1.37

6 1.0 0.136 0.165 0.364 0.335 1.32

2.0 0.587 0.658 0.413 0.342 1.35

3.0 1.076 1.165 0.424 0.335 1.37

8 1.0 0.142 0.169 0.358 0.331 1.29

2.0 0.536 0.605 0.464 0.395 1.33

3.0 0.996 1.096 0.504 0.404 1.37

10 1.0 0.023 0.029 0.477 0.471 1.28

2.0 0.951 1.046 0.549 0.454 1.33

3.0 0.921 1.028 0.579 0.472 1.37

and 100 g/l. The limiting concentration of cresols in the aque-

ous phase, therefore, increased linearly with the increase in

MEA concentration, but was not directly proportional to it,

as also reported earlier (Pratt, 1967).

The effect of cresol isomer ratios on the separation factor

is shown in Fig. 1. The same-phase volumes were used

Fig. 1. The effect of cresol isomer ratios (m-cresol : p-cresol)

on their separation, using 4% monoethanolamine

(MEA) as the aqueous phase and 150 g/l total cresols

in carbon tetrachloride as the organic phase.

1.45

1.40

1.35

1.30

1.252 4 6 8 100

RATIO (m-cresol / p-cresolRatio m-cresol : p-cresol

Se

pa

rati

on

fa

cto

r

242 A. S. Ahmed et al.

Table 2. Distribution of the cresol mixture (3 g) between 20 ml carbon tetrachloride (organic phase) and 20 ml aqueous

monoethanolamine (MEA) (aqueous phase) system

Aqueous MEA Cresol ratio (%) Organic phase (g) Aqueous phase (g) Separation

concentration m-cresol p-cresol m-cresol p-cresol m-cresol p-cresol factor

(w/w, %)

2 90 10 2.260 0.261 0.440 0.039 1.303

70 30 1.762 0.786 0.338 0.114 1.323

50 50 1.228 1.292 0.272 0.208 1.370

30 70 0.73 1.798 0.170 0.302 1.386

10 90 0.250 2.366 0.050 0.334 1.417

4 90 10 2.108 0.247 0.592 0.053 1.309

70 30 1.631 0.740 0.469 0.160 1.330

50 50 1.152 1.230 0.348 0.271 1.371

30 70 0.682 1.708 0.218 0.392 1.393

10 90 0.223 2.170 0.077 0.530 1.414

6 90 10 1.989 0.236 0.711 0.064 1.318

70 30 1.531 0.704 0.569 0.196 1.335

50 50 1.076 1.165 0.424 0.335 1.370

30 70 0.636 1.616 0.264 0.484 1.386

10 90 0.208 2.057 0.092 0.643 1.415

8 90 10 1.865 0.223 0.835 0.077 1.297

70 30 1.381 0.646 0.719 0.254 1.324

50 50 0.996 1.096 0.504 0.404 1.373

30 70 0.589 1.519 0.311 0.581 1.380

10 90 0.197 1.920 0.113 0.780 1.412

10 90 10 1.690 0.206 1.010 0.094 1.310

70 30 1.297 0.614 0.803 0.286 1.329

50 50 0.92 1.030 0.580 0.470 1.382

30 70 0.546 1.454 0.344 0.656 1.396

10 90 0.173 1.777 0.127 0.923 1.413

with 4% MEA in the aqueous phase and 150 g/l total cresols

(in different ratios) initially present in carbon tetrachloride.

The separation factor increased from 1.309 to 1.414 as the

ratio of m-cresol / p-cresol in the system decreased from

90 : 10 to 10 : 90. The variation in separation factors was

virtually the same with other MEA concentrations in the

range from 20 g/l to 100 g/l. In all cases, the total cresols

loading in the aqueous phase was not significantly affected

by the change in isomer ratios (Table 2). Niewoudt (1997),

using boric acid in the aqueous phase, separated phenolic

compounds and neutral oil and reported a similar finding

for his system. These results show that the separation of

m-cresol and p-cresol with the weak organic base MEA is

practicable using the dissociation extraction technique. The

separation factors ranging from 1.28 to 1.37 (Table 1) im-

plied that the number of equilibrium stages necessary to

achieve a high degree of separation should not be small.

Recovery of m-cresol from the aqueous MEA was easily

achieved by contacting it with toluene.

Separation of m-cresol and p-cresol by aqueous caustic soda.

The equilibrium distribution of the mixture of m-cresol and

p-cresol, between carbon tetrachloride and various concen-

trations of caustic soda, is given in Table 3. The volume of

each phase was 10 ml and the total weight of cresols in the

system was 3 g. The data given in Table 3 include isomer

ratios of the total cresols, the normality of caustic soda in

the aqueous phase, the equilibrium contents of each isomer

in each phase, and the separation factors. Separation fac-

tors were greater for caustic soda than those for the MEA

reagent. The separation factors ranged from 1.40 to 1.52 as

the total cresols isomer ratio of m-cresol / p-cresol decreased

from 9 to 0.11 (90 : 10 to 10 : 90) for 0.5 N caustic soda.


Separation factors were not much affected by the caustic

soda normality. The total cresol concentration in the aque-

ous phase remaind approximately constant for a particular

normality of caustic soda, 0.6 molar (64.7 g) for 0.5 N, 1.1

molar (117.4 g) for 1 N, and 2.0 molar (220.1 g) for 2 N. The

loading of total cresols in the aqueous phase was thus

slightly greater than the stoichiometric equivalence of the

caustic soda and the total cresol loading in the caustic soda

was also greater than that of the weak basic reagent MEA.

The separation of m-cresol and p-cresol with 2 N caustic

soda, as the reagent, is shown in Fig. 2.

It was established in the previous studies that carbon tetra-

chloride, toluene and benzene were good solvents for this

system, whereas octan-1-ol had higher affinity for cresols

(Ahmed et al., 2003; 2002) . Therefore, octan-1-ol was selected

as the secondary solvent to exploit the extraction of m-cresol

from caustic soda and to regenerate caustic soda for recy-

cling. The most important influencing factor on the back-ex-

traction of m-cresol from the aqueous caustic soda was found

to be pH of the aqueous phase. Therefore, variation of pH of

the aqueous phase, along with the concentration of m-cresol

in the aqueous phase, concentration of cresol in each phase,

Table 3. Distribution of the cresol mixture (3 g) between 10 ml carbon tetrachloride (organic phase) and 10 ml aqueous caustic

soda (aqueous phase)

NaOH Cresol ratio (%) Organic phase (g) Aqueous phase (g) Separation

(normality) m-cresol p-cresol m-cresol p-cresol m-cresol p-cresol factor

0.5 90 10 2.103 0.250 0.597 0.050 1.42

70 30 1.613 0.745 0.487 0.155 1.45

50 50 1.136 1.231 0.364 0.269 1.47

30 70 0.667 1.702 0.233 0.398 1.49

10 90 0.217 2.156 0.083 0.544 1.52

1.0 90 10 1.612 0.203 1.088 0.097 1.43

70 30 1.220 0.600 0.880 0.300 1.44

50 50 0.843 0.978 0.657 0.522 1.46

30 70 0.501 1.340 0.399 0.760 1.47

10 90 0.159 1.667 0.141 1.033 1.49

1.5 90 10 1.123 0.151 1.577 0.149 1.42

70 30 0.839 0.440 1.264 0.460 1.44

50 50 0.574 0.712 0.926 0.788 1.46

30 70 0.327 0.962 0.573 1.138 1.48

10 90 0.104 1.192 0.196 1.509 1.49

2.0 90 10 0.681 0.096 2.019 0.204 1.40

70 30 0.505 0.280 1.595 0.620 1.43

50 50 0.343 0.449 1.157 1.051 1.44

30 70 0.193 0.599 0.707 1.501 1.46

10 90 0.061 0.738 0.239 1.962 1.47

Fig. 2.Equilibrium distribution of the cresol mixture

between 2 N NaOH (aqueous phase) and carbon

tetrachloride (organic phase).

100

80

60

40

20

0

0 20 40 50 80 100

CRESOL IN THE AQUEOUS PHASE (%)

Cre

so

ls i

n t

he

org

an

ic p

ha

se

(%

)

Cresols in the aqueous phase (%)

p-cresol

m-cresol


Table 4. The effect of pH on the back-extraction of m-cresol from the aqueous phase caustic soda with octan-1-ol

Concentration of pH of the Equilibrium concentration of Percentage

m-cresol in the aqueous phase m-cresol in conjugate phases extraction

aqueous phase (g/l)

(g/l) Aqueous phase Organic phase

12.5 13.23 12.5 - -

25.0 13.20 22.905 2.095 8.38

37.50 13.18 33.261 4.239 11.30

50.00 13.15 40.755 9.245 18.49

63.00 13.12 50.850 12.150 19.29

75.00 13.06 58.754 16.246 21.66

87.00 12.98 63.753 23.247 26.72

108.50 12.27 74.431 34.069 31.40

125.00 11.53 75.500 49.500 39.60

221.00 11.18 76.453 144.547 65.41

and percentage extraction of cresol was also investigated

(Table 4). The results of this study show that aqueous phase

was still alkaline even when m-cresol concentration was more

than twice the stoichiometric equivalence of caustic soda.

Moreover, a reduction in pH proved supportive to the extrac-

tion of m-cresol by octan-1-ol and the percentage extraction

improved in favour of the organic phase. At the equivalence

point of m-cresol (108 g/l), the pH was 12.3 and the extraction

was 31.4%. These results also indicate that there was a lower

limit on the proportion of m-cresol per mole of caustic soda,

about 25 g/l m-cresol, below which the extraction was not

practicable because the aqueous phase was too alkaline.

Conclusions

The process described is capable of separating m-cresol and

p-cresol with aqueous caustic soda as the reagent giving

high values of separation factors and also showing subse-

quent recovery of m-cresol from the aqueous phase by con-

tacting it with the organic solvent, octanol. The process

avoids the continuous consumption of chemicals and still

benefits from the maximum phase loading associated with

strong aqueous reagents. The weak organic acids or bases

may be employed as extractants for the separation of close

boiling tar distillates. The monoethanolamine (MEA), a weak

organic base, employed in this study as the extractant was

noted to be capable of separating cresol isomers. The sepa-

ration factors were, however, rather low as compared to caus-

tic soda. It has been also shown that the separation factor

was a function of the concentration of solutes and their ra-

tios in the organic phase and the concentration of the

extractant in the aqueous phase.

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Determination of the Level of PCBs in Small Fishes from Three Different

Coastal Areas of Karachi, Pakistan

A. B. Munshi, A. S. Hina* and T. H. Usmani

Centre for Environmental Studies, PCSIR Laboratories Complex, Karachi-75280, Pakistan

(received March 31, 2004; revised December 2, 2005; accepted January 5, 2005)

Pak. J. Sci. Ind. Res. 2005 48(4) 247-251

Abstract. The level of congener polychlorinated biphenyls (PCBs) was determined in small fishes. These fishes were

collected from the beaches of Korangi Creek, Hawksbay and Clifton, all located in Karachi, Pakistan. The contamination

status of PCBs was followed by their concentration in tissues. Higher PCBs were found to be the most persistent

organopllutants, present in all the fishes studied.The samples were spiked with 5µl surrogate internal standard solution

containing IUPAC numbers 194 and 198. A total of 14 PCBs were screened in each sample. The concentrations of PCBs

found were 1.25 ± 0.02 ng/g dry weight of fishes collected from Clifton, 1.01 ± 0.52 ng/g of fishes collected from Hawksbay,

and 1.00 ± 0.43 ng/g of fishes collected from Korangi Creek. The congener PCBs 138,153, and in some small fishes 118, were

found to be predominant among all the PCBs tested in this study.

Keywords: organic pollutants, PCBs, small fishes, Karachi coast, polychlorinated biphenyls


Introduction

The coastline of Karachi, Pakistan, stretches along the shores

for about 135 kilometers. Its growing pollution level is not

only due to the increasing volume of trade via the shipping

routes through the Karachi port, but is also due to its intake of

toxic effluents from a number of industries. This is severely

contaminating the mangrove forests and the marine life in the

area. The use of polychlorinated biphenyls (PCBs) in Pakistan

is mainly for industrial purposes. PCBs are the mixture of upto

209 individual chlorinated compounds and are classified as

persistent organic pollutants (Langanathan and Kannan, 1991).

They are used in the manufacturing of electrical equipment,

heat exchangers, hydraulic systems, and several other

specialized applications. PCBs do not readily breakdown in

the environment and thus remain there for a very long period

of time. PCBs may enter the air, water and soil. In water, a small

amount of PCBs may remain dissolved, but most of it sticks to

the organic particles, bottom sediments, and binds strongly to

the soil. PCBs are taken-up by small organisms and fishes

through the contaminated water and may thus get into the

food chain of other animals. Through the food chain, therefore,

PCBs accumulate in fishes and marine mammals, reaching a

level that may be immensly higher than that in the surrounding

water. The consumption of PCBs is hazardous as these are

suspected to be human carcinogens (CEPA, 2003; ATSDR,

2000).

There are no known natural sources of PCBs, but these have

been ragularly found in fishes, wildlife, and humans

(Erickson, 1997). Earlier studies on fish have indicated that

PCBs are unlikely to affect the growth of cultured fish at

concentrations typically found in the environment (Kan-

Atireklap et al., 1997; Duinker et al., 1980). Commercially

produced fish feeds often contain low concentrations of PCBs.

However, cultured fish biomagnify PCBs from feeds, resulting

in their concentration in the fish tissue to exceed that of fish

feed itself (Carline et al., 2004). The U.S. Food and Drug Admi-

nistration (US-FDA) has set the tolerance level of 0.2 ppm (200

ng/g) of PCBs in the finished fish feeds and 2.0 ppm in the fish

sold for human consumption.

The objective of the present study was to provide baseline

data on organochlorine concentrations in the fishes commonly

counsumed as food in Karachi. Samples for the present study

were collected from three different locations along the Karachi

coast.


Sampling locations. The sampling locations included

Korangi Creek, Hawksbay and Clifton (Fig. 1). Korangi Creek

is situated in the South of Karachi, encircled by muddy

creeks. This creek receives effluents from industries and

oil refineries. The creek accommodates a few channels used

by fishermen (Beg et al., 1984). A long stony wall lies on the

East of Hawksbay, receiving intake of effluents, particu-

larly oil, released during loading and unloading of the oil

from tankers. Slicks and tarry deposits were observed dur-

ing the collection of samples from this area. Clifton is one

of the sandy shores where fauna is extremely rich and seems

to be rather free of pollution. Water currents may, however,

247

transport a part of the industrial and domestic wastes to

this beach.

Sample preparation and analysis. Different species of fishes

were collected using catching nets with the help of fishermen

on small trawlers. These samples were washed with sea water

and then placed in glass bottles, brought to the laboratory,

and were deep-frozen to −20 °C. The fishes were identified,

measured and their wet weight was determined. Individuals of

the same species were packed in aluminium foil and stored at

−20 °C until their analysis. The biometric data related with fish

length and body weight, with the number of specimens col-

lected from different locations, is given in Tables 1-3. The

most dominant fish species found in these areas were mullet

and Otolithes rubber, locally known as mushka. Both are

commonly consumed in Karachi. The congener PCBs were

analysed according to the procedures given below.

Extraction of lipids. Two samples of each group of fishes

were selected for their trace chemical analysis. About 20 g

(wet weight) of the edible portion of fish was dried and

ground to a homogeneous powder with about 120 g AR

grade anhydrous sodium sulphate. The homogenized

sample was then extracted in a Soxhlet apparatus for 16 h

by using n-hexane. The sample extracts were reduced in

volume using a Kuderna-Danish (K-D) apparatus and lipid

contents were calculated.

Purification and separation. Lipids in the extracts were re-

moved using the alumina (Al2O

3) dry column chromatographic

technique (Duinker and Hillebrand, 1997). The sample extract

was then concentrated and subjected to silica gel column chro-

matography for clean-up and the separation of PCB congeners

(Duinker et al., 1980).

Quantification of polychlorinated biphenyls. The PCBs were

analysed using a Fisons (Carlo-Erba) 8000 series gas chro-

matograph (GC) with an on-column auto-sampler/injector. The

GC was equipped with a DB-5 and DB-35 capillary column

(30 m length; 0.32 mm internal dia; 0.25 mm film thickness)

and a 63Ni electron capture detector. The column oven

temperature programme was 40 °C, holding for 1 min, increas-

ing at 4 °C/min to 140 °C, followed by increasing at 4 °C/min

to 290 °C, then maintained at that temperature for 10 min. The

detector temperature was set at 310 °C. Hydrogen gas at 2 ml/

min and nitrogen gas at 30 ml/min were used as the carrier

and make-up gases, respectively. The PCB Mix 7 and Mix 19

(obtained from Ehrenstorfer Reference Materials, Dr.

Ehrenstorfer GmbH, bgm-Schlosser- Str. 6, A-86199, Augsburg,

Germany) was used in a dilution series as the external cali-

Fig. 1. Sampling locations of fish for their PCBs load along the coastline of Karachi, Pakistan.

Karachi

Clifton

MalirBin Qasim

ShahBandar

Hawks Bay

Korangi Creek

Marho Kohri

Gullel Kohri

Keti Bunder North

Keti Bunder South

N

10 0 10 20 30 km

248 A. B. Munshi et al.

bration standard for the quantification of polychlorinated

biphenyl congeners.

Quality assurance and quality control measures included the

use of reagent blanks, and surrogate and matrix spike recovery.

Calibration and calibration verifications were routinely checked

at the beginning and the end of each batch of 6 samples. All

laboratory batches of samples contained one procedural blank,

one laboratory control sample, and a duplicate sample. The

procedural blank was spiked with the solvent and surrogate

internal standard PCB congeners 198 and 194. All samples

were spiked with the surrogate compound to determine effi-

ciency. Results were calculated on the basis of the mean value

of specimens.


The concentration of polychlorinated biphenyls (PCBs) in

the analysed fish samples are shown in Tables 1-3.

Fourteen dominant PCB congeners were studied. The

level of different PCBs in the various fish species studied

ranged from 0.001-8.86 ng/g of tissue, which was a

relatively uncontaminated value, when compared to the

data on fishes from the USA (Langanathan and Kannan,

1991). The composition of PCB congeners in each type

of fish, for every location, was found to be different from

any commercial PCB preparation, implying their altera-

tion by various factors (for example, metabolism, bio-

logical transport, hydrodynamic transport, adsorption,

and deposition rates).

A review of Tables 1-3 shows that the fishes from Hawksbay

and Korangi Creek were found to be less contaminated by

PCBs. The most contaminated species by ΣPCBs (the sum of

significant values of PCBs concentrations) were Sardinella

sindensis and Pristiopoma hasta for Hawksbay and Korangi

Creek, respectively, probably due to the higher lipid con-

tents (5.37- 7.23%) of these species. At Hawksbay, Lyari river

is the main source for draining PCBs at the northern side.

However, at Korangi Creek the main source of PCBs is

industrial effluents generated by oil refineries situated in this

region. The Clifton beach was found to be highly

contaminated by PCBs, as compared with the other two

locations. Cynoglossus sindensis was the most contaminated

species at this location. Main source for PCBs at this area is

the effluents discharge from the nearby located plastics,

paints and polymer industries.

On an overall basis, the concentration of lower-chlorinated

congeners was found to be higher than that of the higher-

chlorinated ones. This may be probably due to the

indiscriminate discharge of effluents from a variety of PCB

Table 1. Biometric data of the fish species caught from Clifton beach, Karachi, Pakistan and the various polychlorinated biphenyls

(PCBs) detected from their bodies (ng/g tissue)

Chlorobiphenyl IUPAC Cynoglssus Equula Arius Pristiopoma Otolithes

species number sindensis splendens serratus hasta rubber

(PCB congener) n = 8 n = 12 n = 5 n = 2 n = 10

l = 15 (10-20) l = 8 (5-15) l = 25 (20-35) l = 22 (18-25) l = 122 (80-150)

w = 80 (70-89) w = 100 (80-120) w = 21 (15-25) w = 365 (319-410) w = 20 (18-25)

2,2,5-Trichlorobiphenyl 18 4.45 2.90 0.25 0.01 0.19

2,4,4- Trichlorobiphenyl 28 6.18 nd 0.69 0.02 0.03

2,4,5- Trichlorobiphenyl 31 5.06 nd nd 0.03 0.04

2,2,3,5- Tetrachlorobiphenyl 44 1.09 0.24 0.37 0.01 nd

2,2,5,5- Tetrachlorobiphenyl 52 8.26 nd 0.56 0.03 nd

2,2,4,5,5- Pentachlorobiphenyl 101 7.89 nd 0.71 0.10 0.07

2,3,3,4,4- Pentachlorobiphenyl 105 0.93 0.37 0.12 0.01 0.02


2,2,3,4,5,6- Hexachlorobiphenyl 138 6.28 0.18 0.61 0.02 0.05



2,2,3,3,4,4,5- Heptachlorobiphenyl 170 1.52 nd 0.12 0.003 0.01

2,2,3,,4,4,5,5- Heptachlorobiphenyl 180 2.46 nd 0.20 0.01 0.002

2,2,3,3,4,4,5,5- Octachlorobiphenyl 194 0.02 nd 0.02 0.001 nd

n = number of fish catch; l = mean length (cm), within paranthesis is the range; w = mean weight (g), within paranthesis is the range; nd = not

detected

PCBs in Coastal Small Fishes 249

Table 2. Biometric data of the fish species caught from Hawksbay, Karachi, Pakistan and the various polychlorinated biphenyls

(PCBs) detected from their bodies (ng/g tissue)

Chlorobiphenyl IUPAC Stromateus Scomberoides Gerres Engraulis Sardinella

species number sinenbis commersannianus filamentosus purava sindensis


l = 10 (9-12) l = 30 (25-35) l = 22 (15-25) l = 20 (18-25) l = 18 (15-20)

w = 60 (54-70) w = 120 (110-155) w = 130 (110-155) w = 45 (40-55) w = 85 (80-100)




2,2,3,5-Tetrachlorobiphenyl 44 0.003 0.67 0.01 0.27 0.40

2,2,5,5-Tetrachlorobiphenyl 52 0.002 n.d 0.02 0.43 3.22

2,2,4,5,5-Pentachlorobiphenyl 101 0.01 0.46 0.16 0.44 2.90



2,2,3,4,5,6-Hexachlorobiphenyl 138 0.01 0.37 0.44 0.17 2.73



2,2,3,3,4,4,5-eptachlorobiphenyl 170 0.002 0.09 0.18 0.20 0.30

2,2,3,,4,4,5,5-eptachlorobiphenyl 180 0.002 0.09 0.33 0.16 1.09

2,2,3,3,4,4,5,5-tachlorobiphenyl 194 0.001 0.01 0.30 0.03 0.09

n = number of fish catch; l = mean length (cm), within paranthesis is the range; w = mean weight (g), within paranthesis is the range

Table 3. Biometric data of the fish species caught from Korangi Creak, Karachi, Pakistan and the various polychlorinated

biphenyls (PCBs detected from their bodies (ng/g tissue)

Chlorobiphenyl IUPAC Otolithes Pristiopoma Lepturacanthus Sphyraena Mugil

species number rubber hasta savala foresteri cephalus


l = 20 (1921) l = 28 (26-30) l = 22 (16-25) l = 10 (8-12) l = 9 (3-12)

w = 72 (64-80) w =232 (210-255 w = 187 (150-225)w = 27 (20-35) w = 45 (40-50)


2,4,4- Trichlorobiphenyl 28 0.077 0.031 0.011 0.033 0.076

2,4,5- Trichlorobiphenyl 31 0.086 0.023 0.014 0.014 0.077

2,2,3,5- Tetrachlorobiphenyl 44 0.073 0.023 0.019 0.017 0.032

2,2,5,5- Tetrachlorobiphenyl 52 0.108 0.022 0.028 0.031 0.072







2,2,3,3,4,4,5- Heptachlorobiphenyl 170 0.017 0.035 0.005 0.004 0.019

2,2,3,,4,4,5,5- Heptachlorobiphenyl 180 0.03 0.07 0.005 0.008 0.032

2,2,3,3,4,4,5,5- Octachlorobiphenyl 194 0.002 0.005 0.001 0.002 0.002

n = number of fish catch; l = mean length (cm), within paranthesis is the range; w = mean weight (g), within paranthesis is the range

A. B. Munshi et al.250

sources. The difference in molecular size of PCBs may play a

role in the uptake of chlorinated PCBs (Goutner et al., 2000).

However, for a greater understanding of the pattern of

accumulation of congeners in the fishes of the region, further

detailed research with a broader perspective is suggested.

At Hawksbay, ΣPCBs ranged from 0.069-27.73 ng/g of tissue.

PCB congeners 28 and 153 were observed to be in higher

concentration generally in all the species. The PCB congener

194 was found at its highest concentration of 0.30 ng/g of

tissue in Gerres filamentosus than all the other species, and

all other locations.

Levels of ΣPCBs in the Korangi Creek area ranged from 0.21-

0.84 ng/g of tissue in all fishes. In every species, PCBs 194 and

180 were observed at the lower concentration of 0.001-0.005

and 0.01-0.07 ng/g of tissue, respectively. At the lower

congener numbers of 18-52, the average concentration ranged

from 0.01-0.11 ng/g of tissue in the various fish species

collected from this location.

The most polluted location for PCBs was Clifton, where ΣPCBs

level ranged from 0.294-63.98 ng/g of tissue, which values

were higher than the concentrations reported from other areas

in different species (Bopp et al., 1998). At this location, the

PCB congener 194 was found to be at the lower concentrations

of 0.001-0.02 ng/g of tissue, than the other PCB congeners.

Some fishes from this region contained lower PCBs, even below

the levels reported in the fish from USA (US EPA, 1992).

Levels of PCBs in fishes from all the three locations were found

to be lower than the United State Food and Drug

Administration (US FDA) permissible limits for human

consumption (Macauley et al., 1992).

It may be emphasized in conclusion that PCBs emanating from

industrial effluents should be controlled at the source,

otherwise their increased intake by marine life is likely to pose

a serious biological threat.

References

ATSDR. 2000. Toxicological Profile for PCBs, Agency for

Toxic Substances and Disease Registry, GA, US

Department of Health and Human Services, Atlanta, USA.

Beg, M.A.A., Basit, N., Siddiqui, F., Mahmood, I., Siddiqui,

M.A. 1984. Studies on the biological contamination of the

coastal environment of Karachi. Pak. J. Sci. Ind. Res. 27:

206-210.

Bopp, R.F., Chillrud, S.N., Shuster, E.L., Simpson, H.J.,

Estabrooks, F.D. 1998. Trends in chlorinated hydrocar-

bon levels in Hudson River Basin sediments. Environ.

Health Perspec. 106 (S4): 1075-1081.

Carline, R.E., Barry, P.M., Ketol, G.H. 2004. Dietary uptake of

polychlorinated biphenyls (PCBs) by Rainbow Trout. J.

Aquaculture 66: 91-99.

CEPA. 2003. Polychlorinated Biphenyls, 2003, Pop’s Global

Monitoring Programme Proceedings, Reports and

Documents, Canadian Environmental Protection Agency

(CEPA), Ottawa, Canada.

Duinker, J.C., Hillebrand, M.T.J. 1997. Minimizing blank values

in chlorinated hydrocarbon analysis. J. Chromatogr. 150:

195-199.

Duinker, J.C., Hillebrand, M.T.J., Palmork, K.H., Wilhemsen, S.

1980. An evaluation of existing methods for quantitative

estimation of polychlorinated biphenyls in environmen-

tal samples and suggestion for an improved method based

on measurement of individual components. Bull. Environ.

Contam. Toxicol. 25: 956-964.

Erickson, M.D. 1997. Anlytical Chemistry of PCBs, Lewis

Publishers, New York, USA.

Goutner, V., Albanis, T.A., Konstantinou, I., Paoakons-

tantinou, K. 2000. PCBs and organochlorine pesticide

residues in eggs of Audouin’s gull (Larus audouinii) in

the North-eastern Mediterranean. Marine Pollut. Bull.

42: 377-388.

Kan-Atireklap, S., Tanabe, S., Sanguansin, J.,Tabucanon, M.S.,

Hungspreugs, M. 1997. Contamination by butyltin com-

pounds and organochlorine residues in green mussel

(Perna viridis L.) from Thailand coastal waters. Environ.

Pollut. 97: 79-89.

Langanathan, B.G., Kannan, K. 1991. Time perspectives of

organochlorine contamination in the global environment.

Marine Pollut. Bull. 22: 562-584.

Macauley, J.M., Summers, J.K., Heitmuller, P.T., Engle, V.D.,

Brooks, G.T., Babi-Kow, M. 1992. Statistical Summary

EMAP-Estuaries Louisiana Provin Lee, EPA/620/R-94/

002, Environmental Research Laboratory, Gulf Breeze, FA,

USA.

US EPA. 1992. National Study of Chemical Residues in Fish,

823-R-92-008a, Office of Science and Technology Stand-

ards and Applied Science Division, Washington DC,

USA.

PCBs in Coastal Small Fishes 251

Laboratory Evaluation of Plant Extracts as Antifeedant Against the LesserMealworm, Alphitobius diaperinus and Rice Weevil, Sitophilus oryzae

M. Kamruzzamana, M. Shahjahan*b and M. L. R. Mollaha

aBangladesh Institute of Nuclear Agriculture, P O Box 4, Mymensingh-2200, BangladeshbDepartment of Entomology, Bangladesh Agricultural University, Mymensingh, Bangladesh

(received November 19, 2003; revised January 11, 2005; accepted April 27, 2005)

Pak. J. Sci. Ind. Res. 2005 48(4) 252-256

Abstract. Studies were conducted on leaf, seed and bark extracts (acetone, ethanol and water) of Ipomoea fistulosa, Datura

fastuosa, Eucalyptus citridora, Helitropium indicum, Hedyotis corymbosa and Sapium indicum for their antifeedant effects

against the lesser mealworm, Alphitobius diaperinus and rice weevil, Sitophilus oryzae. The results showed that all the

extracts of test plants had antifeedant effect on both the insects. Among the six plant extracts tested, Sapium indicum

extracts had the highest antifeedant effect on the lesser mealworm and rice weevil. The ethanol extracts of leaf and seed were

more effective than those obtained in the other two solvents. The coefficient of deterrency increased proportionally with

increase in doses. The seed extract was more effective than leaf extract.

Keywords: plant extracts, antifeedant, lesser mealworm, rice weevil, Alphitobius diaperinus, Sitophilus oryzae


Introduction

Insect infestation in stored grains and their products is a seri-

ous problem throughout the world. Approximately, 200 spe-

cies of insects and mites attack stored grains and stored prod-

ucts (Maniruzzaman, 1981). Among these species, the lesser

mealworm (Alphitobius diaperinus) and rice weevil

(Sitophilus oryzae) are the most common and destructive

pests. Chemical control of insects during storage has been in

practice for a long time, but it has serious drawbacks (Sharaby,

1988). Indiscriminate use of chemical insecticides has given

rise to many serious problems, including genetic resistance in

pest species, toxic residues, increasing costs of application,

environmental pollution, and hazards to human beings and

animals during its handling and afterwards (Khanam et al.,

1990; Ahmed et al., 1981). This situation is indicative of the

need for safe, locally available and less expensive materials for

pest control during storage. Locally available plants and min-

erals have been widely used in the past to protect stored prod-

ucts against insect infestations (Golob and Webley, 1980).

The advantages of botanical products for this purpose are

that they are less expensive, non-hazardous and can be easily

produced by farmers. In the rural areas of South-Asia, includ-

ing Bangladesh, farmers traditionally mix leaves, barks, seeds,

roots or oils of certain plants with stored grains to protect

them from insect attacks. The use of antifeedants obtained

from indigenous plants in plant protection is still in the experi-

mental stages in Bangladesh. Very little work has been re-

ported on their efficacy against insect pest (Shahjahan and

Amin, 2000; Akhtar et al., 1998). The present study was under-

taken with some locally grown plants, such as Ipomea fistulosa

(vern. dholkalmi), Datura fastuosa (vern. datura), Eucalyptus

citridora (eucalyptus), Helitropium indicum (vern. hatisur),

Hedyotis corymbosa (vern. khetpapri) and Sapium indicum

(vern. urmoi) to investigate their compatibility with the pest

management programme by determining their antifeedant ef-

fects against Alphitobius diaperinus and Sitophilus oryzae.


The present studies were conducted on the evaluation of some

plants for their antifeedant effects against the lesser meal-

worm, Alphitobius diaperinus and rice weevil, Sitophilus

oryzae in the laboratory of the Department of Entomology,

Bangladesh Agricultural University, Mymensingh during the

period from July 1999 to June 2000. The test insects were reared

separately in plastic jars (12.0 x 23.0 x 6.5 cm). The lesser meal-

worms were fed on wheat grains and rice weevils on rice grains.

The jars were kept in laboratory at 18.70-28.9 °C temperature

and 73.34-87.90% relative humidity.

Preparation of plant extracts. The plant samples (leaf, seed,

bark) of “dholkalmi”, “datura”, eucalyptus, “hatisur”,

“khetpapri” and “urmoi” were collected from different areas of

Bangladesh. Fresh leaves, seeds and barks of these plants

were washed with water, air-dried, and followed by drying in

oven at 60 °C. The dried plant samples were ground manually

and passed through a 25-mesh sieve to obtain fine powder of

each plant part. The powders were preserved in airtight con-

tainers. Thirty grams of the fine powder of each plant sample

was taken in a 600 ml beaker to which were separately added

300 ml of different solvents (acetone, ethanol and water). The

252

mixtures were then stirred for 30 min with a magnetic stirrer (at

6000 rpm) and left to stand for 24 h. The mixture was then

filtered through a fine cloth and then filter paper. The filtered

extracts were concentrated by evaporation of solvents in a

waterbath at the temperature of 45 °C, 55 °C and 80 °C for

acetone, ethanol, and water, respectively. The extracts were

preserved in tightly-corked, labelled bottles and stored in re-

frigerator until their use for insect bioassays. Different con-

centrations (7.5, 10.0, 12.5 and 15.0% for the lesser mealworm,

and 2.5, 5.0, 7.5 and 10.0% for rice weevil) of each category of

plant extracts were prepared by dissolving them in water, prior

to insect bioassay.

Antifeedant test. The potency of antifeedant effects of the

plant extracts, against the lesser mealworm and rice weevil,

were determined by the method originally described by Nawrot

et al. (1986), later modified by Talukder and Howse (1995). Six

wheat wafer disks (15 mm dia) were used in each case as the

test food. The disks were oven-dried and saturated by dip-

ping either in solvents (control: C) or diluted extracts (treated

disk: T). They were dried in air through the night and their

individual weight was taken before feeding them to 10 insects

as the sole feed for 10 days. Two blank disks in each case

(treated with solvent only, but not fed to insects) were also

prepared. The feeding of insects was recorded under the fol-

lowing three condition: (i) on pure food, composed of two

untreated disks (control: cc), (ii) on food with a possibility of

choice between one treated (T) and the other untreated (C)

disks (choice test), and (iii) on food with two treated (TT)

disks (no choice test). Each treatment was replicated thrice.

After ten days, all the disks were reweighed. The disks were

observed for increase in weight because of the absorption of

moisture from the surrounding air, which was provided for the

normal growth and development of the insects. Therefore, a

correction procedure was applied. Disk weight loss, which

was the amount of food consumed (FC), was calculated by the

following formula given by Serit et al. (1992).

FC = IW-[(FWs x IW

b)/FW

b]

where:

IW = initial weight of the disk after being treated with extract

or solvent

FW = final weight of the wafer disk

b = weight of the blank disk (treated with solvent only and

where no insects were released)

s = weight of treated or control (treated with solvent only)

disks, which were given to insects as food

Therefore, according to the amount of the food consumed by

the insects in the control (CC), choice test (CT) and no choice

test (TT), three feeding deterrent activity coefficients were

calculated using the following formulae as described by Nawrot

et al. (1986).

(i) Absolute coefficient of deterrency (control and no-choice

test):

A = (CC - TT/CC + TT) x 100

(ii) Relative coefficient of deterrency (choice test):

R = (C - T/C + T) x 100

(iii) Total coefficient of deterrency:

T = A + R

where:

T = total coefficient of deterrency

A = absolute coefficient of deterrency

R = relative coefficient of deterrency

The total coefficient values served as an index of antifeedant

activity expressed on a scale between 0 and 200 (index values

between 200 and < 0 might be considered as insect

phagostimulants). The index zero (0) designated an inactive

compound, and the index for 200 for a compound with maxi-

mum activity. Indices were expressed as 151-200, + + + + ; 101-

150, + + + ; 51-100,+ +; and 0-50, +. All the experimental data

were analysed by analysis of variance (ANOVA).


The results of antifeedant effects of “dholkalmi”, “datura”,

eucalyptus, “hatisur”, “khetpapri” and “urmoi” plant extracts

on the lesser mealworm and rice weevil are presented in Tables

1-4. For both the insects, the differences of coefficient deter-

rent values between plant, plant part, solvent and dose were

found to be significant at 1% level of probability.

The lesser mealworm. It may be noted from Table 1 that among

the six tested plant extracts, “urmoi” (Sapium indicum) had

the highest antifeedant effect (120.99), whereas eucalyptus

had the least antifeedant effect (98.79). The seed extracts of

“urmoi” showed the highest total feeding deterrent effect

(123.60), while the leaf extract of eucalyptus possessed the

least total feeding deterrent effect (92.22), as may be noted

from Table 2. The results also showed that the seed extracts

were more effective than leaf extracts. Considering the effect

of solvents (Table 3), ethanol extract showed the highest total

antifeedant effect (123.65), which was significantly different

from water extract (105.13) and acetone extract (103.24). The

coefficient of deterrent values increased proportionally with

the increase of doses (Table 4).

Rice weevil. All the plant extracts had moderate inhibitory

effects on the feeding activities of rice weevil. Among the six

tested plant extracts, “urmoi” had the highest feeding deter-

rent effect (115.47), whereas eucalyptus possessed the least

253Plant Extracts as Antifeedant Against Insects

Table 2. Antifeedant effect of extracts of different plant parts on Alphitobius diaperinus and Sitophilus oryzae

Plant Plant Alphitobius diaperinus Sitophilus oryzae

species parts Coefficient of deterrency* Coefficient of deterrency*

Absolute Relative Total Efficacy** Absolute Relative Total Efficacy**

Ipomea fistulosa Leaf 56.69 b

60.64 cd

117.33 bc

+ + + 47.58 d

50.03 d

97.61 d

+ +

(“dholkalmi”) Bark 59.61 a

63.00 ab

122.61 a

+ + + 54.60 b

60.49 b

115.09 b

+ + +

Datura fastuosa Leaf 52.28 de

58.32 e

110.60 d

+ + + 41.67 f

44.77 f

86.44 f

+ +

(“datura”) Seed 56.22 bc

58.59 e

114.81 c

+ + + 44.88 e

47.35 e

92.23 e

+ +

Eucalyptus citriodora Leaf 45.21 f

47.01 h

92.22 h

+ + 28.14 i

32.16 i

60.30 i

+ +

(“eucalyptus”) Seed 50.21 e

54.55 f

104.76 f

+ + + 38.73 g

44.15 f

82.88 g

+ +

Helitropium indicum Leaf 51.56 de

54.43 f

105.99 ef

+ + + 38.35 g

42.08 g

80.43 g

+ +

(“hatisur”) Seed 54.64 c

59.97 de

114.61 c

+ + + 42.21 f

44.86 f

87.07 f

+ +

Hedyotis corymbosa Leaf 46.62 f

51.35 g

97.97 g

+ + 34.73 h

38.00 h

72.73 h

+ +

(“khetpapri”) Seed 52.67 d

55.94 f

108.61 de

+ + + 47.31 d

50.40 d

97.71 d

+ +

Sapium indicum Leaf 56.99 b

61.39 bc

118.38 b

+ + + 52.08 c

55.69 c

107.77 c

+ + +

(“urmoi”) Seed 59.46 a

64.14 a

123.60 a

+ + + 59.20 a

63.96 a

123.16 a

+ + +

S xc 0.61 0.44 0.84 0.40 0.56 0.75

* probability level = 0.01; different letters in the same column denote significant differences by DMRT; ** on a scale of 0 - 200 index (inactive

- highly active), + + + = 101 - 150 index, + + = 51 - 100 index

254 M. Kamruzzaman et al.

Table 1. Antifeedant effect of different plant extracts on Alphitobius diaperinus and Sitophilus oryzae

Plant Alphitobius diaperinus Sitophilus oryzae

species Coefficient of deterrency* Coefficient of deterrency*


Ipomea fistulosa

(“dholkalmi”) 58.15 a

61.82 a

119.97 a

+ + + 51.09 b

55.26 b

106.35 b

+ + +

Datura fastuosa

(“dutura”) 54.25 b

58.45 b

112.70 b

+ + + 43.28 c

46.06 c

89.34 c

+ +

Eucalyptus citridora

(eucalyptus) 48.01 d

50.78 e

98.79 e

+ + 33.44 e

38.15 e

71.59 e

+ +

Helitropium indicum

(“hatisur”) 53.10 b

57.00 c

110.10 c

+ + + 40.28 d

43.47 d

83.75 d

+ +

Hedyotis corymbosa

(“khetpapri”) 49.64 c

53.65 d

103.29 d

+ + + 41.02 d

44.20 d

85.22 d

+ +

Sapium indicum

(“urmoi”) 58.22 a

62.77 a

120.99 a

+ + + 55.64 a

59.83 a

115.47 a

+ + +

S xc 0.43 0.31 0.60 0.29 0.40 0.53

*probability level = 0.01; different letters in the same column denote significant differences by DMRT; ** on a scale of 0 - 200 index (inactive

- highly active), + + + = 101 - 150 index, + + 51 - 100 index

deterrent effect (71.59) on rice weevil (Table 1). The absolute

and relative coefficient of deterrency represented the no choice

and choice tests, respectively. When the insects had no op-

portunity to choose between the treated and the control disks

(no choice test), adults consumed either a small amount of the

treated disks or a large amount of the control disks, which

gave low absolute coefficient values. However, when they

had the opportunity to choose between the treated and the

control disks (choice test), the adults directed their feeding

activity to the control disks, which produced high relative

coefficient values. The results showed that the seed extracts

were better than leaf extracts (Table 2). The seed extracts of

S. indicum (“urmoi”) had the highest feeding deterrent effect

(123.16), while the leaf of E. citridora (eucalyptus) had the

least feeding deterrent effect (60.30). Among the three sol-

vents, the highest total coefficient of deterrency was observed

with ethanol extract (109.26), which was significantly different

from water (79.26) and acetone (86.96) extracts (Table 3). The

coefficient of deterrency increased proportionally with the

increase of doses (Table 4). Similar feeding deterrent effects of

different plant extracts, like Aphanamixis polystachya (vern.

pithraj) on Tribolium castaneum (Talukder and Howse, 1995;

1993), Polygonum hydropiper (vern. bishkatali), Vitex

negundu (vern. nishinda) and Aphanamixis polystachya on

Rhizopertha domonika (Akhter et al., 1998); Asclepias

calotropis (vern. akanda), Polygonum hydropiper (vern.

bishkatal), Azadirachta indicum (vern. neem) on Sitophilus

oryzae (Shahjahan and Amin, 2000); castor (Ricinus commu-

nis), Azadirachta indicum, Aphanamixis polystachya on

Alphitobius diaperinus (Rahman et al., 2001); and Melia

azedarach and eucalyptus (E. citridora) plant extracts on

Plecopetra reflexa (Meshram, 2000) have been also reported.

The results obtained suggest that Sapium indicum (vern.

urmor) leaf and seed extracts are useful for inhibition of feed-

ing by the lesser mealworm and rice weevil during storage, as

the extracts have shown strong antifeedant characteristics.

255Plant Extracts as Antifeedant Against Insects

Table 4. Mean antifeedant effect of different plant extracts in different dose levels on Alphitobius diaperinus and Sitophilus oryzae

Alphitobius diaperinus Sitophilus oryzae

Doses Coefficient of deterrency* Doses Coefficient of deterrency*

(%) Absolute Relative Total Efficacy** (%) Absolute Relative Total Efficacy**

7.5 33.91 d

37.28 d

71.19 d

+ + 2.5 26.06 d

29.00 d

55.06 d

+ +

10.0 48.28 c

51.72 c

100.00 c

+ + 5.0 37.54 c

41.00 c

78.54 c

+ +

12.5 60.56 b

64.83 b

125.39 b

+ + + 7.5 50.77 b

55.06 b

105.83 b

+ + +

15.0 71.50 a

75.82 a

147.32 a

+ + + 10.0 62.12 a

66.24 a

128.36 a

+ + +

S x- 0.351 0.254 0.486 0.233 0.325 0.433

* probability level = 0.01; ** on a scale of 0 - 200 index (inactive - highly active), + + + = 101 - 150 index, + + = 50 - 100 index; different letters

in the same column denote significant difference by DMRT

* probability level = 0.01; ** on a scale of 0 - 200 index (inactive - highly active), + + + = 101 - 150 index, + + = 50 - 100 index; different letters

in the same column denote significant difference by DMRT

Table 3. Antifeedant effect of different solvents, used for obtaining plant extracts, on Alphitobius diaperinus and Sitophilus oryzae

Solvent Alphitobius diaperinus Sitophilus oryzae

Coefficient of deterrency* Coefficient of deterrency*


Acetone 49.71 b

53.53 c

103.24 c

+ + + 41.64 b

45.32 b

86.96 b

+ +

Ethanol 59.98 a

63.67 a

123.65 a

+ + + 53.02 a

56.24 a

109.26 a

+ + +

Water 50.10 b

55.03 b

105.13 b

+ + + 37.71 c 41.91

c 79.62

c + +

S x 0.304 0.220 0.421 0.202 0.281 0.375

References

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256 M. Kamruzzaman et al.

Introduction

Garcinia kola (bitter kolanut), Family Guttiferae, is a native of

Southwestern African countries where it grows wild. It has

not attracted the due attention of plant breeders to cultivate it

and improve the wild strain. The plant, however, has gained

recognition in Western Africa, though it is not a crop of com-

merce (Irvine, 1963). Its fruit is normally about 7.5 cm in dia

and contains 4 to 6 light brown seeds embedded in the pulp.

G. kola is a popular seed, eaten on social and other occasions

in most parts of Western African countries and is an impor-

tant ingredient in medicinal preparations (Nwafor and

Ogbeneaga, 1992). In some parts of Africa, however, the seeds

are believed to cause impotency in man. The nuts are eaten

raw as a stimulant to resist hunger and sleep (Adeyeye and

Ayejuyo, 1994), and also used for the treatment and manage-

ment of cough and asthma (Nwafor and Ogbeneaga, 1992).

The root cuttings are used as chewing stick and for medicinal

purposes, such as in the treatment of cough.

The antibiotic Septrin (30 mg trimethoprim and 400 mg of

sulfamethaxazole) is effective against a wide range of gram-

negative and gram-positive bacteria (Hitchings, 1967). Septrin

is indicated for the treatment of bacterial infections of the

respiratory tract, urinogenital tract including gonnorhea,

prostatitis, gastrointestinal tract (typhus, paratyphus, chol-

era), skin and soft tissues, and those following surgical and

stomatological interventions. A potentiating effect of Septrin

on Garcinia kola (or vice versa) has been reported, presum-

ably due to the sites of action of the inhibitors being at

different steps in the same biosynthetic pathway (Nwafor

and Ogbeneaga, 1992). Although the combination of Septrin

with G. kola is extensively used, there are no reports on the

quantitative biochemical effects of such uses, as related to

chemotherapeutics. Earlier studies have shown the syner-

gistic action of the drug combinations, using serial-dilution

techniques, disc-sensitivity test, or experimental infections

in animals (Burchall, 1969). In addition to the differences in

the definitions of synergism and additivity of the drug-ac-

tion among various authors/workers (Garrett and Brown,

1993; Garrett, 1978a), these methods do not seem practicable

for the quantification of the chemotherapeutic effect. A lin-

ear relationship between inhibition and the inhibitor cannot

be presumed and should not be expected to exist. In addi-

tion, the additivity of killing of the numbers of bacteria is not

necessarily synonymous with an additivity rate of constants

for the killing of bacteria. Diffusion of drugs in culture ex-

periments may not be independent of other laboratory fac-

tors, thus resulting in a false presumption of synergy of

antibacterial action (Toama et al., 1978), while experiments

with live-animals may perhaps relate best to the clinical ef-

fects of the combination, these may show synergy as a re-

Quantification of Bactericidal Action of the Ethanolic Extract of

Garcinia kola Seeds Alone, and in Combination with the Branded

Antibiotic Septrin, on the Culture Isolates from Throat Irritation

Patients by Bacterial Growth Kinetics

V. A. Ajibade*a, I. G. Adanlawob, T. T. Akandea

aScience Technology Department, Federal Polytechnic, PMB 5351, Ado-Ekiti, NigeriabBiochemistry Department, University of Ado-Ekiti, PMB 5363, Ado-Ekiti, Nigeria

(received September 24, 2002; revised May 12, 2005; accepted May 21, 2005)

Abstract. The relationship of growth rates of Streptococcus pyogenes, isolated from patients with protracted throat

irritations and tonsillitis, with Garcinia kola extract and the branded antibiotic Septrin was investigated. A steady state was

obtained shortly after the addition of Septrin, whereas a lag phase of about five generations elapsed for G. kola. The

inhibitory effect of Septrin was about five times greater than the effect of G. kola. The inhibitory effect of G. kola was only

bacteriostatic, whereas Septrin caused bacterial death if a certain threshold of concentration (1 mg) was passed, as

evidenced by a decrease in the number of bacterial cells. Combination of Septrin and G. kola at concentrations where both

acted merely bacteriostatically, led to effects considerably greater than would be expected from simple additivity. It is

justifiable to conclude that the combination of G. kola extract and Septrin had a synergistic effect.

Keywords: Garcinia kola, antibiotic Septrin, bacterial growth kinetics, ethanolic extract, antibiotic synergism, antibiotic

Septrin, Streptococcus pyogenes, throat irritation


Pak. J. Sci. Ind. Res. 2005 48 (4) 257-262

257

sult of pharmacologic interactions rather than antibacterial

effects. For these reasons, it was felt appropriate to study

the antibacterial action of the combination in an experimen-

tal design not subject to these limitations. Garrett (1978b)

has outlined theoretical/mathematical models for the differ-

entiation of drug synergism, antagonism and additivity. Ex-

perimental methods that use the kinetics of bacterial growth

and the effects of antibiotic combinations have been devel-

oped. In studies with such methods, the activity of the drug

is defined in terms of decrease in the growth rate caused by

the drug, as a function of the concentration of the drug.

Techniques for viable and total count have been used to

quantify the decrease in the growth rates of Escherichia

coli generations as a function of concentrations of the anti-

biotic, and to differentiate between inhibition, uninhibited

growth simultaneous with killing, and inhibition of growth

simultaneous with or followed by killing (Garrett and Miller,

1995; Garrett and Brown, 1993; Garrett, 1978b).

The aim of the present work, therefore, was to use this general

approach to determine whether the combined action of G. kola

and Septrin will be additive or more than additive, i.e., synergis-

tic. However, for such studies to be meaningful, it was also

necessary to quantify the relative potencies of the two test

materials by the same methods, and in particular to determine if

they were acting by pure inhibition or inhibition with killing.

Thus, the growth dependency of isolates of Streptococcus

pyogenes, from sputum and throat swabs in the presence of the

individual drug/plant extract was also evaluated.


Specimens of sputum and throat swabs, obtained from fif-

teen patients (ten from patients at the State Specialist Hos-

pital, Ado-Ekiti and five from the Federal Polytechnic Cam-

pus, Ado-Ekiti) were used for the research study. The ten

samples from the hospital were clinical specimens of patients

referred to the laboratory for sputum microscopy, while the

other five were from students complaining of cough and

throat irritations. The sputum was collected in sterile dis-

posable plastic bottles. Each specimen was inoculated on

an agar base medium with 5% lysed blood and incubated

anaerobically at 37 °C for 24 h. The culture isolates were

identified according to Edwards and Ewing (1962) using the

gram-staining procedure, blood haemolysis, catalase test and

antibiotic susceptibility.

Garcinia kola seeds were obtained from the local market at

Ibadan, Oyo State, Nigeria. The dried, mature nuts were

ground into powder. Active principles were extracted from

30 g of the ground samples by agitating with 10 ml ethanol.

The mixture was allowed to stand for 24 h and filtered. The

extract was completely dried by using vacuum rotary evapo-

rator at 40 °C.

The Septrin tablets (marketed as Septran in several coun-

tries) were obtained from a pharmacy at Olorunsogo area,

Ibadan, Nigeria. The tablets were powdered and stored in

sterile plastic bottles. Seven g of the crude extract of G. kola

and Septrin were separately dissolved in 50 ml distilled wa-

ter. The mixtures were shaken vigorously to make the stock

solutions.

Various concentrations of G. kola extract (0.5-400 mg/ml) were

added to the bacterial culture isolates in the logarithmic-

growth phase. Samples were taken at appropriate time inter-

vals and the number of viable cells (plate count) and/or the

total number of cells (coulter counter or turbidity measure-

ments) were counted during incubation at 37 °C using

spectrophotometre. Similar experiments were done in the

presence of graded concentrationsof Septrin (0.125-4.0 mg/

ml) alone and in combination with the G. kola extract samples.

Because of the lag time in G. kola action (Ajibade and

Adanlawo, in press), the extract samples were added to the

bacterial culture isolates at different times of the logarithmic

growth phase. Thus, G. kola extract was added to the bacte-

rial culture isolates first, which was followed by the addition

of Septrin 1.5 h later.


The bacterial culture isolates from the sputum of 15 patients

were noted to be predominantly gram-positive cocci arranged

in chains, with β haemolysis on blood agar, catalase activity

negative, and having sensitivity to the antibiotic bacitracin.

The bacterial culture isolates were identified as predominantly

Streptococcus pyogenes. Other colonies were not substantial

enough to cause infection, and therefore ignored for further

investigations.The effect of G. kola extracts on the isolated

bactrial culture on their viable count and total count are shown

in Fig. 1 and Fig. 2. A lag phase of about five generations

existed before there was any significant effect of any G. kola

extract concentration on the growth of S. pygonese at 37 °C

(Ajibade and Adanlawo, in press). This lag phase appeared to

be independent of the specific activity of G. kola. Subse-

quently, the technique for viable count (Fig. 1) and total count

(Fig. 2) showed, respectively, that at 400 mg/ml the bacterial

population decreased from over 1010 in the control to less

than 105 cells/ml in the viable count (Fig. 1), while the bacterial

population decreased from 108.5 in the control to 105 cells/ml

at 100 mg/ml (Fig. 2). Apparent first-order generation rate con-

stants were obtained (Kapp/sec) from the slope of these new

growth curves in accordance with the general expression rates

as below:

258 V. A. Ajibade et al.

Kapp x tlog N = ———— + constant (1)

2.303

where:

Kapp

= apparent first-order generation rate

N = bacterial population

t = time

The coincidence of experiments for total and viable count sup-

ports the well known theory that G. kola extract is bacterio-

static and not bactericidal. Even at very high concentrations of

G. kola, the rate constants were positive, and no decrease in

viable cells was observed. It is obvious that there was no linear

logarithmic relationship among these two parameters.

A linear relationship, therefore, appears to exist with a

Lineweaver-Burk type plot as below:

1 Ka

——— = —— + Kb

(2)

Ko – K

app C

where:

Ko = generation rate constant in the absence of G. kola

Ka and K

b = constants

Ka = the measure of the molar activity of G. kola extract tested

C = concentration of G. kola extract

If this equation (2) is multiplied by the concentration of the

extract, the following is obtained:

C————— = (K

a + K

b) x C (3)

Ko – K

app

Fig. 3a is a plot of this type, showing a fit of the experimental

data to equation (3).

The effect of Septrin (Fig. 3a) in contrast to G. kola (Fig. 3b)

showed only a very short phase before the onset of inhibition

was observed with Septrin. Apparently, depending on the cul-

ture conditions and concentrations of Septrin, the onset of

inhibition was obtained almost immediately (Fig. 4). The rea-

sons for this difference in the lag phase could be consistent

with the sites of actions of the two inhibitors (Hitchings, 1967).

Cellular division was required before the G. kola activity was

observed. During the approximately five cellular divisions,

before the onset of G. kola inhibition, the stored folates in the

cells, such as dihydrofolic acid (Hitchings and Burchall, 1992),

were depleted, while new synthesis was inhibited by the G.

kola extract. Only after the stored folate was depleted did the

inhibition of folate system became critical for cell division.

On the other hand, Septrin blocked the systhesis of tetra-

hydrofolate directly at the level of dihydrofolic acid (Baker

and Beng-Thong, 1994; Hitchings and Burchall, 1992). Appar-

ently, Septrin blocked beyond the site of folate storage, and a

relatively rapid onset of action was observed (Fig. 3a). After

the short lag phase, cells grew in the presence of low concen-

trations of Septrin at reduced logarithmic rates. The same rela-

tionship was observed between the antibacterial activity and

the rate constants for bacterial growth as for G. kola (Fig. 3b).

From these results it appears that under the experimental con-

ditions of this study, the ratio of activities (Ka’s

) of G. kola to

Septrin was about 1:5; because KG.k

= 4.254 x 10-3 /ml/sec, and

KSp

= 0.785 x 10 –3 /ml/sec. This ratio is similar to the ratio of

MIC values for the two antibiotic agents obtained in similar

studies with the same strain of S. pyogenes (Septrin 0.175 mm;

G. kola 0.8 mm) by the present authors during earlier investi-

gations (Ajibade and Adanlawo, in press). G. kola at 2 mg in

the present studies caused a 50% inhibition of growth rate

259

Fig. 1. Typical generation rate curves of Streptococcus

pyogenes at 37 °C in the presence of various con-

centrations of Garcinia kola by the viable count

method.

Via

ble

count

/ m

l (1

0x)

Time (h)

0

2

4

6

8

10

12

1 2 3 4 5 6 7 8 9

Time (hrs)

control

0.5 mg/ml

1.0 mg/ml

2.0 mg/ml

3.0 mg/ml

4.0 mg/ml

5.0 mg/ml

10 mg/ml

400 mg/ml

Fig. 2. Typical generation rate curves of Streptococcus

pyogenes at 37 °C in the presence of various concen-

trations of Garcinia kola by the total count method.

Time (h)

Via

ble

count

/ m

l (1

0x)

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9

control

0.5 mg/ml

3.0 mg/ml

4.0 mg/ml

5.0 mg/ml

100 mg/ml

Bactericidal Action of Garcinia kola and Antibiotic Septrin

V. A. Ajibade et al.

+Viable count

Time (h)

Fig. 3a. Quantitative relations between apparent Strepto- coccus pyogenes growth-rate constant (KA and concentrations of Septrin; the curves are plotted in accordance with the exprssion KoC/Kw = (K. + KJ x C (see equation 3).

1 2 3 4 5 8 Time (h)

Fig. 3b. Quantitative relations between apparent Strepto- coccuspyogenes growth-rate constants (Kapp) and concentrations of Garcinia kola; the curves are plotted in accordance with the exprssion KoC/Kgp = (K, + KJ x C (see equation 3).

1 2 3 4 5 6 7 8

Time (h) Fig. 4. Typical generation rate curves of Streptococcus

pyogenes at 37 OC in the presence of various concentrations of Septrin.

(Fig. 4); one-half of the concentration (1 mg) does not cause 25% inhibition, but rather 33% inhibition. Since neither the activity of Septrin, nor that of G kola was linearly related to the concentration used, estimates of potency of combination based on assumptions of linear relationships between potency and concentration will be subject to error.

The second major difference between the effects of Septrin (Fig. 4) and G kola (Fig. 1 and Fig. 2) was that negative rate constants were obtained from experiments with viable counts at concentration as low as 2 mg. At these concentrations of Septrin, the total and viable counts obtained simultaneously did not coincide. In addition to the inhibition of cell division, killing was caused by Septrin. At the lower concentration of Septrin, viable and total counts did coincide, and no killing was observed. This means that Septrin was bacteriostatic at low concentration, but at higher concentrations killing was observed simultaneously with the inhibition. The threshold concentration seems to lie between 1 mg and 2 mg under the conditions used. The rate of killing was independent of the concentration of Septrin after the threshold concentration had passed. Although the rate constants were independent of the concentration of Septrin, they were dependent on the number of microorganisms.

The combined effects of Septrin and G kola on the growth ofS. pyogenes is shown in Fig. 5. Due to the lag time in G kola action, the extract was added to the culture at different times of the logarithmic growth phase. G kola extract was added first, Septrin was added 1.5 h later. The results showed that G kola extract and Septrin inhibited the growth most at the respective combination ratio of 2: 1, while the least inhibition was evident at single application. It can, therefore, be seen that the effect of the combination was significantly greater than when either sample was used alone. If the effects of the two samples were independently additive, one may predict the effects of the combination by combining the two expressions (equations 2) that were shown to hold for each sample alone, to obtain:

where:

Bactericidal Action of Garcinia kola and Antibiotic Septrin

The calculated value of the generation rate constants obtained clearly show that the combination effects were much greater than those produced by simple additivity (Fig. 6). Since this combination of Septrin and G kola extracts led to an observed effect much greater than that predictable from simple additivity, it is justifiable to say that the combination has a synergistic effect.

The synergism was further demonstrated by the method of continuous variations (Fig. 6). In this study, generation rates

+control + 1.0 mglml

-A- 2.0 mglml

-3.0 mglml -+ 4.0 mglml

0 ! 1

1 2 3 4 5 6

Time (h)

Fig. 5. Typical generation rate curves of Streptococcus pyogenes at 37 OC in the presence of combinations of different concentrations of Septrin and Garcinia kola.

0 4 1 2 3 4 5 6 7 8 9 1 0 1 1

Time (h)

100 90 80 70 60 50 40 30 20 10 0 t Garcinia kola (%)

0 10 20 30 40 50 60 70 80 90 100 Septrin (%) +

Fig. 6. Generation rate constants (KWp) of 1 mg/ml concentration of Garcinia kola and 0.01 5 mg/ml concentration of Septrin present in the culture (+), and when these were present in different percentage combinations (m) to show synergistic effect of the two.

were evaluated for a series of five cultures inhibited by mixtures of Septrin and G kola, as well as for each sample separately. The concentration of each sample chosen was a fraction of its concentration in the mixed culture so that the sum of the two sample fractions always equaled one. Equations 4 and 5 were used to calculate the curve showing the generation rates expected ifthe combinations were acting in an additive manner, The experimentally obtained generation rates were noted to be considerably lower than those predicted by this curve. Maximal synergy was observed when equipotent combinations of Septrin and G kola were used, but appreciable synergy was observed even when the potency ratio of Septrin to G kola was as low as 1 to 9, or as high as 9 to 1. Therefore, differences in the concentrations of two agents obtained in various body tissues should not have a large effect on the synergism.

References

Adeyeye, E.I., Ayejuyo, 0.0. 1994. Chemical composition of Cola acuminata and Garcinia kola seeds grown in Ni- geria. Int. J. FoodSci. Nuk 45: 223-230.

Ajibade, V.A., Adanlawo, 1.G Preliminary antibacterial efficacy of Garcinia kola seeds. World J. Microbiol. Biotechnol. (accepted).

Baker, B.R., Beng-Thong, H. 1994. Differential inhibition of dihydrofolic reductase from different species. J. Pharm. Sci. 53: 1137-1 138.

Burchall, J.J. 1969. Comparative studies of dihydrofolate reductase. Postgrad Med J. 45 (SuppL): 29-32.

Edwards, P.R., Ewing, W.H. 1982. IdentiJication of Entero- bacteriaceae, 2nd edition, Burness, Minneapolis, Minn., USA.

Garrett, E.R. 1978a. Classification and evaluation of combined antibiotic activity. Antibiot. Chemother. 8: 8-20.

Garrett, E.R. 1978b. The use of microbial kinetics in the qualification of antibiotic action. Arzneim. Forsch. 16: 1364-69.

Garrett, E.R., Brown, M.R.W. 1993. The action oftetracycline and chloramphenicol alone and in admixture on the growth of E. coli. J. Pharm. Sci, 54: 427-431

Garrett, E.R., Miller, GH. 1995. Kinetics and mechanisms of action of antibiotics on microorganisms. 111. Inhibitory action of tetracycline and chloramphenicol on Escheri- chia coli established by total and viable counts. J. Pharm. Sci. 54: 427-43 1.

Hitchings, GH. 1967. Role of inhibitors in studies of comparative enzymology. Fed Proc. 26: 1078- 108 1.

Hitchings, G.H., Burchall, J.J. 1992. Inhibition of folate

bisosynthesis and function as a basis for chemotheraphy.In: Advances in Enzymology, F.F. Nord (ed.), vol 27, pp.417-468, John Willey and Sons, New York, USA.

Irvine, F.R. 1963. West African Botany, Oxford University Press,London, UK.

Toama, M.A., Issa, A.A., Ashour, M.S. 1978. Effect of agar

262 V. A. Ajibade et al.

percentage, agar thickness and medium constituents onantibiotic assay by disc diffusion method. Pharmazie 33:100-102.

Nwafor, A., Ogbeneaga, I.E. 1992. Influence of Garcinia kolaon in vivo secretion of gastric juice acid. Afric. J. Pharm.Sci. 22: 107-109.

Introduction

Probiotics are the food supplements containing viable mic-

robes, which have the potential to beneficially influence the

health of the host (Schrezenmeir and De Vrese, 2001).

Metchnikoff (1907) observed, at the beginning of the last

century that the consumption of fermented foods was helpful

in controlling autointoxication caused by the putrefactive

microbial species that produce toxic compounds in the

intestine. Attempts have been made to replace this putrefactive

microbiota with the saccharolytic species. Lactobacillius,

Streptococcus, and Bifidobacterium species have been found

to play a useful role in this respect (Fuller, 1989). Species

belonging to Lactobacillus have been reported to play an

important role in the maintenance of the intestinal ecosystem

(Sandine, 1979). These organisms have been shown to possess

antagonistic effect towards the growth of pathogenic bacteria,

such as Listeria monocytogenes, Escherichia coli, and

Salmonella spp. (Drago et al., 1997; Chateau et al., 1993;

Ashenafi, 1991). The inhibitory properties of the Lactobacilli

have been linked with metabolic products, such as organic

acids (mainly lactic acid), hydrogen peroxide, and bacteriocins

(Juven et al., 1992).

An essential determinant in the choice of a probiotic

microorganism is its ability to reach, survive, and persist in

the environment in which it is intended to act (Marteau et al.,

1992). Some workers have demonstrated the survival and

Pak. J. Sci. Ind. Res. 2005 48(4) 263-267

Assessment of the Safety of Wild Strains of Lactobacillus as ProbioticsOrogastrically Administered to Rats

V. O. Oyetayo

Department of Microbiology, Federal University of Technology, PMB 704, Akure, Nigeria

(received December 30, 2003; revised May 5, 2005; accepted May 26, 2005)

Abstract. The safety of four wild strains of Lactobacillus, isolated from fresh cow milk and faeces of albino rat (Rattus

norvegicus) was studied. Some biochemical parameters of the serum in the orogastrically-dosed rats were used as the index.

A reduction in the levels of serum cholesterol and of serum aminotranferases in the rats orogastrically-dosed with

Lactobacillus isolates, as compared with the control group was noted. There was no significant difference (P > 0.05) in the

alkaline phosphatase levels of the control and the orogastrically-dosed rat groups. Serum globulin and bilirubin levels

showed a significant difference (P < 0.05) among the control and the Lactobacillus-dosed groups. The control group

recorded the highest weight gain among all the groups studied, but it was not significantly different (P > 0.05) from other

treatments except in the rats dosed with the Lactabacillus casei strain isolated from cow milk. The rats dosed with

Lactobacillus displayed beneficial effects as probiotics in terms of reduced serum cholesterol and liver function improvement

in terms of reduction in the serum aminotransferase levels.

Keywords: Lactobacillus isolates, serum cholesterol, albino rats, cow milk, probiotics, Lactobacillus safety

temporary colonisation of the human gastrointestinal tract by

some lactic acid bacteria (Jacobsen et al., 1999; Alander et al.,

1997). Walker and Duffy (1998) pointed out that the current

perspective on biotechnical applications of probiotic products

requires further in vitro and in vivo investigations to evaluate

the safety of using wild-type organisms, or those obtained by

genetic engineering. The safety and health promoting effect

of Lactobacillus casei isolated from different sources, mainly

the European cheeses, have been reported by using

biomarkers of serum and faeces (Bertazzoni et al., 2001). The

relationship between diet and disease/health can be revealed

by these biomarkers, since they provide a link between the

consumption of specific foods and the biological outcome

(Branca et al., 2001). Major biomarkers, such as plasma

enzymes, and their changes during the disease are related in

many ways to cell pathology (Baron et al., 1994). The aim of

the present study was, therefore, to assess the safety of wild

strains of Lactobacillus, isolated from fresh cow milk and

faeces of albino rats, when orogastrically-dosed to rats using

biochemical markers in the serum as the index.


Lactobacillus cultures. Four Lactobacillus strains, isolated

from fresh cow milk and faeces of albino rats, were used in the

present study (Table 1). These isolates were characterised

using the colony, morphological and biochemical chara-

cteristies. The isolates were observed to show antagonistic

effects against some pathogens and had the ability to adhereE-mail: [email protected]

263

to the ileac epithelial cells of the albino rats (Oyetayo et al.,

2004). The Lactobacillus isolates were cultured in MRS broth

(LAB M) and incubated at 37 ° C in an anaerobic environment

for 48 h to obtain high cell concentration. The method

described by Fujiwara et al. (2001) was used to harvest the

cells by centrifuging at 10,000 xg for 15 min at 0 °C. The cells

were then washed twice with sterile water. The washed cells

were resuspended in dehydrated skim milk (10% w/v, Marvel

brand), lyophilised in a lyophiliser, and stored at -20 °C until

use. The concentration of viable cells in the final powder was

determined by serial dilutions and plating on MRS agar medium

in triplicate (Table 1).

Animals and diet. Twenty Wistar rats (Rattus norvegicus),

5-6 weeks old, were obtained from Physiology Department,

University of Ibadan, Nigeria. They were housed at the

rathouse of the Federal University of Technology, Akure,

Nigeria and maintained at 27±1°C. The rats were fed on basal

diet, a product of Bendel Feed, Edo State, Nigeria, for one

week ad libitum before the treatment. The composition of the

basal diet is given in Table 2.

Orogastric administration of the Lactobacillus strains. The

rats were divided into five groups of four rats each. The

Lactobacillus isolates were reconstituted by suspending 1 g

of the lyophilised culture containing approximately 1010 cfu/g

in 10 ml of sterile water. A single dose of 0.3 ml of the culture

suspensions of the four isolated strains (1M, 2M, 1A, 2A)

was separately administered to 16 rats (4 rats for each

Lactobacillus strain) by orogastric-dosing, while the remaining

4 rats that served as the control were administered sterile skim

milk (Bertazzoni et al., 2001). This treatment was repeated

again on the second day. Post-ingestion period of 18 days

was allowed to elapse after the administration of the

Lactobacillus strains. The initial weight and final weight gain

were recorded. At the end of the feeding test, the rats were

killed by cervical dislocation and their blood aseptically

collected into ethylenediaminetetraacetic acid (EDTA) bottles,

specified for conducting the biochemical analyses of the

serum.

Biochemical assays. Reflotron M06. 02 < 06.00 kit (Boehringer

Mannheim Company) was used for biochemical analyses of

serum to study the effects of the administered Lactobacillus

strains. The biomarkers assayed were: total bilirubin (Bil), total

cholesterol (Chol), aspartate aminotransferase (Ast), alanine

aminotransferase (Alt), alkaline phosphatase (Alp), and

globulin (Glob) levels of the serum. Standardized amount of

the sample was applied on to the test zone of the appropriate

test strip. The strip was inserted into the test chamber and the

flap closed. The analytical results were monitored after some

seconds on the computer monitor. All the tests were carried

out at 25 °C.

Statistical analysis. Data were analysed by one-way ANOVA

followed by Dunnet tests using SPSS 7.5 package. The level

of significance was taken as P < 0.05. The results were reported

as mean values.


The strains used for this feeding trial were selected on the

basis of their inhibitory potential as shown on some pathogenic

bacteria and their ability to adhere to the intestinal epithelial

cells of the experimental albino rats (Oyetayo et al., 2004).

These strains of Lactobacillus were expected to exert a

beneficial effect on the maintenance of the intestinal micro-

flora of the host animals (Chang et al., 2001).

Table 2. Composition of basal diet used to feed the test

albino rats*

Ingredients Level in diet

Crude protein 14.5 %

Crude fat 4.8 %

Crude fibre 7.2 %

Crude ash 8.0 %

Calcium 0.8 %

Phosphorus 0.62 %

Sodium 0.15 %

Manganese 30 mg

Zinc 30 mg

Lysine 0.6 %

Methionine 0.29 %

Vitamin A 8,000 iu

Vitamin D-3 2,400 iu

Vitamin E 15 mg

Vitamin B-2 40 mg

Vitamin C 50 mg

Metabolisable energy 2,300 kcal/kg

*source: Bendel Feed, Edo State, Nigeria

cfu/g = colony forming units per gram; *mean of three replicates

Table 1. The source and strain designation of Lactobacillus

species used for orogastric administration in rats

Source of Strain Lactobacillus Cell count

strains designation species (cfu/g)*

isolated identified

Fresh cow milk 1 M L. acidophilus 5 x 1010

Fresh cow milk 2 M L. casei 4 x 1010

Rat faeces 1 A L. acidophilus 9 x 1010

Rat faeces 2 A L .brevis 9 x 1010

264 V. O. Oyetayo

The final weight gain was slightly higher in the rats of the

control group than the rats in all the four test groups that were

orogastrically administered the isolated Lactobacillus strains

(Fig.1). There was no significant difference (P > 0.05) in the

weight gain of rats in the control group and the test groups

1A, 2A and 1M, however, differences were significant (P <

0.05) in the group 2M. The control group consumed more feed

(401 g) as compared with the other test groups which consumed

394-398 g of the feed. This difference in the feed consumption

was not reflected in the weight gain difference between the

control and the other test groups, except in the 2M group.

The level of alanine aminotransferase (Alt) has been reported

to decline with weight loss (Johnston, 1999). The present

report shows that there was a correlation between the weight

gain and the Alt level in the serum of the rats. Rats in the

control group had higher weight and higher serum Alt than

the other test groups (Table 3). The serum cholesterol was

observed to be higher in the control group (107.75 mg/100 ml)

than the other groups (Fig. 2). It has been suggested that

bacterial metabolites in the fermented milk inhibited

cholesterol synthesis (Fuller, 1989). Bertazzoni et al. (2001)

recently reported a slight reduction in the serum cholesterol

of rats treated with Lactobacillus casei, sourced from the

European cheese. The Lactobacilli used in this study also

showed the ability to reduce serum cholesterol, and in effect

it may be concluded to have the potential of reducing the

incidence of coronary heart disease (CHD). Studies have

indicated that serum cholesterol level is a risk factor in the

incidence of CHD and that persons with elevated serum

cholesterol values develop CHD with greater frequency

(Branca et al.,2001; Kannel, 1978). The levels of

aminotransferases (both Alt and Ast) were higher in the

control group as compared with test groups. The increased

levels of Ast and Alt reflected active hepatocellular damage

(Baron et al., 1994). The level of Alt was generally lower than

Table 3. Levels of some biochemical markers in the serum of albino rats, orogastrically-dosed with various strains of Lactoba-

cillus species isolated from cow milk and rat faeces

Rat test Biochemical markers in the rat serum*

group Alt (iu/l) Ast (iu/l) Alp (iu/l) Bil (mg/l) Glob/mg/l)

lA (L. acidophilus) 25.87ab

94.98a

32.92a

0.47c

24.75a

2A (L. brevis) 13.06a

168.20a

32.67a

0.85ab

23.50a

1M (L. acidophilus) 18.08bc

132.00a

32.67a

0.83ab

21.00ab

2M (L. casei) 14.93cd

135.00a

32.92a

0.83ab

21.00ab

Control 29.65ab

251.03a

32.90a

0.63bc

23.50a

Alt: alanine aminotransferase; Ast: aspartate aminotrasferase, alkaline phosphatase; Bil: bilirubin; Glob: globulin; 1A and 2A: isolated from rat

faeces; 1M and 2M: isolated from cow milk; i u: international units; *values are mean for four rats/treatment and the same superscripts along

the column are not significantly different at P < 0.05

0

5

10

15

20

25

30

35

40

45

C 1A 2A 1M 2M

a

a

a

a

ab

We

igh

t g

ain

(g

)*

Fig. 1. Final weight gain of rats orogastrically-dosed with Lac-

tobacillus isolates from various sources (C: control; 1A

and 2A, respectively, L. acidophilus and L. brevis iso-

lated from rat faeces; 1M and 2M, respectively, L. acido-

philus and L. casei isolated from cow milk;

*bars with the same letter are not significantly different

(P > 0.05).

Lactobacillus strains

45

40

35

30

25

20

15

10

5

0 C 1A 2A 1M 2M

Fig. 2. Level of serum cholesterol in rats orogastrically

dosed with Lactobacillus isolates; *bars with the same

letter are not significantly different (P < 0.05).

Lactobacillus strains

C 1A 2A 1M 2M

b

ab

b

a

b

115

110

105

100

95

90

85

Seru

m c

hole

ste

rol (m

g/

100 m

l)*

265Wild Strains of Lactobacillus as Probiotics

Ast. Alanine aminotrasferase (Alt) is more specific for

monitoring the liver damage, since it is principally found in

the liver (Johnston, 1999; Baron et al., 1994; Cheesborough,

1991), while other organs such as kidney, cardiac muscles,

skeletal muscles, etc., can produce Ast. The lower Alt and Ast

levels founds in the serum of the rats dosed with the

Lactobacillus species is an indication of improved liver

function. These observations indicated that Lactobacillus

species can still persist in the gostrointestinal tract of the rats

for up to three weeks. There was no significant difference (P <

0.05) in the level of Alp in all the rat trial groups. An increase

in osteoblastic activity has been linked with a rise in the Alp

level (Baron et al., 1994). A rise in Alp level can also occur

when there is a lack of bile flow (cholestasis). In the present

study, there was no significant difference (P > 0.05) in the

level of Alp in the serum of the experimental rats, when

compared with the control group (Table 3).

Immunoglobulins are most often investigated in children

sufferring from recurrent infections, or a combination of

infections with allergy (Branca et al., 2001). The globulin

level was slightly higher in the serum of rats dosed with

Lactobacillus acidophilus isolated from albino rat faeces. The

ability of Lactobacilli to cause a non-specific immune resp-

onse, when ingested, has been reported (Kimura et al., 1997).

This immunostimulatory potential was, however, not displayed

by the other Lactobacillus isolates.

The bilirubin level in serum is also a useful indicator of

pathological effects. Over-production of bilirubin has been

linked with an excess breakdown of red blood cells

(Cheesborough, 1991). This may be due to many factors, one

of which is the toxins produced by bacteria. The lowest level

of bilirubin was observed in rats treated with strain 1A (L.

acidophilus) isolated from albino rat faeces. The lower bilirubin

level in rats, dosed with the strain 1A, may be an indication of

the specificity of the isolate to the host; hence it is amiable.

Chang et al. (2001) reported recently that Lactobacillus reuteri

BSA 131 from pigs had a better potential as probiotic agent

for piglets, especially after weaning.

The results obtained during the present study reveal that oral

administration of wild strains of Lactobacillus isolated from

fresh cow milk and albino rats is safe. These isolates have

also been noted to show beneficial effects due to their anticho-

lesterolaemic activity and reduction in the aminotransferase

levels, which is an indication of improved liver function. These

observations indicate the probiotic potential of these strains

of Lactobacillus species.

References

Alander, M., Korpela, R., Saxelin, M., Vilpponem-Salmela, T.,

Mattila-Sandholm,T.,von Wright, A. 1997. Recovery of

Lactobacillus rhamnosus GG from human colonic

biopsies. Lett. Appl. Microbiol. 24: 361-364.

Ashenafi, M. 1991. Growth of Listeria monocytogenes in

fermenting tempeh made of various beans and its

inhibition by Lactobacillus plantarum. Food Microbiol.

8: 303-310.

Baron, D.N., Whicher, J.T., Lee, K.E. 1994. A New Short

Textbook of Chemical Pathology, pp. 151-156, 5th edition,

ELBS, The Barth Press, Avon, UK.

Bertazzoni, M.E., Benini, A., Marzotto, M., Hendriks, H.,

Sbarbati, A., Dellaglio, F. 2001. Preliminary screening of

health-promoting properties of a new Lactobacillus

strain: in vitro and in vivo. HEALFO abstracts, Italy.

Branca, F., Hanley, A.B., Pool-Zobel, B., Verhagen, H. 2001.

Biomarkers of exposure and effect in relation to quality of

life and human risk assessment. Brit. J. Nutri. 86 (Suppl.

1): S 55-S 92.

Cheesborough, M. 1991. Medical Laboratory Manual for

Tropical Countries, vol. 1, pp. 494-526, 2nd edition,

Tropical Health Technology and Butterworth Scientific

Limited, London, UK.

Chateau, N.T., Castellanos, I., Deschamps, A.M. 1993.

Distribution of pathogen inhibition in the Lactobacillus

isolates of a commercial probiotic consortium. Lett. Appl.

Microbiol. 18: 42-44.

Chang, H., Kim, J., Kim, H., Kim, W., Kim, Y., Park, W. 2001.

Selection of Potential Probiotic Lactobacillus Strain

and Subsequent in vivo Studies, pp. 193-199, Antonie

van Leeuwenhoek, Kluwer Academic Publishers, The

Netherlands.

Drago, L., Gismondo, M.R., Lombardi, A., de Haen, C., Gozzini,

L. 1997. Inhibition of in vitro growth of enteropathogens

by new Lactobacillus isolates of human origin. FEMS

Microbiol. 153: 455-463.

Fuller, R. 1989. Probiotics in man and animals: a review. J.

Appl. Bacteriol. 90: 345-352.

Fujiwara, S., Seto, Y., Kimura, A., Hashiba, H. 2001. Establish-

ment of orally administered Lactobacillus gasseri

SBT2055SR in the gastrointestinal tract of humans and

its influence on intestinal microflora and metabolism. J.

Appl. Microbiol. 153: 455- 463

Jacobsen, C.N., Nielsen, V.R., Hayford, A.E., Moller, P.L.,

Michaelsen, K.F., Paerregaard, A., Sandstrom, B., Tveds,

M., Jakobsen, M. 1999. Screening of probiotic activities

of forty-seven strains of Lactobacillus spp. by in vitro

V. O. Oyetayo266

techniques and evaluation of the colonisation ability of

five selected strains in humans. Appl. Environ. Microbiol.

65: 4949-4956.

Johnston, D.E. 1999. Special considerations in interpretting liver

function tests. The American Academy of Family

Physicians, April 15, 1999.

Juven, B.J., Schved, F., Linder, P. 1992. Antagonistic compounds

produced by chicken intestinal strain of Lactobacillus

acidophilus. J. Food Prot. 55: 157-161.

Kannel, W.B. 1978. Status of coronary heart disease factors. J.

Nutri. Educ. 10: 10-15.

Kimura, K., McCatney, A.L., McConnel, M.A., Tannock, G.W.

1997. Analysis of faecal population of Bifidobacteria and

Lactobacilli and investigation of the immunological

responses of their human hosts to the predominant strains.

Appl. Environ. Microbiol. 63: 3394 -3398.

Marteau, P., Pochart, P., Bounik, Y., Goderel, I., Rambaud, J.C.

1992. Survival of Lactobacillus acidophilus and Bifidobac-

terium spp. ingested in fermented milk in the small

intestine: a rational basis for the use of probiotics in

man. Gastroenterologie Clinique et Biologue 17: 25-

28.

Metchnikoff, E.1907. The Prolongation of Life: Optimistic

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Oyetayo, V.O., Adetuyi, F.C., Akinyosoye, F.A. 2004. In vitro

assessment of the probiotic properties of Lactobacillus

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Sandine, W.E. 1979. Role of Lactobacillus in the intestinal

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Schrezenmeir, J., De Vrese, M. 2001. Probiotics: prebiotics

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Wild Strains of Lactobacillus as Probiotics 267

Introduction

The development of a root system capable of anchoring the

shoot and the ability to uptake sufficient water and nutrients

from the soil is essential for the survival of most terrestrial

plants. The root zone soil constraints prevent the development

of root system and eventually the crop yield (Rengasamy and

Vadakattu, 2002). The growth of roots and shoots are often

slower when plants are grown in soil of large bulk density

(Voohees, 1992). In such soils, various physical (availability

of oxygen and water, and mechanical impedance) and biotic

factors may limit root and shoot growth. However, the mecha-

nical impedance (resistance pressure encountered by growing

roots) of the soil is often the single most important factor that

can limit root and shoot elongation. It increases with the in-

crease in soil dry bulk density (e.g., due to compaction) and

also increases as the soil matric potential decreases. Unless

roots are able to exploit the soil structural features, their growth

rate is reduced as mechanical impedance is increased (Townend

et al., 1996). Clark et al. (2001) showed that pea roots are

capable of sensing a partial increase in mechanical impe-

dance that can increase the turgor of seedlings but there was

still some reduction in root growth. In natural conditions, plant

roots invariably encounter some degree of mechanical resis-

tance to their penetration through the soil.

It used to be generally believed that roots are unable to pene-

trate into rigid pores that were narrower than their normal dia-

meter. More recent studies have revealed that roots can grow

into rigid pores that are smaller than their diameter (Bengough

et al., 1997). In soils, roots can often exploit cracks, voids and

larger pores, or enlarge smaller pores by displacing soil par-

ticles. On encountering mechanical impedance, root cell divi-

sion and elongation are decreased (Eavis, 1967). Root dia-

meter just behind the apex can increase and the production of

lateral roots may also be increased, with laterals emerging

closer to the apex (Atwell, 1988; Goss, 1977). Restricting the

soil volume explored by roots reduces shoot growth (Young,

et al., 1997; Passioura, 1991; Carmi and Heuer, 1981). This is

often accompanied by an increased root : shoot mass ratio

(Cook et al., 1996; Blaikie and Mason, 1993). Slower shoot

growth of wheat seedlings was reported in compacted soils

while plants were still in the seed reserve-dependent growth

stage (Nabi and Mullins, 2001; Masle et al., 1990). Dawkins

et al. (1983) observed smaller shoot : root ratio in peas when

roots were growing in compacted than in loosened soil. Masle

and Passioura (1987) grew wheat seedlings for 22 d in small

cores of compacted soil and found that shoot growth and

development were severely restricted. Andrade et al. (1993)

also found that strong soil affected shoot growth early in sun-

flower. Montagu et al. (2001) found that soil compaction

decreased root growth in broccoli.

Roots experience mechanical impedance as they elongate in

the soil and the decrease in their growth rate is due to the

force required to displace soil particles. Strong soil can be a

Pak. J. Sci. Ind. Res. 2005 48(4) 268-273


Restriction to Root and Shoot Growth Limits Their Growth Rates and

Changes the Morphology of Cotton Seedlings During Emergence

Ghulam Nabi*a and C. E. Mullins

b

aLand Resources Research Programme, National Agricultural Research Centre, Islamabad-45500, PakistanbDepartment of Plant and Soil Science, Cruickshank Building, University of Aberdeen, AB24 3UU, UK

(received October 7, 2003; revised April 20, 2005; accepted July 7, 2005)

Abstract. Pre-germinated cotton seedlings were grown under laboratory conditions to determine the affect of root and/or

shoot impedance on their growth. The treatments studied were: (i) both shoot and root unimpeded, (ii) shoot impeded and

root unimpeded, (iii) root impeded and shoot unimpeded, and (iv) both root and shoot impeded. Impeding the root alone,

or root and shoot together, significantly (P < 0.05) reduced axial root length, total root length, and increased root diameter.

The axial root length was reduced by 55%. The number of root laterals was not affected by impedance but lateral spacing

was reduced significantly. Root diameter was increased in treatments where only roots had been impeded. Shoot diameter

was significantly (P < 0.05) greater in the root and shoot impeded treatments. Shoot length was reduced by 15% when only

the shoots were impeded, while 38% reduction was noted when both root and shoot were impeded. Shoot impedance did

not cause any significant effect on the root growth rate when roots were unimpeded. In terms of shoot length, root

impedance had no effect on shoot length, although the combined effect of root and shoot impedance was greater than

shoot impedance alone.

Keywords: mechanical impedance, root/shoot length, root/shoot diameter, restricted root elongation, root development

268

serious problem in agriculture, as it can restrict access of root

system to water and nutrients and can thus decrease crop yields.

In the field, topsoil may be strong due to lack of tillage, while

tillage operations can compact soil just beneath the plough

layer and can lead to the formation of hardpans. Some sub-

soils are naturally strong due to the presence of gravely hori-

zons (Babalola and Lal, 1997), or clay pans (Clark et al., 1998).

Also, under field conditions, emerging shoots may encounter

compaction such as that due to raindrop impact and soil crus-

ting. The present study was, therefore, conducted to deter-

mine the effect of soil compaction on the growth of shoots as

well as roots in cotton during emergence.


Pre-germinated seedlings of cotton, MNH-147, were grown

for 72 h in 300 mm long cylinders having 75 mm internal dia

in a growth cabinet maintained at 32 °C. Long cylinders (150

mm) were packed with soil, either at a bulk density of 0.88

Mg m-3 (mega gram per cubic meter) to represent unimpeded

soil, or 1.25 Mg m-3 to represent an impeding soil. Two cylin-

ders, either of the same, and/or different bulk densities (as

required by the treatment), were joined one above the other to

make a 300 mm length. The four treatments, three replicates

each, were: (i) both shoots and roots unimpeded (RuSu), (ii)

roots unimpeded and shoots impeded (RuSi), (iii) roots impe-

ded and shoots unimpeded (RiSu), and (iv) both roots and

shoots impeded (RiSi).

A sandy clay-loam (Carpow Series) topsoil (0-10 cm) was used

as the growth medium. The air-dried soil was sieved and agg-

regates between 1 and 3.35 mm diameter were retained. The

prepared soil contained 0.21% organic matter and its particle

size distribution was 20.6% clay, 18.0% silt (2-60 mm) and

61.4% sand. Water retention curve of the soil was determined

following standard procedures using a tension table and

pressure plate apparatus (Townend et al., 2001). Based on

this curve, the soil was wetted with nutrient solution to a

matric potential of -10 kPa (kilo Pascal) before packing. The

cylinders were packed at bulk densities of 0.88 and/or 1.25

Mg m-3. They were packed in layers, in 20 mm increments, to

the required bulk density. After packing, two pre-germinated

seedlings of cotton (5 mm long radicle) were transplanted at

the juncture of the two cylinders, before joining them. Bead

thermistors were placed inside the cylinders, and attached to

a data logger (Skye Data Hog) to log temperature at 5 min

intervals and record hourly averages. The cylinders were kept

in a growth cabinet maintained at 32 °C. After 72 h, the cylin-

ders along with intact seedlings were removed from the cabi-

net and penetration resistance (PR) in each cylinder was re-

corded with a portable cone pentetrometer (300 mm long

recessed shaft, steel cone of 3 mm dia and 15° semi-angle).

Two trials were run for each treatment in duplicate cylinders

and mean values were computed. After the penetration resis-

tance measurements, the contents of each cylinder along with

seedlings were removed carefully and the soil was gently

separated from the seedlings. The soil was then placed in jars

to determine its gravimetric water content and matric potential

by the filter paper method (Deka et al., 1996). The roots were

separated from the shoots. The shoot length, diameter, fresh

weight, axial root length, number of root laterals, root dia, and

fresh weight of roots were recorded.

The roots were stained in a 0.01% methyl violet solution and

used for measuring the total root length using a DIAS image

analyser with the root measurement system software, Version

1.6 (Skye Ltd., Llandrinod, Wells, UK). High quality photo-

copies of the stained roots were used for measurement. After

root length measurements, the roots were dried at 80 °C to

determine their dry weight.

The data obtained were statistically analysed for the four treat-

ments and three replications with completely randomised

design in statistical software Minitab for Windows version

10.5 (Minitab Corporation Inc., USA). Treatment means were

compared, using the least significant difference test. Where

this was not met, standard error of means and coefficient of

variation were computed to compare the treatment means.


Growth conditions. Average soil temperature, near the seed-

lings, during the 72 h growth period was ~31.8±1 °C. Tempera-

ture remained fairly constant during the growth period. The

soil moisture content at the start of the experiment was 26.82

g/100 g that corresponded to matric potential of -12.87 kPa.

At harvest time, the moisture content was 22.1 g/100 g that

corresponded to - 45 kPa. Since the moisture content changed

only slightly and the matric potential remained in a range in

which water was readily available, it can be assumed that it

had negligible effect on growth. Penetration resistance in the

cylinders, with intact seedlings, was measured before harves-

ting (i.e., after 72 h). Resistance in the unimpeded sections of

cylinders was ~0.02±0.01 MPa (mega Pascal), while in the

impeded sections it was ~1±0.2 MPa.

Effect on root growth. The axial root length was significantly

(P < 0.05) reduced when root systems were mechanically

impeded (RiSu and RiSi; Table 1a). A reduction of about 55%

in the axial root length of RiSu, relative to the control (RuSu)

was observed, which corresponded to a reduction in growth

rate from 2.11 to 0.96 mm/h. Impeding the shoot alone (RuSi)

did not affect root length. However, impeding the root with or

269Restricting Root and Shoot Growth Changes Their Morphology

without shoot significantly (P < 0.05) reduced the total root

length. The total root length was reduced by 37% in seedlings

whose root system was impeded (RiSu) and (RiSi), as com-

pared to the control (RuSu). Impeding the shoot but not the

root (RuSi) also resulted in a significant reduction (17%) in the

total root length, as compared to the control (RuSu). Late-ral

spacing was significantly reduced, but the number of late-ral

roots was not reduced by mechanical impedance compared to

the control.

Effect on shoot growth. Impeding the shoot (RuSi and RiSi)

significantly (P < 0.05) reduced the shoot length (Table 1b).

Seedlings in unimpeded treatment (RuSu) had 1.6 times lon-

ger shoots than in the impeded (RiSi) treatment. When only

the shoot was impeded (RuSi), a reduction of 15% was obser-

ved in its length. However, when both root and shoot were

impeded, it resulted in a 38% reduction. Fresh shoot mass was

significantly (P < 0.05) decreased, as compared to the control,

only in those treatments in which the root system was im-

peded (RiSi and RiSu; Table 1b). A reduction of 18% was ob-

served when the root and shoot systems were impeded (RiSi),

but only 13% when only the root system was impeded (RiSu).

Shoot mass was less affected when only the shoot system

was impeded (RuSi). Shoot diameter was significantly (P

< 0.05) greater in the root and shoot impeded (RiSi) seedlings

than where only the root system had been impeded (RiSu).

However, when the root system alone was impeded, shoot

Table 1. Root Growth (a) and shoot growth (b) of cotton seedings, as affected by root-shoot interactions, with and without

mechanical impedance

a. Root growth

Treatments Axial Root Total Length of Spacing of Root diameterroot length growth rate root length root laterals laterals Dia Performance(mm) (mm/h) (mm) (mm) (mm) (mm) relative to control

RuSu 152a ± 7 2.11 ± 0.01 370

a ± 18 245a ± 16 2.53

a0.94

ccontrol

(control) (11) (11) (11) (16)

RuSi 146a ± 7 2.03 ± 0.01 330

b ± 18 183b ± 21 2.67

a0.97

c103

(12) (12) (13) (28)

RiSu 69b ± 12 0.96 ± 0.16 237

c ± 26 168b ± 16 1.57

b1.11

b118

(41) (41) (27) (24)

RiSi 69b ± 5 0.95 ± 0.07 229

c ± 10 161b ± 14 2.17

a1.23

a131

(18) (18) (10) (21)

LSD (P < 0.05) 24 - ** ** ** 0.12 -LSD (P < 0.01) 32 0.16 - -

b. Shoot growth

Treatments Shoot Shoot Fresh Dry Shoot diameterlength growth rate shoot mass shoot mass Dia Performance(mm) (mm/h) (mg) (mg) (mm) relative to control

RuSu 50a ± 1.2 0.69

a ± 0.02 391 ± 24a

21± 1 3.08 ± 0.04b control(control) (5) (7) (15) (6) (3.4)

RuSi 42b ± 1.76 0.59

b ± 0.04 358 ± 28a

19 ± 2 3.24 ± 0.12ab

106.2(10) (15) (20) (28) (8.6)

RiSu 50a ± 1.76 0.68

a ± 0.04 340 ± 14b

21 ± 2 3.02 ± 0.06b

98(8) (16) (10) (23) (4.6)

RiSi 31c ± 2.26 0.44

c ± 0.03 321 ± 15b

20 ± 1 3.54 ± 0.16a

114.9(17) (18) (12) (8) (0.11)

LSD (P < 0.05) 7.29 0.10 * NS 3.08 ± 0.04b controlLSD (P < 0.01) 9.90 0.14 (3.4)

RuSu = both root and shoot unimpeded; RuSi = root unimpeded and shoot impeded; RiSu = root impeded and shoot unimpeded;

RiSi = both root and shoot impeded; LSD = least significant difference, values are mean ±1 standard error, se (coefficient of

variation, CV%), n = b; values sharing the same letter in each column do not differ significantly at P < 0.05; NS = non-significant

270 G. Nabi and C. E. Mullins

diameter was not reduced substantially. Root diameter was

significantly (P < 0.05) increased in root impeded treatments,

as compared to the control, while shoot diameter was signifi-

cantly (P < 0.05) increased in the shoot impeded treatments

(RuSi and RiSi), as compared to the control seedlings.

Impeding the root alone or root and shoot together, signifi-

cantly reduced the axial root length and total root length, and

increased the root diameter. Impeding the shoot alone reduced

total shoot length, but did not significantly affect axial root

length, the number of root laterals, or the shoot diameter. The

only major effect was reduction in the length of laterals, which

was quite substantial.

Since factors like temperature, nutrients and water availabi-

lity remained unchanged and were not limiting during the expe-

riment, it is clear that these responses were directly related to

the increased mechanical impedance experienced by the roots

and shoots, in agreement with earlier studies which reported

that the growth rates were reduced when plants were grown in

compacted soils (Young et al., 1997; Bennie, 1996; Cook et al.,

1996; Kirkegaard et al., 1992). However, when plants were

grown in compact soil it was difficult to rule out completely,

the transient shortage in water or nutrients, due to reduced

root extension. Such shortages could cause a down regulation

of shoot growth resulting in the shoot maintaining critical

nutrients and water status. Since in this experiment the soil

matric potential ranged between ~ -13 to - 45 kPa, and the soil

used was wetted with nutrients solution and further the growth

was monitored only for 72 h, for which seed reserves can pro-

vide essentially required nutrients for growth, it can be confi-

dently ruled out that decreased supply of water or nutrients to

the root system had caused reduced growth and their elonga-

tion. Further, the study indicated that the root growth reduc-

tion due to impedance was independent of shoot growth. These

observations do not agree with those of Rwehumbiza (1994),

who found that in 8-d old sorghum plants, shoot impedance

increased root elongation rate and number of root laterals.

Masle and Passioura (1987) observed in 22-d old wheat plants

that the extension rate in emerged shoots was significantly

reduced when only the root systems were impeded. However,

these studies do not mention about the effect of root and

shoot impedance on pre-emerged seedlings. In this study,

plants were harvested after a 72 h of growth period. Since it is

likely that plant growth will differ at different growth stages,

depending upon such factors as the availability of water,

nutrients and soil aeration, the growth response of any plant

part to mechanical impedance may not be the same during

the whole of its life-time. The plant response in later stages,

furthermore, may be quantitatively different when factors like

a greater demand for water and nutrients arises for growth

and maintenance of the seedling. Additionally, in the seed-

lings in which growth is totally or mainly dependent on the

release of seed reserves, the mechanism which operates

during the later growth stages to maintain a given allometry

may be inactive.

Root lengths were significantly reduced when roots alone

(RiSu), or roots and shoots (RiSi) were both impeded. Similarly,

shoot lengths were both reduced when shoot (RuSi), or root

and shoot (RiSi), were impeded. These results are in agree-

ment with those of Russell and Goss (1974). Sharp (1990)

suggested an increased demand for photosynthate to sup-

port a preferential growth of roots and, therefore, a continued

exploration of the soil for water with increased mechanical

impedance to roots. Although these plants were pre-emer-

gent, the roots and shoots are still competing for a limited

supply of reserves. Comparing the unimpeded root and the

impeded shoot (RuSi), and the impeded root and shoot (RiSi)

treatment, it is possible that in the RuSi treatment the shoots

were experiencing a low impedance zone before growing into

the impeding soil. This means that the extra reduction in shoot

growth, which is apparently due to root impedance, could be

an experimental artefact.

Mechanical impedance is known to reduce root elongation

rates, thus reducing the volume of soil that the root system can

exploit (Veen, 1982; Russel and Goss, 1974). Reduced shoot

growth of impeded plants has, therefore, often been associ-

ated with a restricted root volume and an inadequacy in sup-

plying water and nutrients to the plant (Rahman et al., 1999;

Oussible et al., 1992; Atwell, 1990; Boone and Veen, 1982).

The studies on the effect of restricted root volume on shoots

under the conditions of no water or nutrients stress have shown

that reductions in shoot growth still occur (Krizeck et al., 1985;

Peterson et al., 1984), and the reduction of certain regulatory

substances has been suggested to be responsible for restric-

ting shoot growth.

Other growth characteristics, like the number of root laterals,

and fresh and dry root biomass were not significantly affected

either in the root or in the root and shoot impeded treatments.

Similar observations have been reported by Tsegaye and

Mullins (1994). This suggested that these parameters are in-

sensitive to mechanical impedance. However, root diameter

was significantly increased in response to mechanical impe-

dance. The increase in root diameter was in line with the

results of Young et al. (1997) and Wilson et al. (1977). When

apical extension of the root is restricted, there are more cells

per unit root length and individual cells become shorter, but

may expand laterally resulting in increased diameter. There

was a significant increase in the shoot diameter in response to


mechanical impedance. Shoot diameter was increased when

shoot alone (RuSi) or shoot and root (RiSi) were impeded.

When only the root system was impeded, shoot diameter was

not affected significantly. The explanations given above for

increase in root diameter hold true for shoot diameter as well.

The length of root laterals decreased when the shoot was

impeded. However, there was no evidence of a “root signal-

ling” response resulting in reduced shoot growth in response

to high soil strength.

Conclusion

Roots that were impeded had the axial growth rate reduced by

55%, irrespective of whether or not shoots were impeded. With

unimpeded roots, shoot impedance did not cause any signifi-

cant effect on the root growth rate. In terms of shoot length,

the root impedance alone had no effect, although the com-

bined effect of root and shoot impedance on the shoot length

was greater than the shoot impedance alone. Similarly, in terms

of the length of the root axis, shoot impedance had no effect,

although there was a considerable reduction in the length of

the laterals.

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Evaluation of Nutritive Properties of the Large African Cricket(Gryllidae sp)

Yemisi A. Adebowalea, Kayode O. Adebowale*b and Michael O. Oguntokunc

aDepartment of Food Science and Technology, Federal University of Technology, Akure, NigeriabDepartment of Chemistry, University of Ibadan, Ibadan, Nigeria

cDepartment of Animal Production and Health, Federal University of Technology, Akure, Nigeria

(received August 19, 2003; revised May 17, 2005; accepted June 23, 2005)

Pak. J. Sci. Ind. Res. 2005 48(4) 274-278

Abstract. The large African cricket (Gryllidae sp) was subjected to standard analytical procedures to determine its

proximate composition, functional properties, amino acids spectrum, in vitro protein digestibility, and nutritionally

valuable minerals. The moisture was low (2.13-3.48%), while the protein content was high (65.95%) in the male cricket and

65.11% in the female cricket). Seventeen amino acids were detected. The essential amino acids contributed 46.1-47.8% of

the total amino acid content. Results of the in vitro protein multienzyme digestibility indicated high digestibility

(90.7-94.7%). The amino acids scores were also favourable. The crude fibre and fat contents were fairly high, while the total

carbohydrates were low (8.26-12.49%). The carbohydrates fraction contained 85.9-88.0% carbohydrates as stored glyco-

gen. Phosphorus was the highest mineral in the ash (180.92 mg per 100 g), while the concentration of zinc was the lowest

(1.46 mg per 100 g). Copper, manganese, nickel and lead were below the detection limits. Observations on the functional

properties revealed low gelation, oil absorption, and emulsion capacity and stability. The effect of pH on the protein

solubility showed that the lowest solubility occurred at the pH value of 4.0, while maximum solubility was recorded at the

pH values of 6 and 7.

Keywords: Gryllidae sp, nutritional properties, functional properties, large African cricket, new protein

source, Gryllidae amino acids

Interoduction

The large African cricket (Gryllidae sp) is one of the most

notorious and highly destructive pest of many economic crops

in the Western, Central and Eastern parts of Africa (Daramola,

1974). Several authors have observed that this pest attacks

animals, herbs and plantation crops at their nursery stages

(Hewuirlt, 1980; Daramola, 1974; Kaufmann, 1965; Chapman,

1962). This field cricket has been reported to feed on a wide

variety of food plants, such as banana, and tea and coffee

seedlings (Toye, 1982), and some commercial crops, includ-

ing Amaranthus sp, Rosa sp, Mangifera indica, Theobroma

cacao and Cola sp. The female insects, which have an elon-

gated banana-shaped abdomen, lay eggs in batches, approx

2000 eggs in a batch. At room temperature (30±2 oC), the eggs

hatch in 10-12 days. The development of the nymphs takes

40-60 days. The adults, which generally live for 2 to 3 months,

are omnivorous. The crickets grow up to 2-3 inches in length.

The females have long ovipositor, about 18 mm in length.

In view of the nutritive properties of similar insects, a study

on the chemical composition of the large African cricket was

given consideration. These studies aimed at solving two prob-

lems. Firslty, to reduce the destructive effects of this insect in

terms of reduction in their population size to alleviate the

problem. Secondly, to overcome the problem of shortage of

high quality protein sources and the prevalence of protein

malnutrition in the developing countries. It was against this

background, that the nutritional properties of the adult male

and female large African cricket (Gryllidae sp) was investi-

gated.


Materials. The adult male and female crickets (Gryllidae sp)

were collected during the wet season (April to August), when

their population is at the peak. They were collected from

Ibadan, Ile-Ife, Ilesa, Akure, Iseyin, Tede and the entire

South-western Nigeria. The insects were demobilised by as-

phyxiating them in deep freezers at - 20 oC. Their alimentary

canals were carefully removed and the samples subsequently

dried to constant weight in an oven at 40 oC. The dried samples

were pulverised into fine powder using a Christy laboratory

mill. The samples were stored in dry air-tight plastic contain-

ers at 4 oC.

Analytical methods. Proximate analysis of the samples for

moisture, crude fibre, protein and ash contents was carried

out according to AOAC (1990). Carbohydrate fractions were

*Author for correspondence;

E-mail: [email protected]

274

analysed using the method described by Esuoso and

Odetokun (1995). Mineral elements were determined by pre-

ashing 2 g of the samples in a silica crucible and heating on a

gas flame for 10 min. The samples were transferred to a muffle

furnace and incinerated at 900 oC for 6 h. The residual white

ash was dissolved in 0.1 M HNO3. The metals were deter-

mined by atomic absorption spectrophotometer (Perkin-Elmer

8650). Phosphorus was estimated by using the vanado-mo-

lybdate method (Vogel, 1978).

The method of Coffman and Garcia (1977), with slight modi-

fication, was employed for the determination of gelation ca-

pacity. Sample suspensions were prepared in distilled water.

Ten ml of each suspension was poured into a test tube and

slowly heated for 1 h in a boiling waterbath, followed by rapid

cooling in a waterbath of cold water for 10 min. All the tubes

were further cooled in a waterbath at 4 oC for 2 h. The least

gelation concentration was determined as that concentration

when the sample from the inverted tube did not fall down or

slip. Water and oil absorption capacities were determined by

the method of Sathe and Salunkhe (1981). The foaming capac-

ity for each sample suspension was determined by the method

described by Narayana and Narsinga (1982), while the emul-

sion capacity and stability was determined by the method of

Yasumatsu et al. (1972). All determinations were carried out in

triplicate.

For amino acids determination, the samples were hydrolysed

with 6 N HCl for 3 h at 150 oC in vacuo.The amino acids solu-

tion was derivatised with an amino acid derivatiser/analyser

model 420A using phenylisocyanate in the presence of

diisopropylethylamine. The derivative was analysed with a

reverse phase high performance liquid chromatography (RP-

HPLC) microseparation system model 130A, consisting of

pump type P680, ASI-100T automated sample injector and PDA-

100 photodiode array detector.

The in vitro multienzyme digestibility was carried out by the

method of Hsu et al. (1977). Fifty ml of aqueous suspension

of the samples (6.25 mg sample per ml) in distilled water was

adjusted to pH 8.0 with 0.1 M HCl and/or 0.1 M NaOH, while

stirring on a waterbath maintained at 37 oC. The multienzyme

solution containing 1.6 mg trypsin, 3.1 mg chymotrypsin and

1.3 mg peptidase was maintained in an ice-bath and adjusted

to pH 8.0 with 0.1 M HCl and/or 0.1 M NaOH. The enzymes

were purchased from Sigma Chemical Company (St. Louis,

MO, USA). A 5 ml sample of the multienzyme solution was

added to the sample suspension with constant stirring at

37±2 oC. The pH of the suspension was recorded for 15 min

after the addition of the multienzyme solution. The in vitro

digestibility was calculated by using the regression equation

of Hsu et al. (1977). The enzyme activity was determined by

using casein of known in vivo apparent digestibility.


The proximate chemical composition of the large African

cricket is shown in Table 1. The moisture content of the crick-

ets was low (2.13-3.48%). The low value may be regarded as

advantageous in terms of the shelflife of the insects. The crude

protein content was very high ( 65.95% in the male crickets

and 65.11% in the female crickets). The values were higher

than that reported for other insects and animal sources. Ter-

mites were reported to contain 37.39%, while the female repro-

ductive termites contained 56.24% crude protein (Ajakaiye

and Bawo, 1990). Similar results have been obtained in similar

insects (Udoh et al., 1985; Mba, 1980). Therefore, based on

the average human protein intake of 23-50 g, as recommended

by the National Research Council (1974), the large African

cricket may contribute significantly in alleviating the problem

of protein malnutrition in the Third World and developing

countries.

The amino acids composition of the male and female crickets

is shown in Table 2. The contant of essential amino acids was

high in samples of both the sexes (448.6 and 491.9 mg/g in

the male and female crickets, respectively). Aspartic acid and

glutamic acid occurred in the highest concentration in the two

categaries of samples. The nutritive value of proteins depends

primarily on their capacity to satisfy the needs of nitrogen

and essential amino acids (Pellet and Young, 1980). The ali-

phatic amino acids (isoleucine, leucine, valine), which consti-

tute the hydrophobic regions of the protein were fairly high in

both the sexes of the cricket samples. This implies better

emulsifying properties of these samples. In addition, the es-

sential amino acid (phenylalanine) and tyrosine were high in

the two types of samples. These amino acids are precursors

for the synthesis of tyrosine, epinephrin and thyroxin, which

are important constituents of the body’s endocrine system

(Robinson, 1987). A comparison of the amino acids composi-

National quality of Large African Cricket (Grylliae sp) 275

Table 1. Proximate chemical composition (g/100 g) of male

and female crickets (Gryllidae sp)

Composition* Male Female

Moisture 2.13 ± 0.05 3.48 ± 0.02

Crude protein (N x 6.25) 65.95 ±0.14 65.11 ±0.07

Crude fat 7.25 ±0.03 10.78 ±0.05

Ash 5.68 ±0.07 4.31 ± 0.04

Crude fibre 6.51 ±0.12 8.06 ± 0.10

Carbohydrates** 12.49 ±0.08 8.26 ± 0.06

*values are given as mean ± sd of triplicate determinations (dry wt

basis); **carbohydrates expressed as glycogen

tion in both the cricket samples with the FAO/WHO reference

values (FAO/WHO, 1985), indicated that the values obtained

during this study were higher than the values recommended

for pre-school and school going children. This implies that

the cricket samples are a good source for essential amino ac-

ids and may be used for the fortification of cereal-based foods,

which are particularly deficient in lysine. The quality of di-

etary proteins can be measured in many ways. There is a gen-

eral acceptance that this value is a ratio of the available amino

acids in the food or diet, as compared with the daily require-

ments. According to the provisional amino acids scoring pat-

tern and amino acids score reported in Table 3, the amino

acids score compared favourably with the suggested refer-

ence standards. Perhaps the limiting amino acids would be

leucine and threonine in the female cricket samples. Other-

wise, the amino acids score was adequate in both the male

and female samples. The bioavailability of the protein was

also examined and the results are presented, along with the

composition of the amino acids, in Table 2. The digestibility of

proteins and bioavailability of their constituent amino acids

is an important factor which determines the protein quality

(Suman et al., 1992; Hsu et al.,1977). This is true as all pro-

teins are not digested, absorbed and utilised to the same ex-

tent. The multienzyme in vitro procedure has shown good

correlation with in vivo methods. As per observations, the

male crickets recorded protein digestibility of 90.7± 1.4%,

while the female crickets had protein digestibility of 94.7±

1.4%. These values are higher than those already reported for

legumes and animal protein sources (Adebowale et al., 2005;

Lawal and Adebowale, 2004; Adebowale and Lawal, 2003;

Oshodi et al., 1995; Robinson, 1987; Pellet and Young, 1980;

Hsu et al., 1977).

The crude fat content of the cricket samples, as reported in

Table 1, indicate that the male crickets contained 7.25%, while

the female crickets contained 10.78% crude fat. These values

are lower than the values obtained for every developmental

stage of the variegated grasshopper as reported by Adedire

and Ayesanmi (1999), and Ajakaye and Banwo (1990). How-

ever, it was higher than those reported for animal foods, such

as periwinkle (Pachilania byronensis), Crayfish (Paramontes

sp), snail (Vivapara quadarato) and dogwhelk (Thais

cattifera) (Udoh et al., 1985; Mba, 1980).

The crude fibre values were fairly high, while the carbohy-

drate values were much lower than the values reported by

earlier authors (Udoh et al., 1995; Ajakaye and Bawo,1990).

The composition of the carbohydrates fraction is presented

in Table 4. Sugars (glucose, sucrose, fructose and maltose)

were detected in concentrations between 0.7-6.1%. The

carbohydrates fraction contained between 85.9-88.0%

glycogen.

The total ash contents of the cricket samples were generally

low (5.68% for the male crickets and 4.31% for the female crick-

ets). These values are comparable to the values obtained for

the termite, Trinevitermes germinatus, and Thais cattifera

Table 2. Amino acids composition and in vitro digestibility

of the male and female crickets (Gryllidae sp)

Amino acid Composition (mg/g crude protein)

Male Female

Aspartic acid 120.5 115.8

Glutamic acid 110.7 100.6

Serine 90.7 81.5

Glycine `42.3 55.8

Histidine 37.6 46.2

Argininea 48.9 51.6

Alanine 50.6 58.4

Tyrosine 37.3 57.6

Valinea 70.9 84.7

Methioninea 20.1 59.6

Cystine 22.0 10.5

Isoleucinea 55.8 50.9

Leucinea 79.6 71.5

Phenylalaninea 51.6 58.5

Threoninea 49.6 41.7

Lysinea 70.1 73.4

Tryptophan 10.5 11.7

Total essential amino acids (TEAA) 446.6 491.9

Total non-essential amino acids (TNEAA) 522.2 538.1

TNEAA (% of total amino acids) 53.9 52.2

TEAA (% of total amino acids) 46.1 47.8

In vitro enzyme digestibility( %)* 90.7±1.4 94.7±1.4

aessential amino acids; *digestibility of casein as reference protein

(standard protein) = 94.3%

*suggested level (FAO/WHO, 1985); **amino acids score = mg amino

acids per g test protein/mg amino acids per g reference protein

Table 3. Provisional amino acid scoring pattern and amino

acids scores of the male and female crickets (Gryllidae sp)

Amino FAO/ Amino acids Amino acids

acid WHO content score**

Present work Present work

Male Female Male Female

Isoleucine 40 55.8 50.9 1.40 1.27

Leucine 70 79.6 71.5 1.14 1.02

Lysine 55 70.1 73.4 1.27 1.33

Methionine

+cystein 35 42.1 70.1 1.20 2.00

Phenyalanine

+tryptophan 60 88.9 116.3 1.48 1.94

Threonine 40 49.6 41.7 1.24 1.04

Valine 50 70.9 84.7 1.42 1.69

276 Y. A. Adebowale et al.

level*

(Udoh et al., 1985; Mba, 1980). The ash contents represented

the mineral elements present in the samples. The ash contained

a number of nutritionally valuable minerals. The Na/K retio in

the ash was particulary favourable. It is desirable that the Na/K

ratio should approach unity to prevent high blood pressure

(Esuoso et al., 1998). The results of mineral composition analy-

sis are presented in Table 5. The concentration of minerals was

generally low in both the male and female crickets. Lead and

nickel contents were below the detection limits, while the con-

centration of manganese was below 0.9 mg/100 g.

The results of the functional properties of the cricket samples

are shown in Table 6. The least gelation concentration of the

crickets was 10.0%. This high value may be advantageous for

some food products. It can thus be incorporated into foods

which require gelling and thickening. The water absorption

capacity of the crickets was 238.47%. This value is beneficial

for food products where the retention of moisture is desirable

during cooking, which include meat and baked products

(Altchul and Wilcke, 1958). The African giant cricket may,

therefore, be useful for these products. The oil absorption

capacity was 202.1%. This value made the cricket samples

appropriate for incorporation in food products involving fat

absorption, such as bakery products.

The emulsion capacity and stability were 46.8% and 8.5%,

respectively. The capacity of protein to aid the formation and

stabilisation of emulsion is important for incorporation in

foods, such as in cake-baking, coffee whiteners, milk mayon-

naise, salad dressing, comminuted meat, and frozen desserts.

The cricket samples may, therefore, be used in these food

products. The foaming capacity and stability was fairly good.

The effect of pH on the protein solubility is shown in Table 7.

The lowest solubility was recorded at the pH value of 4.0,

while the maximum solubility was noted at the pH values

between 6.0 and 7.0. This is advantageous for incorporation

in food products which require shightly acidic-neutral envi-

ronments.

Table 5. Nutritionally valuable minerals and trace matals

(mg/100 g) of male and female crickets (Gryllidae sp)

Minerals/

trace metal Male Female

Calcium 9.45 1.34

Sodium 60.18 73.87

Potassium 56.35 93.50

Magnesium 50.63 67.69

Phosphorus 180.92 141.87

Iron 20.08 18.33

Zinc 1.46 2.75

Copper nd* nd

Manganese < 0.9 < 0.9

Lead nd nd

Nickel nd nd

*nd = not detected (below detection limit)

Table 7. The effect of pH on the protein solubility of the large

African crickets (Gryllidae sp)

pH value Protein solubility (%)

1 25.84

2 24.50

3 19.38

4 12.92

5 19.38

6 28.84

7 28.90

8 27.48

9 27.01

10 26.20

11 25.95

12 27.30

Table 6. Some functional properties (%) of the large African

crickets (Gryllidae sp)

Functional properties Concentration (%)*

Least gelation concentration 10.00 ± 0.00

Water absorption capacity 238.47 ± 0.05

Oil absorption capacity 202.05 ± 0.10

Emulsion capacity 46.81 ± 0.00

Emulsion stability 8.50 ± 0.10

Foaming capacity 6.00 ± 0.00

Foaming stability (after 2 h) 3.05 ± 0.02

*mean±sd of three determinations

Table 4. Percentage carbohydrates in the male and female

crickets (Gryllidae sp)

Carbohydrates Composition (%)*

Male Female

Sugars 6.1 ±0.3 5.2 ± 0.2

Glucose 4.1 ±0.1 2.1 ±0.0

Sucrose 0.7 ±0.0 0.2 ± 0.0

Fructose 0.9 ±0.0 1.0 ± 0.0

Maltose 3.7 ±0.1 4.4 ± 0.4

Polysaccharides** 85.9 ±1.5 88.0 ± 2.6

*mean±sd of three determinations; **carbohydrates expressed as

stored glycogen

National quality of Large African Cricket (Grylliae sp) 277

Acknowledgements

The authors are grateful to The Abdus Salam International

Centre for Theoretical Physics, ICTP, Trieste, Italy, and the

Swedish International Development Agency, SIDA, for their

support.

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278 Y. A. Adebowale et al.

Prevalence of Intestinal Helminth Parasites of Dogs in Lagos, Nigeria

Comfort Adejoke IbidapoaDepartment of Zoology, Lagos State University, Faculty of Science, PMB 1087, Apapa, Lagos, Nigeria

(received September 10, 2004; revised January 15, 2005 accepted May 16, 2005)

Pak. J. Sci. Ind. Res. 2005 48(4) 279-283

Abstract. A survey of 310 dogs (164 males and 146 females) for infections with intestinal helminth parasites was carried

out in Lagos, Southern Nigeria.Out of these, 175 were pet dogs and were routinely checked by veterinary doctors, while

the remaining 135 were strayed-dogs, which did not receive medical check-up. Strayed-dogs were significantly more

infected (77.8%) than the pet dogs (12.0%) at P < 01. The analysis of infection pattern, by age, revealed that among the

strayed dogs, 92.2%, 76.6% and 59.5% of the puppies, young and adult dogs, respectively, were found infected. Corre-

sponding prevalencies among the pet puppies, young and adult dogs were 21.5%, 7.1% and 5.0%. Helminth ova recovered

from the strayed-dogs included Toxocara canis (47.6%), Ancylostoma caninum (41.9%), Dipylidium caninum (37.9%) and

Trichuris vulpis (20.9%). Helminth ova recovered from the care-receiving dogs were Toxocara canis (5.8%), Ancylostoma

caninum (3.2%) and Dipylidium caninum (2.1%). Female strayed-dogs, with a prevalence of 89.7%, were significantly

more infected than their male counterparts (70.9%). Strayed-puppies, with an infection rate of 92.2%, were significantly

more infected than strayed young and adult dogs. Public health implications of these results, with particular reference to

zoonotic transmission, was highlighted.

Keywords: instestinal helminths, dog intestinal parasites, helminth ova, infected strayed-dogs, helminths of dogs

Introduction

Dogs have remained among the closest associates of hu-

man beings since ancient times. The usefulness of dogs for

hunting, anti-crime and security purposes, food, religious

rituals, and as companion have endeared them to man. How-

ever, in spite of the listed usefulness, the dogs, especially the

free-roaming types, have been incriminated in the transmis-

sion of zoonotic diseases, such as cutaneous larva migrans

(Dryden, 2002; Schantz, 2002; Kagira and Kanyari, 2000;

Kazacos, 2000; Burnham, 1998; Blagburn et al., 1997; 1996;

Hendrix et al., 1996). There is paucity of data on the endopara-

sites of dogs in Nigeria. The existing information is limited to

the studies carried out in Ibadan (Olufemi and Bobade, 1979;

Idowu et al., 1977; Oduye and Olayemi, 1977; Oduye and

Otesile, 1977), in Zaria (Dada and Belino, 1979; van veen

Schillhorn and Adeyanju, 1979), and in Abeokuta (Mafiana

et al., 1993). Legislation on the free-roaming animals is not

enforced in Nigeria. The flooding of Nigerian towns and

streets with these animals, therefore, continues unabated.

The objective of the present study was to determine the

prevalence of helminth parasitic fauna of dogs in the Lagos

area, thus adding to the existing information. Enumeration of

the dangers associated with free-roaming dogs and the strat-

egy to promote public elightenment on the dangers of strayed-

dogs are highlighted. The need to emphasize medical atten-

tion for domestic animals, by taking them for appropriate vet-

erinary care, is suggested.

Malterials and Methods

Some private houses located in different parts of Lagos city

were visited to solicit the cooperation of dog owners. These

dogs were given serial numbers, and the information on their

age, sex, breed, degree of restriction and the type of treatment

each dog had received during the last one year was obtained.

Faecal collections of the dogs that had strayed and received

no treatment for the past one year and of the non-strayed

dogs kept under medical-care were obtained. For the ease of

collection and for obtaining fresh samples, the city was di-

vided into sections. The schedule of faecal collection was

conveyed to each section, prior to the day of visit by the

official. Dog owners were asked to restrict their dogs at night

before the day of visit. This ensured that the faeces collected

corresponded to the dog for which information was obtained.

Faecal samples of 135 free-roaming dogs were obtained. Some

private veterinary clinics were contacted and faecal samples

of 175 non-roaming dogs, brought-in for routine check-up,

were obtained. Relevant informations, as stated above, were

obtained for each dog. For both groups of dogs, efforts were

made to examine the faecal samples immediately after collec-

tion. Whenever not feasible, however, samples were refrige-

rated and examined within two days. Information was obtained,E-mail: [email protected]

279

through a questionnaire, on the levels of education, socio-

economic status and public awareness of dog owners about

the implications of their intimacy with dogs.

Age classification of dogs. The dogs were classified into vari-

ous age groups as follows: (a) puppies of less then six months;

(b) young dogs between six months and two years; and (c)

mature/adult dogs above two years.

Examination of faecal samples. Faecal samples were exam-

ined using the test tube floatation method (Hansen and Perry,

1990). Faeces weighing approx 3 g were put into a beaker

containing 50 ml floatation fluid (sodium chloride, 400 g; wa-

ter, 1000 ml; sugar, 500 g; specific gravity, 1.280) and stirred

thoroughly. The resulting faecal suspension was strained

into another beaker and then poured into a test tube in a rack,

filled to the brim. The tube was covered for about 20 min. The

cover was lifted and the strained faecal suspension was im-

mediately placed on a slide and examined under the micro-

scope for ova.


Results of the examination of stool samples of 310 dogs in the

two categories, 135 strayed-dogs and 175 regularly treated

dogs, are shown in Tables 1 and 2, respectively. The strayed-

dogs consisted of 51 puppies, 47 young, and 37 adults. In the

strayed-dogs category, of the 86 males, 61 (70.9%), and of the

49 females, 44 (89.7%) were detected positive for helminth

ova (Table 1). The female strayed-dogs were significantly

more infected than the male strayed-dogs (P < 0.01). Strayed-

puppies which had an infection rate of 92.2%, were signifi-

cantly more infected than the strayed young and adult dogs

(P < 0.01). The four helminth ova recovered from the strayed-

dogs were: Toxocara canis (49.5%), Ancylostoma caninum

(47.6%), Dipylidium caninum (41.9%) and Trichuris vulpis

(20.9%).

The dogs that had received regular treatment consisted of 65

puppies, 70 young and 40 adults. The infection pattern by sex

in this group showed that out of the 78 males, 9 (11.5%), and

of the 97 females, 12 (12.4%) were detected positive for

helminth ova (Table 2). The overall prevalence of non-strayed

dogs with helminth ova was 12.0%. Medically treated pup-

pies had significantly higher infection rate (21.5%) than the

medically treated young (7.1%) and adult dogs (5.0%) at

P < 0.01. All the dogs were cross- and pure-breeds, and each

had a record of regular veterinary clinic attendance. The ova

of the three helminth species detected from the treated dogs

were Toxocara canis (5.8%), Ancylostoma caninum (3.2%)

and Dipylidium caninum (2.1%). Infection with helminth ova

was significantly higher in the strayed-dogs (77.8%), than the

medically treated dogs (12.0%) at P < 0.01. Response to

questions by the dog owners revealed that only 40% were

aware of diseases transmissible between dogs and man. Ra-

bies was the infection generally mentioned, even though 90%

of them were educated and 60% were employed in the upper

salary scales.

This survey revealed a relatively high prevalence of intestinal

helminths among dogs in the Lagos State, especially among

the free-roaming ones. The public health implications of this

cannot be overemphasized, particularly in connection with

the transmission of zoonotic diseases (Samuel et al., 2001;

Kazacos, 2000; Prociv and Croese, 1990; Schantz, 1989; Little

et al., 1983). In spite of the fact that dogs provide meat deli-

cacy, security, hunting, social and economic benefits, their

role in the spread and transmission of zoonotic diseases, such

as cutaneous larva migrans, rabies, echinococcus and myasis

cannot be overlooked (Dryden, 2002). The free-roaming dogs,

that cross the roads at random, are causative agents of fatal

accidents on Lagos streets. Owing to the immense benefits of

dogs to man and the lack of awareness among the dog own-

ers about the dangers of intimacy with dogs, the public at

280 Comfort Adejoke Ibidapo

Table 1. The prevalence of helminth ova* in different age groups and the sex in strayed-dogs, Lagos, Nigeria

Age Males Females Total

group number number number number number number

examined infected examined infected examined infected

Puppies 34 31 (91.2) 17 16 (94.1) 51 47 (92.2)

(< 6 months)

Young 28 19 (67.9) 19 17 (89.5) 47 36 (76.6)

(> 6 months - 2 years)

Adult 24 11 (45.8) 13 11 (84.6) 37 22 (59.5)

(> 2 years)

Total 86 61 (70.9) 49 44 (89.7) 135 105(77.8)

*values within paranthesis are percentage of the total number of dogs examined in the respective age-group eategory

large, particularly the dog owners especially those involved

with children, should be educated on the risks associated

with keeping dogs. The need to take the dogs on regular vis-

its to veterinary clinics for routine check-up and treatment

must be duly emphasized. This suggestion was also made by

Schantz (2002), Kagira and Kanyari (2000), and Blagburn et

al. (1997). During this study, Toxocara canis and Ancylos-

toma caninum were detected as the most highly prevalent

helminth ova. This is in agreement with the results of Samuel

et al., (2001), Payne-Johnson et al. (2000), Blagburn et al.

(1996), Dada and Belino (1992), and Olufemi and Bobade

(1979). Dipylidium caninum and A. caninum have been re-

ported as the principal cause of canine anaemia (Schantz, 2002;

Dryden et al., 1995; Nolan and Smith, 1995; Hansen and Perry,

1990; Olufemi and Bobade, 1979; WHO, 1979; Oduye and

Otesile, 1977).

Fenwick (2000) had noted that D. caninum is one of the prin-

cipal causes of dog diarrhoea. The reports of this study showd

that puppies, among the different groups, were the most vul-

nerable to infrction. Kagira and Kanyari (2000) made similar

observations in their study in Kenya. Possible explanation

for this may be transplacental, resulting in prenatal infection,

and may also be through the lactogenic source (Payne-

Johnson et al., 2000; Payne and Ridley, 1999; Burke and

Roberson, 1985) . The results of this survey showed that the

cross- and pure-breeds of non-strayed dogs, with records of

regular veterinary clinic attendance, had very low helminth

ova infection in all the age groups. Possible genetic resis-

tance to infection due to the exotic breeds coupled with re-

stricted feeding on special diet, regular veterinary clinic check-

ups, and vaccinations may together account for the very low

rate of parasitic infections in these dogs. On the other hand,

the free-roaming dogs were more exposed to infection through

indiscriminate feeding and the lack of veterinary attention. A

similar trend was observed by Mafiana et al. (1993) in

Abeokuta, Ogun State, Nigeria.

In a telephone survey in Atlanta, Georgia, USA, Fontaine and

Schantz (1989) noted that only 63% of the household heads

believed that diseases of pets could be transmitted to hu-

mans, while few could recognise specific zoonotic infections

other than rabies. The result of the survey conducted during

the present study also followed a similar trend in that only

40% of the dog owners were aware of diseases transmissible

between dogs and man, other than rabies. Higher educational

standard and income group did not seem to influence the

understanding of most respondents. This suggests the need

for creating public awareness on the zoonotic diseases of

dogs (Schantz, 2002). Some scientists have listed the follow-

ing as zoonotic diseases of dogs to be of significance: ticks

and tick borne diseases (such as, babesiosis and ehrlichiosis),

rabies, distemper, trypanosomiasis, hookworm and tumbu fly

infections (Bowmann, 1999; van veen Schillhorm and

Adeyanju, 1979) ringworm, infectious diarrhoea, intestinal

helminthes (such as, Toxocara, toxoplasmosis, heartworm, and

parvovirus) ( Kazacos, 2000; Fontaine and Schantz, 1989).

The occurrence of large numbers of infected stray-dogs, dirty

environmental conditions, which perpetrate the development

and spread of these helminthes, intimacy between man and

dogs, and non-challant attitude of dog owners with regards

to veterinary care demand strict legislative measures, which

shoud be strictly adopted by the government, particularly in

the areas of free-roaming dogs.

Acknowledgements

The author is grateful to the directors and staff of the follow-

ing veterinary clinics, Petervet Veterinary Clinic Services,

Abule Egba; Harmony Projects, Amuwo Odofin; Unity Vet-

erinary Clinic, Egbeda; Alphavet Veterinary Centre, Surulere;

and Hillcrest Veterinary Clinic, Alagbado for their coopera-

tion and assistance during this study. Thanks are also due to

different households within Ojo, Surulere and Alagbado ar-

eas.

Prevalence of Intestinal Helminth Prasites of Dogs in Lagos, 281

Table 2. The prevalence of helminth ova* in different age groups and the sex in dogs which received regular veterinary care,

Lagos, Nigeria

Age Males Females Total

group number number number number number number

examined infected examined infected examined infected

Puppies 30 6 (90.0) 35 8 (22.9) 65 14 (21.5)

(< 6 months)

Young 25 2 (8.0) 45 3 (6.7) 70 5 (7.1)

(> 6 months - 2 years)

Adult 23 1 (4.3) 17 1 (5.9) 40 2 (5.0)

(> 2 years)

Total 78 61 (11.5) 97 12 (12.4) 175 21 (12.0)

*values within paranthesis are percentage of the total number of dogs examined in the respective age-group category

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Contin. Educ. Vet. Pract. 18: 483-509.

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ians, pp. 178-184, 7th edition, W.B. Saunders Company,

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mission of Toxocara canis and Ancylostoma caninum:

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Burnham, P.G. 1998. How to avoid zoonoses. Celebrating

Greyhounds 3: 14-16.

Dada, J.O., Belino, E.D. 1979. Prevalence and public health

significance of helminth ova in dog faeces deposited on

the streets of Zaria, Nigeria. Annals Trop. Med. Parasitol.

73: 495.

Dryden, M.W. 2002. Parasitic diseases of greyhounds: diag-

nosis, epidemiology and control of gastrointestinal para-

sites in dogs and cats. In: Proc. 18th Annual Interna-

tional Canine Sports Medicine Symp. pp. 58-60, Orlando,

FL, USA.

Dryden, M.W., Ridley, R.K., Schoning, P. 1995. Epidemiology

and control of intestinal parasites in greyhound kennels.

Incidence of parasitism and parasite contamination of

greyhound farms. Part 2. Greyhound Rev. 233: 441-443.

Fenwick, B. 2000. Causes, treatment and preventtion of infec-

tious enteritis in dogs. In: Proc. 15th Annual Interna-

tional Canine Sports Medicine Symp. pp. 22-26, Orlando,

FL, USA.

Fontaine, R. E., Schantz, P. M. 1989. Pet ownership and knowl-

edge of zoonotic diseases in De Kalb County, Georgia.

Anthrozoos 3: 45-49.

Hansen, J., Perry, B.D. 1990. The Epidemiology, Diagnosis

and Control of Gastrointestinal Parasites of Ruminants

in Africa: A Handbook, pp. 1-121, ILRAAD, English Press

Ltd., Kenya.

Harvey, J.B., Roberts, J.M., Schantz, P.M. 1991. Survey of vet-

erinarians’ recommendations for the treatment and con-

trol of intestinal parasites in dogs: public health implica-

tions. J. Am.Vet. Med. Assoc. 199: 702-707.

Hendrix, C.M., 1998. Diagnostic Veterinary Parasitology, pp.

21-23, 291-295, Mosby Inc., St. Louis, MO, USA.

Hendrix, C.M., Homer, S.B., Kellman, N.J., Harrelson, G., Bruhn,

B.F. 1996. Cutaneous larva migrans and enteric hookworm

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283Prevalence of Intestinal Helminth Prasites of Dogs in Lagos,

Pak. J. Sci. Ind. Res. 2005 48(4) 284-288

Introduction

The yielding ability of a genotype is the result of the rela-

tionship among the yield contributing components (Gupta

et al., 1999). These yield components are interdependent

in expression. Correlation analysis indicates the degree of

association between traits. It cannot, however, provide the

reasons of association. Therefore, simple correlation co-

efficients are not always effective in determining the real

relationship among traits. Even though correlation analy-

sis can quantify the degree of association between two char-

acters, a significant correlation merely indicates the de-

gree of association between the two characters. The method

of path coefficient analysis provides effective means of de-

termining the direct and indirect causes of association. A

path coefficient is a standardized partial regression coeffi-

cient. As such, it measures the direct influence of one trait

upon another and permits the separation of correlation co-

efficient into components of direct and indirect effects

(Dewey and Lu, 1959).

The 1000-grain weight, in modern high grain number culti-

vars, has reduced slightly (Waddington et al., 1986). The

major factor of low yield of varieties, in the era before the

green-revolution, was their tallness that is negatively corre-

lated with yield (Hatam and Akbar, 1995). It was earlier

found that correlations of days to heading with harvest in-

dex, 1000-grain weight with harvest index, and yield with

harvest index were positive and significant (Ihsanullah and

Mohammed, 2001). Thus, the lines with medium height and

higher harvest index would have potential for higher grain

yield. Plant height showed a strong negative genotypic cor-

relation with grain yield (Shahid et al., 2002). Path analysis

identified that 1000-grain weight and days to maturity had

the positive direct effect on grain yield, whereas days to head-

ing and plant height had negative direct influence on the grain

yield (Pawar et el., 1990).

In the common wheat, long vegetative period partly contrib-

uted to higher grain yield (Bingham, 1969). Positive corre-

lation was observed between length of grain filling period

and grain yield in the spring wheat (Spiertz et al., 1971).

Donald and Hamblin (1976) reported that the harvest index

could be considered as a breeding criteria in cereals. To im-

prove grain yield, selection in the F2 population should be

for plants having high harvest index and high biological yield

(Chowdhry et al., 2000; Hakam et al., 1997), since all these

characters are correlated with grain yield. Correlation stud-

ies between seed yield and nine components in the durum

wheat genotypes were carried out (Belay et al., 1993), and it

was found that seed yield exhibited a strong positive asso-

ciation with all the characters studied, except days to head-

ing and harvest index. Besides the seed yield itself, plant

height and 1000-grain weight may be considered good indi-

rect selection criteria. Duration of vegetative period has a

positive influence on grain yield and negative influence on

grain filling period (Razzaq et al., 1986). Cultivars with the

Correlation and Path Analysis in Candidate Bread Wheat (Triticum

aestivum) Lines Evaluated in Micro-Plot Test Trial

Tila Mohammad* , Sajjad Haidar, M. Jamil Qureshi, Abdul Jabbar Khan and Roshan ZamirNuclear Institute for Food and Agriculture (NIFA), Peshawar, Pakistan

(received April 24, 2003; revised March 3, 2005; accepted July 7, 2005)

Abstract. Correlation and path analysis among yield and yield-associated traits of eight candidate bread wheat lines,

including two check varieties, were studied during 2001-02. All the characters studied differed significantly from each

other, except biological yield and harvest index. Positive genotypic and phenotypic correlation was estimated between

plant height and biological yield. Plant height was negatively correlated with harvest index and grain yield, both at the

genotypic and phenotypic levels. It was, however, non-significant at both levels. Significant and positive genotypic

correlations were observed between biological yield with harvest index and grain yield. Path analysis showed that days

to heading, days to maturity, and plant height had negative direct effect on grain yield, whereas biological yield and

harvest index had a high and positive direct effect on grain yield. It may be concluded from the present studies that

biological yield and harvest index may be considered as the best selection criteria in the selection of high yielding

genotypes, at least from the standpoint of the evaluated set of genotypes.

Keywords: genotypic correlation , phenotypic correlation, bread wheat, path analysis, micro-plot trial, Triticum aestivum

∗Author for correspondence; E-mail: [email protected]

284

highest harvest index were found superior and efficient in

apportioning their dry matter into grain yield and vegetative

part in proper proportions.

The aim of the present study was to determine the interrela-

tionship and association of yield, some yield contributing com-

ponents, and to measure the direct and indirect influence of

these component characters on yield.


The experimental material was comprised of eight candidate

wheat lines, along with two standard varieties as check. These

were planted in micro-plot test trials in a randomized complete

block design, with three replications at the experimental farms

of Nuclear Institute for Food and Agriculture (NIFA), Peshawar,

Pakistan, during 2001-02. The genotypes studied were: CT-

97150, CT-99245, IDA-97026, CT-99187, CT-99197, ID-

A97044, CT-99059, and IDA-97107, along with two check

varieties, namely, Bakhtawar-92 and Fakhre-Sarhad. The geno-

types were selected from the International Wheat Screening

Nursery (IBWSN) raised at NIFA. These genotypes originated

from the International Maize and Wheat Improvement Centre,

Mexico for testing and evaluation at NIFA. The genotype num-

bers of the lires were the code numbers assigned to the selected

genotype lines at NIFA. The plot size harvested was 5 m x 1.2 m

(4 rows, 5 m long) with row-to-row spacing of 30 cm. The data

were recorded on days to heading, days to maturity, plant height

(cm), biological yield (kg/plot), grain yield (kg/plot), and har-

vest index (%). The harvest index was computed from the for-

mula: economic yield/total biological yield x 100 (Singa, 1977).

Economic yield was the grain yield obtained by harvesting and

threshing the two central rows of each experimental plot, while

total yield was the weight of the above ground biomass (grain

plus straw) of the same central two rows. A plant was assumed

to be physiologically mature when 75 percent of the glumes of

the primary spike had turned yellow. The 1000-grain weight

was computed by counting the grains with a grain counter and

then weighed on electronic balance.

Analyses of variance were computed for all the traits, and

genotypic and phenotypic correlations coefficients between

all the possible pairs of all characters as described by Steel

and Torrie (1960). Path coefficient analysis was carried out

according to the methodology adopted by Dewey and Lu

(1959) at genotypic level by the solutions of simultaneous

equations. Grain yield was kept as the resultant variable, with

other characters as causal factors.


The data revealed that the genotypes differed significantly

from each other for all the characters studied, except bio-

logical yield and harvest index (Table 1). Although the val-

ues for biological yield and harvest index of the wheat geno-

types did not differ significantly, it had a positive correla-

tion with other characters, specially the grain yield. It was

reported by Hakam et al. (1997) that the selection for plants

with high biological yield, and high harvest index leads to

high yielding lines in wheat. Genotypic and phenotypic cor-

relations coefficients are presented in Table 2, and direct

and indirect effect of each trait contributing towards grain

yield is given in Table 3. Apparently, the variations for the

four characters, namely, days to heading, days to maturity,

plant height, and grain yield, were not great, though signifi-

cant differences among varieties were observed. Similar re-

sults were reported earlier by Gupta et al. (1999) and Singh

et al. (1982).

Days to heading. Genotypic and phenotypic correlations of

days to heading with days to maturity, plant height, biological

yield, and grain yield were positive, while these were nega-

tive with harvest index and were non-significant in all cases.

Path analysis showed that days to heading had negative direct

effect on grain yield (- 0.0248) and positive indirect effect

through biological yield (0.2323), while negative indirect ef-

fect through days to maturity (- 0.0002), plant height (- 0.0064)

and harvest index (- 0.8910). Similar results have been re-

ported by Gupta et al. (1999).

Correlation on Path Analysis in Wheat

Table 1. Mean squares for analysis of variance of different characters in micro-plot trial for correlation and path analysis in

candidate bread wheat lines

Source of variation d.f. Days to Days to Plant height Biological yield Harvest index Grain yield

heading maturity (cm) (kg/plot) (%) (kg/ha)

Varieties 9 7.392** 1.667** 78.955** 0.173n.s. 20.030n.s. 442325.4**

Replications 2 0.234n.s. 1.063n.s. 3.781n.s. 1.895n.s. 4.627n.s. 74016.0n.s.

Error 18 0.382 0.326 6.210 7.493 12.710 55957.3

Total 29

* = significant; ** = highly significant; n. s. = non-significant

285

T. Mohammad et al.

Table 2. Genotypic and phenotypic correlation coefficients matrix of micro-plot trial for correlation and path analysis in candi-

date bread wheat lines

Variables Correlation Days to Days to Plant Biological Harvest Grain

(vg; vp) heading maturity height yield index yield

(cm) (kg/plot) (%) (kg/ha)

Days to heading vg 1

vp 1

Days to maturity vg 0.2968 1

vp 0.2379 1

Plant height (cm) vg 0.1900 - 0.1426 1

vp 0.2058 - 0.0599 1

Biological yield (kg/plot) vg 0.3967 0.3459 0.4535 1

vp 0.2675 - 0.0751 0.4216 1

Harvest index (%) vg - 0.1744 - 0.7403* - 0.4947 0.6781* 1

vp - 0.1316 - 0.1145 - 0.4255 - 0.4719 1

Grain yield (kg/ha) vg 0.1118 - 0.1790 - 0.0253 0.9066** 0.9293** 1

vp 0.0745 - 0.1897 - 0.1034 0.3503 0.6571* 1

* = significant; ** = highly significant; vg = genotypic correlation; vp = phenotypic correlation

Table 3. Direct (in parenthesis) and indirect matrix effect of different traits in micro-plot trial for correlation and path analysis

in candidate bread wheat lines

Variables Days to Days to Plant height Biological yield Harvest index Grain yield

heading maturity (cm) (kg/plot) (%) (kg/ha)

Days to heading (- 0.0248) - 0.0002 - 0.0064 0.2323 - 0.8910** 0.1118

Days to maturity - 0.0074 (- 0.0007) 0.0048 0.2025 - 0.3782 - 0.1790

Plant height (cm) - 0.0047 0.0001 (- 0.0335) 0.2655 - 0.2527 - 0.0253

Biological yield (kg/plot) - 0.0098 - 0.0002 - 0.0152 (0.5855) 0.3464 0.9066**

Harvest index (%) 0.0043 0.0005 0.0166 0.3970 (0.5108) 0.9293**

* = significant; ** = highly significant

Days to maturity. Days to maturity were negatively corre-

lated with plant height, harvest index and grain yield, both at

the genotypic and phenotypic levels, while these were posi-

tively correlated with biological yield at the genotypic level

(0.3459) and negatively correlated at the phenotypic level

(- 0.0751). Negative genotypic correlation of days to matu-

rity was significant only with harvest index (- 0.7403). Simi-

lar results were reported earlier by Singh et al. (1982). Days

to maturity showed low, but negative direct effect on grain

yield (- 0.0007). Its indirect effect was negative through days

to heading (- 0.0074) and harvest index (- 0.3782) and was

positive through plant height (0.0048) and biological yield

(0.2025).

Plant height. Positive genotypic and phenotypic correlations

were observed between plant height and biological yield. Plant

height was negatively correlated with harvest index and grain

yield, both at the genotypic and phenotypic levels, but it was

non-significant at both the levels. Similar results were observed

by Ahmad et al. (1980). Plant height directly affected the grain

yield in a negative direction (- 0.0335) as was earlier reported

by Nabi et al. (1998) and Chowdhry et al. (1986). The pos-

sible explanation is that an adequate amount of dry matter is

partitioned towards the height of the plant in taller plants, af-

fecting the grain yield adversely. Positive indirect effect of

plant height was through days to maturity (0.0001) and bio-

logical yield (0.2655), which was counter-balanced by nega-

tive indirect effect through days to heading (- 0.0047) and

harvest index (- 0.2527).

Biological yield. Significant and positive genotypic correla-

tions were observed between biological yield with harvest in-

dex (0.6781) and grain yield (0.9006). At the phenotypic level,

negative correlation was observed between biological yield

and harvest index (- 0.4719), and positive but non-significant

correlation was observed between biological yield and grain

286

yield (0.3503). Biological yield had the highest and positive

direct effect on grain yield (0.5855). Singh and Singh (2001)

and Chowdhry et al. (2000) also reported highly significant

and positive genotypic correlation between grain yield and

biological yield. It had negative indirect effect through days

to heading (- 0.0098), days to maturity (- 0.0002) and plant

height (- 0.0152), while positive indirect effect through har-

vest index (0.3464).

Harvest index. Harvest index was positively correlated with

grain yield at the genotypic (0.9293) and phenotypic (0.6571)

levels. It was highly significant (p < 0.01) at the genotypic

level and significant (p < 0.05) at the phenotypic level. The

direct effect of harvest index on grain yield was high and posi-

tive (0.5108). Indirect positive effects were found through days

to heading (0.0043), days to maturity (0.0005), plant height

(0.0166) and biological yield (0.3970). These results are in

agreement with those reported by Singh and Singh (2001) and

Shoran et al. (2000).

Grain yield. Highly significant and positive genotypic cor-

relations were observed between grain yield and biological

yield (0.9066) and harvest index (0.9293). At the pheno-

typic level, the association between grain yield and harvest

index (0.6571) was positive and significant, while it was

positive but non-significant with biological yield (0.3503).

Grain yield had negative and non-significant genotypic and

phenotypic correlation with days to maturity and plant height.

Earlier, Ashraf et al. (2002) revealed that grain yield was

positively correlated with plant height. They also observed

that in grain yield, high direct effects were contributed by

biomass and harvest index, although the latter had negative

association with grain yield. They further observed that high

indirect contribution was via the biomass by most of the yield

components and hence the two traits (biomass and harvest

index) should be given emphasis while selecting high yield-

ing wheat cultivars.

Days to heading had positive, but non-significant, geno-

typic and phenotypic correlations with grain yield. Bio-

logical yield (0.5855) and harvest index (0.5108) had

the highest and positive direct effect on grain yield, while

days to heading (- 0.0248), days to maturity (- 0.0007)

and plant height (- 0.0335) had negative direct effects

on grain yield.

Present studies have revealed that the higher biological yield

and higher harvest index may be considered as the best selec-

tion criteria in the selection of high yielding genotypes. Pre-

viously, Razzaq et al. (1986), and Donald and Hamblin (1976)

also reported biological yield and harvest index as importent

selection criteria in wheat.

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T. Mohammad et al.288

Studies on the Utilization of Oscillatoria thiebautii as Food Source for

Artemia sp Culture

S. G. Abbas Shah*, Razia Sultana and Abid AskariFood and Marine Resources Research Centre, PCSIR Laboratories Complex, Karachi-75280, Pakistan

(received May 20, 2004; revised February 1, 2005; accepted May 10, 2005)

Pak. J. Sci. Ind. Res. 2005 48(4) 289-291

Aquaculture is expected to grow at a fast pace. This is neces-

sary to match with the additional demand for high quality

protein in order to keep pace with the anticipated population

growth. The importance of phytoplanktons as the source of

fish and shrimp feed is well known. They provide the basis of

low-technology in the intensive pond culture systems. It has

been observed that Oscillatoria thiebautii and other cyano-

bacteria growing in ponds are consumed by Artemia sp and

many other zooplanktons. O. thiebautii, a eurythermal cyano-

bacterium occurring mainly in tropical seas, performs many

important functions. It has the ability to fix elemental nitro-

gen and uptake combined nitrogen nutrients (Carpenter and

McCarthy, 1975; Sournia, 1968; Desikachary, 1959). It has been

observed during earlier studies that large quantities of cyano-

bacteria, especially O. thiebautii, are consumed by tilapia and

channel catfish (Han and Dickman, 1995; Perschbacher, 1995).

Artemia is a non-selective filter feeder, feeding on particulate

matter of biological origin (organic detritus from mangrove

waters), as well as on living organisms of the appropriate size

and range including microscopic algae and bacteria (Reeve,

1963). Nutritionally, Artemia has high digestibility and app-

ears to meet most of the macro- and micronutrient require-

ments of freshwater and marine fishes, and crustacean larvae.

Phytoplanktons are the basic unit of the food web in aquacul-

ture systems. Maintenance of the phytoplankton bloom is the

most important aspect in Artemia culture and for successful

production of its biomass and cyst. Being important in the

food chain and as a nitrogen fixer, O. thiebautii was cultured

and studies were undertaken in the laboratory to assess the

feasibility of its utilization as feed by Artemia.

The phytoplankton and Artemia culture. O. thiebautii was

cultured in four aquaria measuring 45 x 35 x 26 cm and three

aquaria measuring 29 x 29 x 21 cm. The aquaria were filled with

reconstituted seawater from saltworks where the native

Artemia sp was found to be present. The salinity of the sea-

water was maintained around 50 ppt and the temperature

ranged between 16-30 °C during the period of study. The pH

of water was maintained at 8±1. Dissolved oxygen during the

study ranged between 5-8 mg/l. O. thiebautii inoculum po-

pulation was collected from the intermediate 50 ppt salinity

ponds of solar saltworks at Korangi Creek, Karachi, Pakistan,

with the help of phytoplankton nets with 5 µm mesh and in-

oculated in the study aquaria. After one week of inoculation,

the population of O. thiebautii multiplied and the algal bloom

started. At this stage, the nauplii of Artemia, which were

hatched separately, were introduced at the rate of 25 nauplii

in 10 litre and 50 nauplii in 50 litre of water, respectively, in

small and large aquaria. Total O. thiebautii colonies during

the mentioned period were reported as cells (1.2 x104/ml).

Nutrient supplementation. The salt nutrients, such as KNO3,

Na2HPO

4.2H

2O and urea at the rate of 15 g/1 were supple-

mented weekly to enhance the growth and population of

O. thiebautii, as the Artemia population was feeding on the

phytoplankton.

Population determination. Samples of Artemia and O. thiebautii

were collected weekly from the aquaria for wet weighing and

the recording of population data. For the determination of

O. thiebautii biomass, 5% formaldehyde was added to one

litre of culture medium from the aquaria. The biomass of the

phytoplankton was allowed to settle for 48 h, the supernatant

was decanted and the sedimented material was analysed for

weight and cell count.

Abstract. Growth and population of a native parthenogenetic Artemia sp in relation to Oscillatoria thiebautii was

studied. O. thiebautii was inoculated to confirm its utilization in the Artemia sp culture. The study was carried out

between October-July when the temperature ranged between 16-30 °C. The culture salinity and pH was maintained

at 50 ppt and 8±1, respectively. The Artemia, at these culture conditions, showed good growth and survival in the

presence of O. thiebautii as its food source. The study indicates O. thiebautii as a cost-effective feed for Artemia sp

culture, which is both inexpensive and available in abundance.

Keywords: Oscillatoria thiebautii, Artemia sp, algal bloom, Artemia food, Artemia culture, aquaculture


Short Communication

289

Artemia health status. The Artemia health status was deter-

mined by their swimming activity, which they exhibited well in

a small glass beaker placed near a light source. The Artemia

were noted to concentrate at one spot, showing the crowding

effect. Microscopic inspection revealed that the digestive tracts

were always full with food. However, when the thoracopod

and mouth region was clean, it meant that the animals were

starving. Growth of Artemia was followed by determining the

wet weight of the Artemia present in 15 samples/l of the cul-

ture medium. These data were extrapolated for the calculation

of Artemia biomass in the total culture medium volume of the

aquaria.

Observations on the growth of O. thiebautii and Artemia. The

phytoplankton material was studied under the microscope for

the identification of O. thiebautii. The microalgal cell count

was low in the hotter months May-July (27-30 °C) and high in

cooler months December-April (16-23 °C). These low and high

values of O. thiebautii cell colony count corresponded with

low and high population of Artemia during these months

(Table 1; Fig. 1). The average size of Artemia reached about

8 mm in two weeks. The Artemia biomass harvested from small

aquaria was 25 g/10 litre culture medium, and from large aqua-

ria was 1 kg/50 litre of culture medium during one month of

culture. It was observed that as the Artemia population in-

creased, the O. thiebautii population decreased. It has been

reported that the growth of Artemia was directly proportional

to the population of O. thiebautii (Neelakanta and Papanasam,

1987; Wongart, 1986). It has been also observed that as the

temperature increased above 30 °C the growth of O. thiebautii

declined (Sorgeloos et al., 1986). Our observations, therefore,

are in agreement with those reported in the literature.

The maximum harvest of Artemia averaged 25 kg wet biomass

for the total culture in 230 litres of culture medium in 5 months.

The Artemia population ranged between 3000-15000 indivi-

duals/litre. Sorgeloos et al. (1986) indicated that efficient con-

version of the algal food by Artemia in a flow-through culture

system was attained when the algal cell concentration in the

effluent approximated the critical minimum cell concentration

of about 5000 cells/ml. This could be achieved by adjusting

the retention time of algal culture in the Artemia aquaria (Roels

et al., 1979). The survival rate of Artemia in the present stud-

ies was abserved to be 100%, which was estimated by measur-

ing the wet weight of the Artemia present in several samples

of one litre culture medium and extrapolating the average to

the total aquarium volume in accordance with Vanhaecke and

Sorgeloos (1989). Artemia reached maximum size of 10 mm in

3-week old cultures and started breeding.

The nutritional quality, ingestibility and food value of O.

thiebautii were verified in preliminary growth and survival

tests, by placing freshly hatched nauplii in 25 ml algal cell

suspension in 10 cm petri plates at different cell concentrates

for the culture period of 14 days. The nutritional quantity of

the Artemia fed on O. thiebautii revealed very healthy popu-

lation, with continuous reproduction and good growth con-

dition. The proximate analysis of Artemia showed 35.2%

protein, 9.8% fat, 13.1% carbohydrates and 17.4% ash.

The present studies have shown that O. thiebautii is a good

food source for Artemia. Both these organisms have shown a

positive relationship.

Acknowledgement

The authors are thankful to Dr. Rabia Zuberi, Head, Food and

Marine Resources Research Centre, PCSIR Laboratories Com-

25000

20000

15000

10000

5000

0

Num

ber/

litre

of

Art

em

ia

Average count O. thiebautii

Fig. 1. Regression graph showing Oscillatoria thiebautii

density vs number of Artemia from October to July.

0 5000 10000 15000 20000 25000 30000

S. G. A. Shah et al.290

Table 1. The effect of seasonal variation on the growth of

Oscillatoria thiebautii and Artemia in aquaculture*

Month pH Average Average count Number

temperature of O. thiebautii of Artemia

(°C) per ml ** per litre

October 8.1 24 5000 9000

November 8.0 24 8000 10200

December 8.0 23 10800 12500

January 7.9 20 12000 17500

February 7.9 16 18000 19500

March 7.9 16 20500 20400

April 8.0 25 19500 18500

May 8.0 27 6000 8000

June 8.0 28 5900 7600

July 8.0 30 5400 7100

*salinity was maintained at 50 ppt; **total O. thiebautii colonies

during the respective mentioned period.

plex, Karachi, for her encouragement and the facilities pro-

vided for work during this study.

References

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take of combined nitrogenous nutrients by Oscillatoria

(Trichodesmium) thiebautii in Western Sargasso Sea.

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Desikachary, T.V. 1959. Cyanophyta, Indian Council of Agri-

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Han, X., Dickman, M. 1995. Changes in super (13) content of

the organic component of lake sediments during the last

500 years in Crawford Lake, South Ontario, Canada.

Hydrobiologia 310: 177-187.

Neelakanta, P.R., Papanasam, P.N. 1987. Ecology of Artemia

along the Southeastern Coast of India. In: Artemia Re-

search and its Application, P. Sorgeloos, D.A. Bengston,

W. Decleir and E. Jasper (eds.), vol. 3, pp. 136-139, Uni-

versal Press, Wettern, Belgium.

Perschbacher, P.W. 1995. Algal management in intensive

channel catfish production trials. World Aqua Culture

56: 65-68.

Reeve, M.R. 1963. The filter feeding of Artemia. II. In suspen-

sion of various particles. J. Exptl. Biol. 40: 207-214.

Roels, O.A., Sharfstein, B.A., Tobias, W.J. 1979. Artificial

Upwelling Progress Report (1978-1979): Cultivation

of Brine Shrimp Artemia, Final Report for the Work

Supported by NOAA, National Sea Grant Project,

NA-79-AA, D-00039, 4700 Avenue U, Galveston, Texas

77550, USA.

Sorgeloos, P., Lavens, P., Leger, Ph., Tackaert, W., Vanhaecke,

P., Versichele, D. 1986. Manual for the Culture and Use of

Brine Shrimp Artemia in Aquaculture, pp. 1-319, Artemia

Reference Centre, Ghent, Belgium.

Sournia, A. 1968. La Cyanophycae Oscillatoria (Trichodes-

mium) dans le plankton marine: taxonomic et observation

dans le canal de Mozambique. Nova Hedw. 5: 1-12.

Vanhaecke, P., Sorgeloos, P. 1989. International study on

Artemia. XLVII. The effect of temperature on cyst hatch-

ing, larval survival and biomass production for different

geographical strains of brine shrimp Artemia spp. Annls.

Soc. Zool. Belg. 119: 7-23.

Wongart, L. 1986. Biological Analysis of Artemia Culture from

Salt and Salt cum Artemia, Annual Technical Report, pp.

5-16, National Artemia Reference Centre, Kaesetsart Uni-

versity, Bangkok-10903, Thailand.

Short Communication: Oscillatoria thiebautii as Food for Artemia Culture 291

Isolation, Determination and Characterization of Taro(Colocasia esculenta) Starch

Feroz A. Jafferya, Abid Hasnain*a, Khalid Jamilb and Tahir Abbasb

aDepartment of Food Science and Technology, University of Karachi, Karachi, PakistanbFood and Marine Resources Research Centre, PCSIR Laboratories Complex, Karachi, Pakistan

(received September 28, 2004; revised February 4, 2005; accepted March 14, 2005)

Abstract. The starch content of taro (Colocasia esculenta) was determined using DNS colourimetric, acid-hydrolysis andenzymatic methods. Starch content varied from 80.3 to 81.3% as determined by DNS colourimetry. For the extraction oftaro starch, different techniques were used. It was found that there was a noticeable improvement in the yield of starchusing the freeze-thaw method. The various physicochemical properties of the extracted starch were also compared withthe starch obtained by ammonia and alkali extractions. It was found that the hydration capacity, swelling volume, moistureabsorption, freeze-thaw stability, as well as swelling power, were generally higher while solubility was lower of the freeze-thaw extracted starch than that extracted by ammonia and alkali. The DNS colourimetric method is recommended as asimple method for the determination of taro starch.

Keywords: taro starch, freeze-thaw extraction, starch extraction, DNS colourimetry, Colocasia esculenta, starch characte-rization

Pak. J. Sci. Ind. Res. 2005 48(4) 292-296

IntroductionEdible aroids are starchy tuber crops of the humid tropicaland subtropical regions of the world. They are herbaceousplants (Family: Araceae), consisting of five genera of whichColocasia esculenta is the most important food crop. Thisspecies is commonly known as taro or old cocoyam, (verna-cular: arvi, dasheen). It is an important low-cost starchyfood source (Hong and Nip, 1990). Taro has been reportedto have 70-80% starch on a dry weight basis (Jane et al., 1992;Tu et al., 1979), comprised of small granules, having dia bet-ween 1.4 and 5 μm (Sugimoto et al., 1986). Taro starch, in viewof its small granule size, has been considered to be easily di-gestible, hence it is widely used in baby foods and the dietsof people allergic to cereals and children sensitive to milk(Wang, 1983). In addition to the food use, taro has foundsome industrial applications as well. The small size of tarostarch granules makes them ideal in cosmetic formulations,such as face powder, and in dusting preparations which useaerosol dispensing systems (Griffin and Wang, 1983). Tarostarch has been considered to be a suitable filler in biode-gradable plastics and as a fat substitute (Daniel and Whistler,1990).

The starch content of edible aroids has been determinedusing a variety of analytical techniques. Average starch valueof 679 g/kg, on a dry weight basis, was determined from cor-mels (Colocasia esculenta) grown in Bangladesh, by hydro-lysis of starch (Chowdhury and Hussain, 1979). Using the

glucoamylase hydrolysis and copper reduction method, thestarch content of a single taro cultivar was reported to be 540g/kg on a dry weight basis (Hussain et al., 1984). In anotherstudy, the starch content was calculated to be 70.6%, on a dryweight basis (Agbor-Egbe and Rickard, 1990). Starch valuesin this study were determined using acid hydrolysis and theferricyanide reduction method. Since in the selection of ediblearoid cultivars in a germplasm collection, the starch content isconsidered to be a very important characteristic, there is aneed for the development of an accurate but simple and inex-pensive method of starch analysis.

In spite of the versatile uses of the taro starch, large-scaleextraction and utilization of taro starch is not practiced any-where. This has been probably due to the difficulty in extrac-ting the taro starch from fresh tubers, which also contain a lotof mucilaginous materials. Moorthy (1991) made an attemptto extract taro starch by using dilute solution of ammonia. Inanother study, the starch was extracted by using NaOH solu-tion (Jane et al., 1992). The quality, as well as yield of thestarch, using these methods, was found to be reduced due tothe chemical treatments involved. An easy and convenientmethod is, therefore, also needed to be developed for theextraction of taro starch to produce good quality starch withbetter yield. The purpose of the present study was to explorea new technique for the isolation and determination of tarostarch and to compare the so developed method with thereported methods by evaluating the physicochemical pro-perties of the extracted starch.*Author for correspondence

292

Materials and MethodsMaterials. Taro (Colocasia esculenta) was purchased fromthe local market of Karachi, Pakistan. The chemicals used wereof Analytical grade (E. Merck and Sigma).

Isolation of taro starch. Three different techniques were usedfor the extraction of taro starch as given below.

Extraction by the freeze-thaw method. This is a simple andconvenient method, recently developed by the presentauthors. In this method, peeled taro was ground in a grinder,blended in distilled water in the ratio of 1:2, and mixed well ina mechanical mixer for 1 h. The slurry was filtered throughan 80 mesh sieve, followed by passing through 260 meshsieve. The residue on the sieve was removed and the filtratewas kept in a freezer at -10 °C for 5 days. The frozen extractwas thawed at room temperature for 4 h. The separated waterwas decanted. Residue was carefully dried at 45 °C and thenpulverized.

Extraction with ammonia. The extraction was carried outfollowing the procedure of Moorthy (1991), using 0.03 Mammonia solution.

Extraction with NaOH. The method of Jane et al. (1992) wasused. In this method, 0.05% NaOH solution was used for theextraction of taro starch.

Purification of starch. Defatting of starch was carried out bySoxhelt extraction with 75% aqueous n-propanol for 7 h. Thesolvent was removed by vacuum evaporation and the starchwas air-dried to a moisture content of 10%. Deproteinizationof the starch was done by the method of Jane et al. (1992),using NaCl solution and toluene.

Starch analysis. Three different methods were used for thedetermination of starch present in the dehydrated taro pow-der.

DNS colourimetric method. DNS (3,5-dinitrosalicylic acid)colourimetric method was used for the determination of sta-rch (Geirwyn, 1995). About 0.1 g of the powdered sample washydrolyzed in 10 ml of 1.5 M H2SO4 for 20 min in a boilingwaterbath. 12 ml 10% NaOH was added and the contents werefiltered in 100 ml volumetric flask (hydrolysate). Solutions of0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg/ml of glucose were preparedin 100 ml volumetric flask. To six labelled test tubes was sepa-rately transferred 1 ml of each concentration of the standardglucose solution (0.25 - 1.5 mg/l). A tube containing 1 ml dis-tilled water served as the blank. To each of these test tubeswas then added 1.0 ml of DNS reagent (2,6-dinitro-salicylicacid) and 2.0 ml of distilled water. All the tubes were heated ina boiling waterbath for 5 min and then cooled. The volume ofeach test tube was adjusted to 20 ml with distilled water. Ab-sorbance of each standard glucose solution was measured at

540 nm to prepare the standard curve. The taro starch samplefor analysis was prepared in a similar manner by replacingthe glucose solution and the absorbance taken at 540 nm. Theconcentration of taro starch was determined from the stan-dard glucose calibration curve and then the percentage ofstarch was calculated.

Enzymatic method. The enzymatic method used was in ac-cordance with Jane et al. (1992). Taro flour (50 mg, dsb) wassuspended in 90% dimethyl sulfoxide (3 ml) and boiled in awater bath at 96 °C for 1 h. After cooling, methanol was addedto precipitate the solids. The mixture was centrifuged, andthe supernatant was discarded. A phosphate buffer (pH 6.9,0.1 M, 3 ml) and porcine pancreatic α-amylase (1,330 units)was added to the solid residues. The mixture was incubatedin a shaker waterbath at 35 °C for 4 h. At the end of the incu-bation, glucoamylase (25 units) in an acetate buffer solution(pH 4.3, 0.1 M, 0.55 ml) was added to the digestion mixture,and the pH of the mixture was adjusted to 4.5. The digestionmixture was then incubated in the shaker waterbath at 55 °Cfor 4 h. Glucose produced in the digest was analyzed by mea-suring the absorbance at 520 nm, using a mixture of hexoki-nase and glucose-6-phosphate dehydrogenase.

Acid-hydrolysis method. The starch content of the taro pow-der was determined by the AOAC (1984) direct acid hydroly-sis procedure (Method 22.045), as modified by Rickard andBehn (1987).

Moisture absorption. Moisture absorption by starch was de-termined using the method of Nyqvist (1983), with few modi-fications. The starch powder was dried at 60 °C for 6 h until themoisture level was < 1%. The hygrostate was prepared usinga saturated Na2SO4 solution in the wells of a glass dessicator.A starch sample of 2 g was placed in a watchglass and kept inthe hygrostate. The percentage moisture absorbed was deter-mined from the weight gained after 240 h (10 days).

Hydration capacity. Hydration capacity, or water absorption,was determined by the method of Komblum and Stoopak(1973). 400 mg starch samples were placed in each of the 15 mlplastic centrifuge tubes. Distilled water was added from aburette, the tubes were covered with parafilm and their con-tents mixed on a vortex mixer (Vortex_Gennie Scientific Indus-try, USA) for 2 min. The mixture in each tube was left to standfor an additional 3 min and then immediately centrifuged at2000 xg for 10 min in a Gallenkamp bench centrifuge. Thesupernatant was decanted and the sediment was weighed.The weight of water adsorbed and retained was determinedas the gain in weight of the dry sample.

Swelling power and solubility. Swelling power was determi-ned by using the method of Lauzon et al. (1995). Starch sus-pension (1%, w/v) was heated to 55, 65, 75, 85 and 95 °C and

293Taro (Colocasia esculenta) Starch

was kept at each temperature for 30 min, followed by coolingdown to the room temperature rapidly in an ice waterbath.The cooled sample was centrifuged at 5,000 xg for 20 min. Theswelling power was measured from the precipitate. The solu-bility was determined by using 0.4% (w/w) starch dispersionaccording to the method of Leach et al. (1959). The amount ofsoluble material in the supernatant was estimated from itsvolume and concentration.

Swelling volume. The swelling volume was determined byheating 15 ml of an aqueous (w/v) starch suspension in screwtop 40 ml universal sample bottles. Samples were heated in a95 °C waterbath, with gentle shaking until gelatinizationoccurred, and then left in the waterbath for a further periodof 1 h. After cooling, the samples were transferred into 15 mlconical centrifuge tubes and centrifuged at 2200 rpm (approx1000 xg) for 20 min. The swelling volume was obtained direc-tly by reading the volume of the sediment in the tube. Theswelling volume is the volume of sediment per 100 ml of thestarch sample.

Freeze-thaw stability. A modified method based on Narkrugsa(1996) and Schoch (1968) was used to determine the freeze-thaw stability. Starch sample of 15 g was mixed in 300 ml dis-tilled water in a beaker at 95 °C, stirred with a mechanicalmixer for 20 min. The mixture was poured into a plastic cupand frozen at -10 °C, for 7 days. The frozen mixture was thenthawed in a water bath at 30 °C, for 4 h. From this, 100 ml ofthe mixture was centrifuged at 8000 rpm for 30 min. Theamount of water separated from the mixture, after centrifu-gation, was measured. Results used for calculation were themeans of triplicate measurements.

Viscography. Starch suspension (6%) was subjected toviscography, using a Brabender Viscoamylograph. Starchsuspension was gradually heated from 25 to 92.5 °C at a rateof 1.5 °C/min, held at 92.5 °C for 10 min, and then cooled atthe same rate.

Results and DiscussionThe variations in the taro starch content, using different ana-lytical methods studied, are shown in Table 1. Significant dif-ferences were observed in the quantity of starch determined.The overall mean starch values of all taro samples for DNScolourimetric method (80.3 - 81.3%) were found to be higheras compared with the values obtained using the enzymaticmethod (73.0 - 74.3%) and the acid-hydrolysis method (76.3 -77.1%). These observations indicate that the DNS colouri-metric method is suitable for the determination of taro starchaccurately in a simple procedure.

Taro contains large quantities of mucilagenous materials andit is difficult to extract starch from this tuber. The settling of

starch is too slow because of these mucilagenous materials,leading to a reduction in the yield and also rendering extractsusceptible to microbial degradation during the settling pro-cess over 1-2 days. A noticeable increase in the yield, on extrac-tion with ammonia and sodium hydroxide solution, followedby the conventional settling process, can be used for thelarge-scale taro starch production. It was observed, thatalthough ammonia and sodium hydroxide solution improvedthe settling of taro starch and prevented the microbial decay,yet the starch obtained had a brownish colour, which couldnot be removed even by repeated washing. Ammonia andsodium hydroxide solution also disrupted the starch granu-les to some extent and affected their functional properties.Extraction using the freeze-thaw technique yielded good qua-lity taro starch, with high yield (upto 19.8%), as comparedwith starch extracted using the ammonia and alkali methods(Table 2).

As evident from Table 3, the hydration capacity of the freeze-thaw extracted starch (TS-1) was noted to be generally higherthan that extracted with ammonia (TS-2) and alkali (TS-3). Theabsorbed water was undoubtedly taken up by the granuleson their surfaces (Medcalf and Gilles, 1965). The lower valuesobtained with TS-2 and TS-3 indicate that the ammonia andsodium hydroxide treatment had caused breakage of somestarch granules, and thus the capacity to absorb water by thegranules had decreased. Due to relatively more cleavage ofstarch granules, TS-3 absorbed much less water among thestarches extracted by the three methods. Similarly, the en-hanced moisture absorption of TS-1 showed that it wasmuch more hygroscopic than TS-2 and TS-3 starches. Itshows that greater surface area was available to absorb themoisture in the case of TS-1, which indicates the superiorityof TS-1 over TS-2 and TS-3 starches.

When starch gels were subjected to freeze-thaw cycling, thewater used in the preparation of the gel was noted to get

Table 1. Starch content (%, dry weight basis) of taro flour* asdetermined by different methods

DNS colourimetric Enzymatic Acid-hydrolysismethod method method

80.8 ± 0.5 73.8 ± 0.5 76.7 ± 0.4*mean ± sd (n = 3); DNS = 3,5-dinitrosalicylic acid

Table 2. Yield (%, fresh weight basis) of taro starch* extractedby using different methods

Freeze-thaw Extracted with Extracted withextraction NH3 NaOH

19.3 ± 0.5 16.3 ± 0.5 15.0 ± 0.5*mean ± sd (n = 3)

294 F. A. Jaffery et al.

separated due to the tendency of starch molecules to reasso-ciate, thus forming insoluble aggregates. The stability ofstarch to freeze-thaw cycling makes it suitable for use in thefrozen food products (Jaroslaw et al., 2004). The starches thatare most stable to freeze-thaw cycling are also the most stableduring refrigerated storage (White et al., 1989). The freeze-thaw stability of a starch gel is evaluated by the amount (%)of water released (syneresis) when the starch chains retro-grade (reassociate) during the freeze-thaw cycle. The presentresults show that TS-1 had higher freeze-thaw stability thanthe counterpart TS-2 and TS-3 starches. This is probably dueto the greater degree of reassociation between starch chainsin the TS-1 starch.

The swelling volume of starches is also affected by variouschemicals (Moorthy, 1991). The swelling volume of starchesextracted with ammonia and alkali were, therefore, comparedwith that of the freeze-thaw extracted starch. The swellingvolume of taro starch was noted to increase on using ammo-nia solution and much more in the case of alkali extraction.Higher swelling indicated a lowering of associative forcesbetween the starch granules, and hence the taro starch ap-peared to have undergone some reduction in associativeforces when extraction was done using ammonia and sodiumhydroxide solution.

Solubility of starch is observed to be a function of tempera-ture (60 to 90 °C). At temperature below their gelatinizationtemperature, starches are less soluble. Solubility of starchgreatly increased at higher temperaturs (80 and 90 °C). Solu-bility characterization of starch reportedly depends upon thedegree of substitution and the degree of polymerization. Thepresent results show that the TS-1 starch was the least solublethan the TS-2 and TS-3 (Fig. 1). It is probably due to weake-ning of starch granules during the chemical treatment, whichcaused the ease in solubilization of TS-2 and TS-3 starches.The swelling power of starch was found to be a function oftemperature and it followed a pattern similar to that of solubi-lity characteristics. Prior to gelatinization there is only a slightincrease in the swelling capacity of starches. Once the gelati-nization process sets in, however, swelling increases rapidlywith increasing temperature. The swelling power rises signi-

ficantly at the gelatinization temperature. When the crystalregion in the starch granules begins to melt, it enhances theswelling power. As the temperature of an aqueous suspensionof starch granules is raised above the gelatinization range,hydrogen bonds between polymers continue to be disruptedand water migrates into the interior of the molecule (Shi andBeMiller, 2002). The present results showed that the swellingpower of TS-1 was higher than the TS-2 and TS-3 starches(Fig. 2). It was probably due to weakening of the hydrogenbonds by the ammonia and sodium hydroxide treatments.

Viscosity of taro starch solution (5, 6 and 7%) was determinedby Brabender Viscoamylograph. Important characteristicswere recorded during the heating and cooling cycle, inclu-ding pasting temperature, viscosity at 95 °C, viscosity afterholding at 95 °C for 30 min, and viscosity after cooling to 50 °C(Table 4). The results show that considerable viscosity stabi-lity was observed throughout the heating-cooling cycle. Thisis an evidence of restricted swelling and solubilization, and ofresistance to mechanical disintegration. When the pastedstarch was cooled, setback (retrogradation) was observed.Viscosity can be considered as a measure of the strength ofstarch granules that are intact, while the starch which hasundergone chemical and microbiological damage loses vis-cosity (Moorthy, 1991). The present data indicate that TS-1starch began to swell at a slightly higher temperature thanthe TS-2 and TS-3 starches. This observation may be related

Temperature (°C)

01 02 03 04 05 06 07 0

5 0 6 0 7 0 80 90

TS -1TS -2TS -3

Sol

ubilit

y (%

)

Fig. 1. Solubilities of taro starches extracted by differentmethods (TS-1: freeze-thaw; TS-2: ammonia; TS-3:alkali).

295Taro (Colocasia esculenta) Starch

Fig. 2. Swelling power of taro starches extracted by diffe-rent methods (TS-1: freeze-thaw; TS-2: ammonia;TS-3: alkali).

40

30

20

10

050 60 70 80 90

Sw

ellin

g po

wer

(%

)

Temperature (°C)

Table 3. Moisture absorption, hydration capacity, swellingvolume and freeze-thaw stability of taro starches* extractedby using different methodsStarch Moisture Hydration Freeze-thaw Swelling

absorption capacity stability volume(%) (g/g) (%) (ml/g)

TS-1 14.25 ± 0.35 2.85 ± 0.05 62.8 ± 0.42 34.6 ± 0.50TS-2 9.50 ± 0.28 2.50 ± 0.04 50.5 ± 0.35 27.8 ± 0.45TS-3 7.85 ± 0.24 2.18 ± 0.04 43.2 ± 0.32 19.8 ± 0.42

*mean ± sd (n = 3); TS-1: freeze-thaw method; TS-2: ammoniamethod; TS-3: NaOH method

to the suggested more compact granule structure of the TS-1starch. Similarly, the TS-1 starch had high pasting tempera-ture in contrast with TS-2 and TS-3 starches. The relativelylower viscosity breakdown can be exploited in food useswhere a short non-cohesive texture is required. Chemical treat-ment disrupts the structure of starch granules, rendering themmore susceptible to hydration and swelling and eventuallycomplete disintegration takes place at a lower temperature(White et al., 1989).

ConclusionThe results of the present study have indicated that DNScolourimetric method is an inexpensive and simple method forthe determination of taro starch. The quality, as well as yield,of taro starch can be improved by using the freeze-thawextraction method. Better understanding of the functionaland physicochemical properties of taro starch may, therefore,lead to its new applications in the food industries.

References

Chapman and Hall, Glasgow, UK.Griffin, G., Wang, J.K. 1983. Industrial uses. In: Taro, A Review

of Colocasia esculenta and its Potentials, J.K. Wang(ed.), pp. 1-301, University of Hawaii Press, Honolulu,USA.

Hong, G.P., Nip, W.K. 1990. Functional properties of precookedtaro flour in sorbet. Food Chem. 69: 334.

Hussain, M., Norton, G., Neale, R.J. 1984. Composition andnutritive value of cormels of Colocasia esculenta (L.)Schott. J. Sci. Food Agric. 35: 1112-1119.

Jane, J., Shen, L., Chen, J., Lim, S., Kasemsuwan, T. 1992. Physi-cal and chemical studies of taro starches and flours.Cereal Chem. 69: 528-535.

Jaroslaw, K., Leslaw, J., Mariusz, W., Bohdan, A. 2004. In-fluence of selected hydrocolloids on tritical starch rheo-logical properties. Int. J. Food Sci. Technol. 39: 641-652.

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Medcalf, D.G., Gilles, K.A. 1965. Wheat starches: comparisonof physicochemical properties. Cereal Chem. 42: 558-567.

Moorthy, S.N. 1991. Extraction of starches from tuber cropsusing ammonia. Carbohydrate Polymers 16: 391-398.

Narkrugsa, W. 1996. Changes in some physicochemical pro-perties of tapioca and glutinous rice starches after mic-rowave heating. Kasetsart J. (Nat. Sci.) 30: 532-538.

Nyqvist, H. 1983. Saturated salt solutions for maintainingspecified relative humidities. Int. J. Pharm. Tech. Prod.Mfr. 4: 47-48.

Rickard, J.E., Behn, K.R. 1987. Evaluation of acid and enzymehydrolytic methods for the determination of cassavastarch. J. Sci. Food Agric. 41: 373-379.

Shi, X., BeMiller, J.N. 2002. Effects of food gums on viscosi-ties of starch suspensions during pasting. CarbohydratePolymers 50: 7-18.

Sugimoto, Y., Nishihara, K., Fuwa, H. 1986. Some properties ofthe taro and yam starches. J. Jpn. Soc. Starch Sci. 34: 1-5.

Tu, C., Nip, W.K., Nakayama, T. 1979. Starch and flour fromtaro. In: Small-Scale Processing and Storage of Tropi-cal Root Crops, pp. 1-249, 2nd edition, D.L. PlucknettWestview Press, Boulder, CO, USA.

Wang, J.K. 1983. Taro, pp. 4-7, University of Hawaii Press,Honolulu, USA.

White, P.J., Abbas, I.R., Johnson, L.A. 1989. Freeze-thaw sta-bility and refrigerated-storage retrogradation of star-ches. Starch/Staerke 41: 176-180.

Agbor-Egbe, T., Rickard, J.E. 1990. Evalution of acid and en-zyme hydrolytic methods for the determination of ediblearoid starch content. J. Sci. Food Agric. 53: 313-319.

AOAC. 1984. Official Methods of Analysis, 14th edition, Asso-ciation of Official Analytical Chemists, Washington DC,USA.

Chowdhury, B., Hussain, M. 1979 Chemical composition ofthe edible parts of aroids grown in Bangladesh. Indian J.Agric. Sci. 49: 110-115.

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Geirwyn, S.J. 1995. Analytical Chemistry of Food, pp. 124-125,

296 F. A. Jaffery et al.

Table 4. Viscosity of taro starches extracted using differentmethodsStarch* Starch VP V97 VH VC Pastingtype concentration (BU) (BU) (BU) (BU) temperature

(%) (°C)

TS-1 5 380 350 290 360 84-97TS-1 6 620 590 540 600 84-97TS-1 7 950 800 660 910 82-97

TS-2 5 330 320 270 310 80-83TS-2 6 580 560 470 580 80-83TS-2 7 860 700 610 880 80-84

TS-3 5 310 290 250 300 71-75TS-3 6 550 540 440 540 71-75TS-3 7 730 660 580 720 70-75

VP = peak viscosity; V97 = viscosity at 97 °C; VH = viscosity afterholding at 97 °C for 30 min; VC = viscosity after cooling to roomtemperature; *extraction method used (TS-1: freeze-thaw; TS-2:ammonia; TS-3: NaOH)

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Articles in Journal(In “References”)

Park, S.H. 2005. Fine tuning and cross-talking of TGB-β signalby inhibiting Smads. J. Biochem. Mol. Biol. 38: 9-16.

Aksu, Z., Kabasakal, E. 2004. Batch adsorption of 2,4-dichlo-rophenoxyacetic acid (2,4-D) from aqueous solution bygranular activated carbon. Separation PurificationTechnol. 35: 223-240.

Evans, W.J., Johnson, M.A., Fujimoto, Cy. H., Greaves, J. 2000.Utility of electrospray mass spectrometry for the charac-terization of air-sensitive organolanthanides and relatedspecies. Organometallics 19: 4258-4265.

(In “Text”)Park (2005), Aksu and Kabasakal (2004) and Evans et al. (2000)(Park, 2005; Aksu and Kabasakal, 2004; Evans et al., 2000)

BooksCinar, A., Parulekar, S.J., Undey, C., Birol, G. 2003. Batch Fer-

mentation: Modeling, Monitoring, and Control, MarcelDekker, Inc., NY, USA.

Chapters in Edited BooksNewby, P.J., Johnson, B. 2003. Overview of alternative rapid

microbiological techniques. In: Rapid MicrobiologicalMethods in the Pharmaceutical Industry, M.C. Easter(ed.), pp. 41-59, 1st edition, Interpharm/CRC, Boca Raton,Florida, USA.

Articles in Proceedings of Conferences, Symposia, Semi-nars, WorkshopsMarceau, J. 2000. Innovation systems in building and con-

struction, and the housing industry in Australia. In: Proc.Asia-Pacific Sci. Technol. Mangmt. Sem. National Inno-vation systems: How to Maintain a Sustainable Growthof the Asia-Pacific Region, 6th, pp. 129-156, Japan Int. Sci.Technol. Exchange Centre, Saitama, Japan.

Technical/Department ReportsSIC-PCSIR. 2002. Biannual Report, 2000-2001; 2001-2002,

Scientific Information Centre, Pakistan Council of Scien-tific & Industrial Research, PCSIR Laboratories Campus,Off University Road, Karachi, Pakistan.

ThesisSaeed, A. 2005. Comparative Studies on the Biosorption of

Heavy Metals by Immobilized Microalgal Cultures, Sus-pended Biomass and Agrowastes. Ph. D. Thesis, pp. 1-248, University of the Punjab, Lahore, Pakistan.

PatentsYoung, D.M. 2000. Thermostable Proteolytic Enzymes and Uses

Thereof in Peptide and Protein Synthesis, US Patent No.6,143,517, 7th November, 2000.

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