ISSN 0030-9885 Coden: PSIRAA 48 (5) 297-370 (2005)
Vol. 48, No. 5, September-October, 2005
Pakistan Journal of Scientificand Industrial Research
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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
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Editorial Board
Pakistan Journal of Scientific and Industrial Research
Pakistan Journal of Scientific and Industrial Research
Vol. 48, No. 5 Contents September - October 2005
Physical Sciences
On Methods Derived from Hansen-Patrick Formula for Refining Zeros of Polynominal
Equation
S. Uwamusi 297
Studies of Molecular Interactions of ααααα-Amino Acids in Aqueous and Cationic Surfactant
Systems Investigated from Their Densities and Apparent Molal Volumes at 283.15,
288.15 and 293.15K
M. Singh 303
Liquid-Liquid Equilibrium Data of Ternary Systems of Water, Acetic Acid and Alkanols
(1-Butanol, 1-Pentanol and 1-Hexanol)
M. A. Rahman, M. S. Rahman, M. N. Nabi and M. A. K. Azad 312
Synthesis and Antimicrobial Activity of Some Heterocycles: Part-V
M. M. H. Bhuiyan, K. M. M. Rahman, M. K. Hossain and M. A. Rahim 318
Combined Aerobic and Physicochemical Treatment of Pharmaceutical Industry Sludge
I. O. Asia and C. M. A. Ademoroti 322
Essential Oil Composition of Green Peel of the Inter-Varietal Mandarin Hybrid, Kinnow
Orange
S. Mahmud, A. Waheed, T. Nazir and R. Khanum 329
Biological Sciences
Biochemical Changes Induced in Some Rabbit Tissues on the Administration of an
Antimalaria Drug, Fansidar
O. I. Oloyede and M. F. Asaolu 334
Interspecific Variations in the Fecundity of Some Dominant Fish Populations in
Ikpoba River, Nigeria
L. I. N. Ezemonye and F. A. Osiezaghe 338
Numerical Taxonomy of Two New Mite Species of the Genus Caloglyphus Berlese
(Acaridae) from Pakistan
M. Sarwar, M. Ashfaq and S. Akbar 345
Cause and Effect Relationship for Some Biometric Traits in Bread Wheat
N. M. Cheema, M. A. Mian, M. Ihsan, M. A. Tariq, G. Rabbani and A. Mahmood 354
Combining Ability Analysis of Seed Cotton Yield and its Components in Cotton
(Gossypium hirsutum)
M. T. Azhar and A. A. Khan 358
Effect of Storage Fungi on the Seed Quality Parameters of Different Mustard Varieties
T. Khan, K. Khan and M. Z. Haq 362
Short Communications
Evaluation of Commercial and Candidate Bread Wheat Varieties for Durable Resistance to
Rusts in Pakistan
S. J. A. Shah, T. Muhammad and Farooq-e-Azam 366
Multiple Shoot Bud Formation and Plantlet Regeneration from in vitro Cultured Pistacia vera
Seeds
S. Jabeen and N. Zaidi 368
Pak. J. Sci. Ind. Res. 2005 48(5) 297-302
On Methods Derived from Hansen-Patrick Formula for Refining Zeros of
Polynominal Equation
Stephen UwamusiDepartment of Mathematics, University of Benin, Edo State, Nigeria
(received March 29, 2004; revised March 28, 2005; accepted March 31, 2005)
Abstract. A one-parameter family of iteration functions as derived by Hansen and Patrick (1977) was studied. The
Halley’s method was of particular interest, which was modified by using the Taylor polynomial equation of order two
to obtain the well-known Chevbyshev’s iteration formula. Further, using the Laguerre’s disk, two new methods were
constructed out of the Chevbyshev’s functional iteration fomula. The obtained methods may, and often will, depend on
the already calculated values.
Keywords: Hansen-Patrick formula, binomial series, Taylor polynomial equation, Laguerre’s disk, polynomial zeros,
Chevbyshev’s functional iteration
E-mail: uwamusi [email protected]
Introduction
The principal objective of this study was to draw attention to
the family of one-parameter iteration formulae derived by
Hansen and Patrick (1977) for finding zeros of polynomial
equation.
P(z) = 0 (1.1)
It was assumed that P was real or complex, and possessed a
certain number of derivatives necessary in the neighbourhood
zeros of p. For convenience, p was specified to be the func-
tion of z with simple zero, ζ.
Then for h (ζ) ≠ 0
where h was the reduced polynomial of p
the equation (1.1) will thus assume the form:
P = (z - ζ) h (1.2)
By taking log of both sides of (1.2) and differentiating the
resulting expression with respect to z, the following was ob-
tained:
Continuing, after some serious but rigorous analysis, Hansen
and Patrick (1977) obtained a family of functional iterative
formulae in the form:
zi
(k+1) = Φ(zi
(k)) (k = 0, 1, .....) (1.4)
where Φ is a rational map given as:
P′
P
1
z - ζ
h′
h= + (1.3)
(α + 1)pΦ(z
i
(k)) = zi
(k) _ (1.5)
α p′ ± [(p′)2 (α + 1)pp′′]½
It is remarked that α appearing in (1.5) is a variable param-
eter that rules the governing equation (1.4), which is based on
the approximation of second order derivative of h to the square
of its first derivative.
Interest was motivated by organizing the remaining parts of
the present study in the Results and Discussion section as
follows.
a. Some cubically convergent methods that could be obtained
when the values of α were in the region of (−1.1) was investi-
gated as a class of Hansen-Patrik iterative formulae. This was
done in particular, by neglecting some order of approxima-
tions higher than the term pp′′ appearing in the denominator
parts of these methods, and if it was assumed further that |p|
was sufficiently small in magnitude, then a limiting case of
Halley’s iterative formula of third order was obtained.
One disadvantage of Laguerre’s method, as well as the Euler
and Ostrowski formulae, is that they may occasionally branch
off into complex plane even if the roots of the polynomials
are real.
b. A functional iterative method was derived from the substi-
tution of Halley’s correction formula into Taylor polynomial
of order two for a function p. The obtained method was famil-
iar to the third order convergent Chevbyshev’s formula. More
useful information may be seen from Jarratt (1968).
In a second approach, the p′′ appearing in the Chevbyshev’s
formula was approximated by the first finite difference
297
approximation wherein the bound due to Laguerre was used
(Braess and Hadeler, 1973). In this way, new iterative meth-
ods were obtained. The obtained methods may, and often will,
depend on the already calculated values.
c. Finally, a sample numerical example was illustrated with
these methods and the results so obtained were noted to be
quite accurate as the solutions were approximated within 10-5
in the infinity norm.
Results and Discussion
a. A class of Hansen-Patrick iterative formulae. The val-
ues of α are crucial factors in establishing a family of meth-
ods that are iterative in nature for the determination of meth-
ods (Hansen and Patrick, 1977). For example, for α = 0, a
method due to Ostrowski (1966) was obtained from (1.4) in
the form:
zi
(k+1) = zi
(k) − p
(2.1) ± [(p′)2
− p′′]½
(k = 0, 1,.......)
By letting α = ∝, a limiting case of the class of methods (1.4)
is the Newton’s second order method:
zi
(k+1) = zi
(k) −
p(2.2)
p′
If α = 1, a method due to Euler is obtained in the form:
zi(k+1) = z
i(k) −
2p
p′ + [(p′)2 − 2pp′′]½
(k = 0, 1,.......) (2.3)
In the case of α = −1, after some minor rearrangements of
(1.4), the Halley’s method (Davies and Dawson, 1975) is
obtained in the form:
zi
(k+1) = zi
(k) − p
(2.4)
p′ −
pp′′
2p′
(k = 0, 1,.......)
Setting α = , a method due to Laguerre (Ostrowski, 1966),
and Wilkinson (1965), for instance, can be obtained in the
form:
zi
(k+1) = zi
(k) − np
(2.5) p′ ± [(n−1)2 p′2
− n(n−1)pp′′]½
(k = 0, 1,.......)
One advantageous point about method (2.4) is, that it is free
of the square root sign, which is a disadventage for methods
(2.1), (2.3) and (2.5). The presense of square root signs, in
these methods, may lead to complex roots even though the
roots of the polynomial equation are real, especially when pp′′
> (p′)2. Furthermore, the cost of evaluating the square root
signs may be prohibitively expensive.
The foregoing preliminary discussion was the source of moti-
vation for the present research study.
It was further intended to investigate as to what would happen
if α takes on rational values on the interval (−1, 1), i.e., −1< α
< 1 and the expansion of (1.4) by binomial series. A set of
methods was hoped to be obtained out of method (1.4), and
as a limiting case of these methods, it was hoped that Halley’s
formula will be obtained.
zi
(k+1) = zi
(k) − ½p
(2.6) (− ½p′) ± [(p′)2
− ½pp′′]½
First, a plus sign in the term was taken:
− ½p′ ± [(p′)2 − ½pp′′]½
The formula (2.6) was then simplified by multiplying through
the denominator and numerator parts of the weight function
by a factor of 2 to have:
zi(k+1) = z
i(k) −
p(2.7)
− p′ + [4(p′)2 − 2pp′′]½
Then the term:
[4(p′)2 −2pp′′]
½
was rewritten as:
2p′
1
−
pp′′ ½
2p′2
thus, (2.7) becomes:
zi
(k+1) = zi
(k) − p
(2.8)
− p′ + 2p′
1
− pp′′ ½
2(p′)2
(k = 0, 1,.....)
on expanding:
1 −
pp′′ ½
by the binomial series and writing: 2(p′)2
pp′′ as:
(p′)2
Q
R
1 n-1
298 S. Uwamusi
then:
1
−
pp′′ ½
= 1 − R
+ R2
+ 0(.)3 2(p′)2
4Q 32Q2
In view of the substitution pp′′
= R
it is
(p′)2
Q
,
1
−
pp′′ ½
= 1 − pp′′
+ (pp′′)2
+ 0(.)3 (2.9) 2(p′)2
4(p′)2 32(p′)4
Hence, the method (2.8) after using the right hand side of
(2.9), becomes:
zi
(k+1) = zi
(k) − p
(2.10)
p′
−
pp′′ +
p2 pp′′2
2p′ 16(p′)3
Similarly, by using the same procedure of (2.10), with α =
- 0.1, - 0.2, - 0.3, - 0.4, - 0.6, - 0.7, - 0.8, - 0.9, and 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, the following iterative
methods were obtained.
zi
(k+1) = zi
(k) − p
(2.11)
p′
−
pp′′ +
9 p′2 pp′′
2
2p′ 80 p′3
(α = - 0.1)
zi(k+1) = z
i(k) −
p (2.12)
p′
−
pp′′ +
p′2 pp′′
2
2p′ 10p′3
(α = - 0.2)
zi
(k+1) = zi
(k) − p
(2.13)
p′
−
pp′′ +
7 p′2 p′′
2
2p′ 80 p′3
(α = - 0.3)
zi
(k+1) = zi
(k) − p
(2.14)
p′
−
pp′′ +
3 p′2 p′′
2
2p′ 40 p′3
(α = - 0.4)
zi
(k+1) = zi
(k) − p
(2.15)
p′
−
pp′′ +
p′2 p′′
2
2p′ 20p′3
(α = - 0.6)
zi
(k+1) = zi
(k) − p
(2.16)
p′
−
pp′′ +
3 p′2 p′′
2
2p′ 80 p′3
(α = - 0.7)
zi
(k+1) = zi
(k) − p
(2.17)
p′
−
pp′′ +
p′2 p′′
2
2p′ 40p′3
(α = - 0.8)
zi
(k+1) = zi
(k) − p
(2.18)
p′
−
pp′′ +
p′2 p′′
2
2p′ 80p′3
(α = - 0.9)
zi
(k+1) = zi
(k) − p
(2.19)
p′
−
pp′′ +
11 p′2 p′′
2
2p′ 80 p′3
(α = 0.1)
zi
(k+1) = zi
(k) − p
(2.20)
p′
−
pp′′ +
3 p′2 p′′
2
2p′ 20 p′3
(α = 0.2)
zi
(k+1) = zi
(k) − p
(2.21)
p′
−
pp′′ +
13 p′2 p′′
2
2p′ 80 p′3
(α = 0.3)
zi
(k+1) = zi
(k) − p
(2.22)
p′
−
pp′′ +
7 p′2 p′′
2
2p′ 40 p′3
(α = 0.4)
zi
(k+1) = zi
(k) − p
(2.23)
p′
−
pp′′ +
3 p′2 p′′
2
2p′ 16 p′3
(α = 0.5)
zi
(k+1) = zi
(k) − p
(2.24)
p′
−
pp′′ +
p′2 p′′
2
2p′ 5p′3
(α = 0.6)
zi
(k+1) = zi
(k) − p
(2.25)
p′
−
pp′′ +
17 p′2 p′′
2
2p′ 80 p′3
(α = 0.7)
zi
(k+1) = zi
(k) − p
(2.26)
p′
−
pp′′ +
9 p′2 p′′
2
2p′ 40 p′3
(α = 0.8)
299Derivations from Hansen-Patrick Formula for Refining Zeros
zi
(k+1) = zi
(k) − p
(2.27)
p′
−
pp′′ +
19 p′2 p′′
2
2p′ 80 p′3
(α = 0.9)
zi
(k+1) = zi
(k) − p
(2.28)
p′
−
pp′′ +
p′2 p′′
2
2p′ 2p′3
(α = 1.0)
On further delection of additional terms of order higher than
pp′′, in each of these methods (2.10 to 2.28), the Halley’s
formula was obtained (Hansen and Patrik, 1977).
b. Derivation of Chevbyshev’s third order formula. A
method using the truncated Taylor polynomial, which followed
the Taylor series expansion, was developed for function p given
by the relation:
0 = p(zi
(k)) + (zi
(k+1) − zi
(k))p′ (zi
(k)) + ½(zi
(k+1) − zi
(k))2 p′′(zi
(k)) (3.1)
The Halley’s correction is:
H(zi
(k)) =
p(zi
(k))
p′(z
i
(k))
− p(z
i
(k))p′′(zi
(k))
2p′(zi
(k))
The following iterative formula is obtained on substituting
the term (zi
(k+1) − zi
(k))2 in (3.1) by the Halley’s correction:
zi
(k+1) = zi
(k) − p
1 + 2pp′
2 p′′
(3.2) p
(2p′2 − pp′′)2
If it is assumed that |p| is sufficiently small, and if the addi-
tional term pp in the denominator part of (3.2) is neglected,
then (3.2) will result in Chevbyshev’s method (Jarratt, 1968):
zi
(k+1) = zi
(k) − p
1 + pp′′
(3.3) p
2p′2
(k = 0, 1, .......)
It is notewortly that the substitution of (zi(k+1) = z
i(k))2 by the
Chevbyshev’s correction in the Taylor polynomial (3.1) was
not profitable, as it may be recollected that terms higher than
pp′′ were ignored in method (3.3). Thus, the optimal method
that one can obtain from methods (2.10) − (3.2) is the method
(3.3).
c. A new set of methods derivable from Chevbyshev’s
formula. The presently proposed formulae for finding zeros
of non-linear equation of single variable, referred to in the
introduction, will now be described in detail.
The term p′′(zi
(k)), appearing in the Chevbyshev’s formula,
was approximated in the form:
p′′(zi
(k)) = p′ (z
i
(k)) − p′(zi
(k-1))(4.1) z
i
(k) − z
i
(k-1)
Following carefully such ideas (Braess and Hadeler,1973), it
is known that Laguerre’s disk:
|z(k) − zi
(k-1) | < n| p(z(k))
| (4.2) | p′(z(k)) |
which contained at least one zero of p. Then using this
connection, for optimal z, the inequality (4.2) was satisfied
with equality, and the disk was thus in contact with the circle
(Braess and Hadeler, 1973):
|zi
(k) − zi
(k-1) | = n| p(z(k)) | (4.3)
| p′(z(k)) |
In view of the equality expressed in (4.3), method (4.1) may
be rewritten in the form:
p′′(zi
(k)) =
p′(zi
(k)) − p′(zi
(k-1))(4.4)
n p(z(k))
p′(z(k))
Because of (4.4), the method (3.3) takes the form:
zi
(k+1) = zi
(k) − p(z
i
(k))
1 + (p′(z
i
(k)) − p′(zi
(k-1)(4.5)
p′(zi
(k)) 2np′(zi
(k))
(k = 0, 1,....)
Since the method (4.5) made use of nonlinear information of
the degree of polynomial, there was a unique similarity with
Laguerre’s method.
Furthermore, an implicit method from method (3.4) can be
created.
Suppose, it is instead set as:
p′′(zi
(k)) = (p′(z
i
(k)) − p′(zi
(k+1)(4.6)
(z
i
(k)) − (zi
(k+1))
then, as before, an implicit iterative formula is obtained:
zi
(k+1) = zi
(k) − p(z
i
(k))
1 + p′(z
i
(k)) − p′(zi
(k+1))(4.7)
p′(zi
(k)) 2np′(zi
(k))
(k = 0, 1,....)
Method (4.7) is not self-starting. It thus requires the
results of other methods. For this purpose, we introduced
the use of Newton’s second order method that served this
300 S. Uwamusi
purpose as the predictor, while method (4.7) acted as the
correction. Hence, in this case, the case of predictor-
corrector formula was obtained.
Further information on the use of predictor-corrector formu-
lae may be gained from Lambert (1974), and Kung and Traub
(1974). As it were, when the computed zi
(k) from the corrector
method was sufficiently close to the result computed from the
predictor formula, then it may be noted from the computation
that the term:
p′ (zi
(k)) − p′(zi
(k+1))
2np′(zi
(k+1))
was sufficiently close to the origin. In such a circumstance,
the method (4.7) differs only very little from Newton’s
formula:
zi
(k+1) = zi
(k) −
p′(zi
(k))
p′(zi
(k))
(k = 0, 1,....)
and is hence uniformly bounded, away from the origin, on the
compact interval containing the zero of p.
d. Numerical experiment. Consider the following numerical
test problem.
p(z) = z7 − 28z6 + 322z5 − 1960z4 + 6769z3 −1312z2 + 13068z
− 5040 = 0
By taking the initial starting root to z(0) = 8
Based on this inital starting root, the values obtained using
various methods are shown in Table 1. It may be noted from
Table 1 that each of these methods is condensing to the
numerical value of 7, which is the true zero of p. The conver-
gence of these methods is monotonic, i.e., Φ(z(k+1)) ⊆ Φ (z(k)),
where z(k+1) ⊆ z(k) as k →∝.
Because || Φ(z(k+1))|| ⊆ || Φ (z(k))||
then z = ∩ cl Φ (z(k))
where z is the inductive limit of the sequence (zi
(k+1))
Φ(z(k)) is bounded below on D0, z(0) ⊂ D
0
then limk→∝
[Φ(z(k)) − Φ (z(k+1)] = 0 on the basis of inverse func-
tion theorem.
hence [Φ (z(k)) + c (z(k+1)) − z(k))] ≤ Φ (z(k)), c ∈ [0,1]
thus z(k) ∈ c(0)[Φ (z(0))]
where it might be defined as c(0) [φ(z(0))] = z∈ D: φ(z(k)) ≤ φ
(z(0))
It is remarked that the orders of convergence of methods
(4.5) and (4.7) cannot be less than three in the sense of Alefeld
and Herzberger (1974), as well as Kung and Traub (1974).
Of all the methods tested, Laguerre’s method is the fastest,
but one drawback of Laguerre’s method is that it requires
the computation of square root, which is quite expensive.
Amoung the presently proposed methods, method (4.5)
appears to be stable, as it does not exhibit the problem of
cycling, whereas both Halley’s and Chevbyshev’s formulae
have this tendency to “cycle”. Methods (4.5) and (4.7) also
do not require second order differentiation of a polynomial
equation. As can be observed from the foregoing discussion,
the advantages and disadvantages of each of these methods
as listed in Table 1 may swing either way.
Table 1. Values for various methods derived from Hansen-Patrik formula (Hansen and Patrik, 1977) for refining zeros of
polynomial equation
Number of Halley’s Chevbyshev’s Proposed Proposed Newton’s Laguerre’s
iterations method (2.4)* method** method (4.5)* method (4.7)* method (2.2)* method (2.5)*
0 8 8 8 8 8 8
1 7.614325063 7.464851443 7.58677686 7.59846325 7.614325069 7.05471012
2 7.162354128 7.137917042 7.335685949 7.302129212 7.568219771 7.000036151
3 7.0009143675 7.011492649 7.151255677 7.1103923 7.291817563 6.999999990
4 7.000001835 7.000015521 7.045419265 7.06378217 7.110719918 7.000000009
5 7.000000529 6.999999646 7.005235768 7.00745791 7.022458237 7.000000019
6 6.999999535 7.00013132 7.000114957 7.001156183 7.000000000
7 6.999999712 7.000000906 7.00000328
8 7.000000517 6.99999903
*reference to equation numbers in the text; **Chevbyshev’s method (Jarratt, 1968)
301Derivations from Hansen-Patrick Formula for Refining Zeros
Conclusion
The present study investigated the family of iteration formu-
lae for refining zeros of a polynomial presented by Hansen
and Patrick (1977), from which the Halley’s method was a
particular case study. It was noted that when |p(z)| was suffi-
ciently small in magnitude, a family of methods obtained from
equation (1.4) for the variable parameter α [α ∈ (−1,1)] was
reduced to the well known Halley’s method (2.4). By using
the Taylor polynomial equation of order two, and substituting
the Halley’s correction formula, the well known Chevbychev’s
formula for finding zeros of a polynomial was rediscovered.
It is further remarked that the Chevbychev’s formula obtained
through this process is the optimal method. Furthermore, two
new formulae were derived from this Chevbychev’s functional
iteration method by using Laguerre’s disk. Thus, the obtained
methods may, and often will, depend on the already calcu-
lated values. These methods were illustrated on a polynomial
and the results obtained were quite accurate in comparison
with other known formulae.
References
Alefeld, I.G., Herzberger, J. 1974. On the convergence
speed of some algorithms for the simultaneous approxi-
mation of polynomial roots. SIAM J. Numer. Anal. 11:
237-243.
Braess, D., Hadeler, K.P. 1973. Simultaneous inclusion
of the zeros of a polynomial. Numer. Math. 21:
161-165.
Davies, M., Dawson, B. 1975. On the global convergence
of Halley’s iteration formula. Numer. Math. 24:
133-135.
Hansen, E., Patrick, M. 1977. A family of root finding meth-
ods. Numer. Math. 27: 257-269.
Jarratt, P. 1968. The use of comparison series in analysing
iteration functions. Comp. Journal 11: 314-316.
Kung, H.T., Traub, J.F. 1974. Optimal order of one-point
and multipoint iteration. J. Comp. Machinery 21:
643-651.
Lambert, J.D. 1974. Computational Methods in Ordinary
Differential Equations, Chapters 1-4, John Willey and
Sons, New York, USA.
Ostrowski, A.M. 1966. Solutions of Equations and Sys-
tems of Equations, Chapter 15, Academic Press, Lon-
don, UK.
Wilkinson, J.H. 1965. The Algebraic Eigenvalue Problem,
Chapter 7, Oxford University Press, Ely House, London
W.1., UK.
302 S. Uwamusi
Introduction
The limiting density (ρ0) and apparent molal volume (Vφ
0) are
fundamental properties of binary and ternary solutions of
non-ideal systems of amino acids and surfactants (Ali and
Nain, 2002; Singh et al., 2001). Such thermodynamic func-
tions of biomolecules in mixed solvents are of significant
interest as they influence their activity to a great extent
(Apelblat and Manzurola, 1999). These properties can be
used to obtain the desired polarity of solvents with surfac-
tants (Creighton, 1990; Dill, 1990; Frank, 1973). The surfactant
systems have been, therefore, drawing attention for the deter-
mination of their activity, and the activity coefficients of
amino acids and peptides in aqueous and non-aqueous sol-
vents (Mohammad and Wahab, 2002; Cabani et al., 1972).
Surfactants are of immense significance due to their interac-
tion with hydrophobic and hydrophilic parts of the amino
acids (Barbosa et al., 2001). They also assist in the partition of
amino acids, if added to immiscible solvents in combination
(Sandberg and Edholm, 2001; Key and Weitzman, 1987;
Monica and Bufo, 1977; Frank, 1973). Further, the size of the
anion associated with N+ ion of the pyridine ring of the surfac-
tants is seen to influence the activity of the surfactant itself,
and of biomolecules (Mohammad and Wahab, 2002; Sandberg
and Edholm, 2001). The polar head of α-amino acids (hydro-
philic) is confined to one end, and the alkyl chain (hydropho-
bic) to another, similar to the alignment of hydrophilic and
hydrophobic parts in surfactants (Singh, 2001). How does an
increase in the alkyl chain of amino acids affects their interac-
tion with water and aqueous surfactant solutions is of primary
interest. Studies undertaken elsewhere, on different solvents,
do not elucidate the mechanism of interactions of surfactants
with amino acids (Frank, 1972-1983). As amino acids are also
related to acetyl coenzyme-A (acetyl-CoA) of the Kreb’s cycle
(Key and Weitzman, 1987), it may be of interest to understand
how the enzyme functions with amino acids in surfactant
solutions in biological processes, since the enzyme catalyzes
cellular reactions and amino acid interactions. Therefore,
molar volumes of acids can be rationalized with the stability of
proteins and the activity of an enzyme to recognize and bind
its substrate. How do molal volumes of amino acids vary in
surfactant solutions is not yet known (Barabosa et al., 2001;
Frank, 1972-1983), since molar expansion/contraction affect
interactions of biofluids/biochemicals (Rialdi and Blitonen,
1973). Therefore, the density (ρ) and molal volume (Vφ) proper-
ties of amino acids are helpful in elucidating the structural
interactions and reorientations of enzymes with their substra-
tes (Singh and Kumar, 2004; Goss, 2003; Sharma et al., 1992;
Crawford et al., 1977; Nemethy and Scheraga, 1974). Also, the
ρ and Vφ being state-related functions, these can depict transi-
tion of systems that may be of some use for non-ideal solu-
tions (Singh, 2005; Parmar and Dhiman, 2002; Singh, 2001;
Pandey et al., 1998). Thermodynamic investigations on mac-
romolecules and biomolecules in aqueous systems have
always been a matter of interest (Singh, 2004), so as to focus
the structural reorientation and conformational states regar-
Pak. J. Sci. Ind. Res. 2005 48(5) 303-311
Studies of Molecular Interactions of α-Amino Acids in Aqueous and
Cationic Surfactant Systems Investigated from Their Densities and
Apparent Molal Volumes at 283.15, 288.15 and 293.15 K
Man SinghChemistry Research Laboratories, Deshbandhu College, University of Delhi, New Delhi-110019, India
(received October 21, 2003; revised April 21, 2005; accepted May 20, 2005)
E-mail: [email protected]; Tel: 091-011-26217579
Abstract. Density (ρ/103 kg m-3) and molal volumes (Vφ/10-6 m3 mol-1) of glycine, valine and leucine from 0.03 to 0.07 mol
kg-1, and cetyl pyridinium chloride (CPC) and cetyl pyridinium bromide (CPB) were measured in 0.0497 mol kg-1 aqueous
surfactant solution systems at different temperatures. The data were regressed against molality, and constants were
referred to as the limiting density (ρ0) and apparent molal volumes (Vφ
0) and denoted as solute-solvent interactions, while
their slope constants indicated molecular interactions and influence of composition. It was observed that amino acids with
a shorter alkyl chain, such as glycine, had weaker affinity to interact with cationic surfactants, in comparison with the
longer alkyl chain amino acids, such as leucine. The CPB with larger-sized anion showed greater molecular interaction with
amino acids.
Keywords: pyridinium ring, intermolecular forces, hydrophobic interactions, transfer volume, cationic surfactants
303
ded as valuable for the determination of activity and activity
coefficients (Singh and Chand, 2005; Rialdi and Biltonen,
1973). Thus, the present studies were understood to have
biological, biotechnological and biophysical significance, as
such kinds of chemical species enter the cellular and intracel-
lular matrix where their size could exert some pressure on the
membrane systems. The molecules with hydrophobic-hydro-
philic ends, like valinomycine protein, monitor the transport
of various cations such as Na+ and K+ from aqueous to lipid
phase through permeable membrane in the cells. The present
studies may, therefore, be of some use to understand the car-
rier and channel mechanism for transporting of several ions
performed by such molecules in the body.
Materials and Methods
Glycine, valine and leucine (AR, Sigma), and cetyl pyridinium
chloride (CPC) and cetyl pyridinium bromide (CPB) (E. Merck)
were dried and stored on P2O5 in a dessicator, before use.
Solutions (w/w) were prepared in triple-distilled, deionised and
degassed water, which was prepared by distilling deionized
water in the presence of KMnO4 and KOH twice and degas-
sed by boiling. Densities and volumes were measured with a
25x10-3dm3 capacity bicapillary pyknometer at constant tem-
perature within ±0.1 °C. The temperature was maintained by
an automatic electric relay attached to a contact thermometer
and a 25-watt immersion rod, together with circulation of cold
water through a copper coil immersed in a waterbath. The
pyknometer with solutions was weighed within an accuracy
of 0.01 mg using Dhona balance, model 100 DS. The pykno-
meter was calibrated with water of 1x10-7Ω
-1conductivity. The
reproducibility in weights was checked immediately after ta-
king the measurement and was found to be 1x10-5/103kg/m3.
Results and Discussion
The density (ρ) and molal volume (Vφ) data were calculated
from weights as:
ρ = W x ρsolv
/W0 + 0.0012 (1- W / W
0) (1)
Vφ = 1/ρ [M
2- (1000/m) (W - W
0) / (W
0- W
e)] (2)
where:
ρ and ρ
solv = densities of the solution and solvent, respectively
0.0012 (1-W / W0) = buoyancy correction of air
m = molality of the solution
M2 = mol wt of the solute
We, W
0, W = wts of empty, with solvent-, and with solution-
filled pyknometer, respectively
Uncertainty in Vφ data was computed from equation (3) given
below:
Vφ = (1000/m)∆ρ (3)
where:
m = molality
∆ρ = a calculated value of 1/10-5, representing uncertainty in
wt measurements
Uncertainties were found within the permissible range of the
standard deviation. The obtained data were regressed as:
ρ = ρ0 + Sd m (4)
Vφ =
V
φ
0 + Sv
m (5)
where:
ρ0 (103 kg m-3) and V
φ
0 (10-6 m3 mol-1) = limiting values of density
and molal volumes, respectively
Sd (103 kg2 m-3mol-1) and S
v (10-6 m3 kg mol-2) = the slope cons-
tants of density and molal volumes, respectively, as given in
Tables 1 and 2
Vφ
0 and Sv = solute-solvent dipole-dipole interactions, respec-
tively, depending on the amount of charge of the solute and
the nature of solvent molecules
The transfer limiting densities (ρ0tr) and transfer limiting molal
volumes (Vφ
0tr) from aqueous to aqueous surfactant solution
systems were calculated as:
ρ0tr = ρ0 (ternary) - ρ0 (binary) (6)
Vφ
0tr = Vφ
0 (ternary) - Vφ
0 (binary) systems (7)
The calculated values are given in Table 3 and the contribu-
tion of the -CH2 group to ρ0 and V
φ
0 was evaluated from equa-
tion (8) given below:
ρ0 = ρ0 (leucine) - ρ0 (valine); and
Vφ
0 = Vφ
0 (leucine) - Vφ
0 (valine) (8)
The values calculated from equation (8) are shown in Table 4.
The constants of regression of the limiting data against tem-
peratures are given in Table 5. For binary systems, the density
of water was taken from literature (Apelblat and Manzurola,
1999). The Vφ
0 of the amino acids in the surfactant solutions
are drawn in Fig. 1. The ρ of 0.05 to 1.25 mol kg -1 aqueous
NaCl was measured for Vφ
0 values, which were reproduced
to ±0.05/10-6 m3 mol-1 as compared to the data available in lite-
304 M. Singh
rature (Apelblat and Manzurola, 1999), which authenticated
the measurements. The water densities were taken from litera-
ture and an increase and decrease in the ρ values with concen-
tration and temperature indicated the forming and breaking
of the hydrogen bonds. The ρ0 values of aqueous glycine as
related with temperature was noted as 293.15 > 288.15 > 283.15
K, with reverse order of Sd, and ρ0 values of the amino acids
at each temperature were noted as glycine > valine > leucine.
The order of ρ0 of amino acids proved that α-amino acids with
the increase in their alkyl chain resulted in lowering in ρ0 due
to larger hydrophobic interactions with the addition of -CH2
group in the amino acid molecules. It proved that α-amino
acids in aqueous solutions had greater force of attraction
between dipoles of water and their polar group, as compared
to the hydrophobic part, which lacked the possession of such
forces. Similarly, the trend of Sd values was seen as leucine >
glycine > valine, which predicted a greater composition de-
pendence of leucine at each temperature. It indicated stron-
ger hydrophobic interactions of the amino acids with the
larger alkyl chain. However, prominent hydrophilic interac-
tions of glycine seemed to be strengthened with composition,
yet still remained weaker than that of leucine and valine. This
behaviour of amino acids could be attributed to the distance
between hydrophobic and hydrophilic heads due to the size
of the alkyl chain.
The surfactants in aqueous systems was observed to have
an order of ρ as CPB > CPC, which showed that pyridinium
surfactants of the same composition with Br- anion destabi-
lized the water structure to a larger extent as compared to Cl-
(Table 2). It showed that an induced dipole of larger anion
remained effective even when it was associated with N+ ion
of the pyridinium ring of the cationic surfactants. On transfer
of amino acids in aqueous cationic surfactant solutions, the
order of their ρ0 values differed from the values of aqueous
systems and were noted as leucine > valine > glycine at each
temperature, which was reverse as compared to the values for
amino acids in the aquous systems. From this it may be con-
cluded that there existed a stronger force of attraction bet-
ween water and glycine, indicating glycine to be a stronger
structure breaker in the aqueous medium, as dipoles of water
and zwitterion of glycine are of smaller size, thereby stronger
hydrophilic interactions occurred. But with surfactant sys-
tems, the glycine was found to attain the lowest and leucine
the highest ρ0 values, which proved that surfactants dubbed
the hydrophilic interaction of glycine and strengthened the
hydrophobic interaction of leucine in the same proportion.
This explains how the cetly chain (alkyl chain of sixteen car-
bon atoms) of surfactants developed considerably stronger
Table 1. Regression analysis of density (ρ) and molal volume
(Vφ) data of aqueous amino acids and surfactants against
molality, and of amino acids-aqueous surfactant systems,
their limiting constants at infinite dilution and slope constants
at different temperatures
Tempera- Limiting Limiting molal Slopeture density volume constant
(K) (1x104)
ρ0 S
dVφ
0 Sv
SV
Aqueous glycine
283.15 0.99965 0.0043 41.92 0.46
288.15 0.99916 0.0065 42.37 0.47
293.15 0.99887 0.0066 42.91 0.53
Aqueous valine
283.15 0.99900 -0.2003 170.56 -763.21
288.15 0.99930 0.0053 147.38 -197.78
293.15 0.99870 -0.0030 141.99 -158.63
Aqueous leucine
283.15 0.99740 0.0489 344.17 -4112.90
288.15 0.99650 0.0477 413.53 -4766.70
293.15 0.99680 0.0147 579.04 -13256.00 10.38
Glycine in 2% CPC
283.15 0.99740 0.0415 4741.80 -114280.00 83.58
288.15 0.99640 0.0484 2970.40 -65957.00 44.49
293.15 0.99590 0.0405 1597.80 -35713.00 23.93
Valine in 2% CPC
283.15 0.99790 0.0237 3932.10 -81435.00 53.65
288.15 0.99740 0.0025 2641.80 -53626.00 35.80
293.15 0.99600 0.0455 1606.80 -36142.00 24.65
Leucine in 2% CPC
283.15 0.99820 0.0134 3909.60 -80883.00 53.90
288.15 0.99770 0.0199 2392.90 -48723.00 32.07
293.15 0.99690 0.0231 1239.30 -26720.00 18.56
Glycine in 2% CPB
283.15 0.99840 0.0426 3608.30 -147682.00 200.00
288.15 0.99710 0.0417 2821.40 -119075.00 200.00
293.15 0.99700 0.0436 1892.80 -79585.00 100.00
Valine in 2% CPB
283.15 0.99960 0.0334 3401.80 -144219.00 200.00
288.15 0.99830 0.0133 2512.20 -105186.00 100.00
293.15 0.99800 0.0158 1684.70 -70365.00 95.67
Leucine in 2% CPB
283.15 0.99880 0.0347 3718.80 -152616.00 200.00
288.15 0.99850 0.0320 2247.50 -91844.00 100.00
293.15 0.99740 0.0512 1663.30 -66873.00 81.69
ρ0 = 103 kg m-3; S
d = 103 kg2 m-3 mol-1; Vφ
0 = 10-6 m3 mol-1; Sv = 10-6 m3
kg mol-2; SV´ = 10-6 m3 kg2 mol-3; ρ0 = limiting density; S
d = slope
constant for limiting density; Vφ
0 = limiting molal volume; Sv = slope
constant for limiting molal volume; SV´ = slope constant of m, mola-
lity of surfactants
305Interactions of Amino Acids in Aqueous/Cationic Surfactant Systems
hydrophobic interactions with the alkyl chain of leucine than
of hydrophilic chain of glycine. These states of hydrophobic
interactions among them developed stronger force of attrac-
tion between the dipoles of water and glycine, and the change
in the behaviour of leucine in the surfactant systems was
attributed to the -CH2 group.
Comparison of the values of ρ0 of amino acids in the surfac-
tant systems have shown that they were higher by 0.00043/
103 kg/m3 for CPB than for CPC at each temperature. The
values of ρ0 of the aqueous amino acids were higher by
0.00022/103 kg/m3 than of surfactant solutions, which
indicated that surfactants weakened the intermolecular
forces that existed between the amino acids and water. Thus,
surfactants behaved as structure breakers for amino acids in
aqueous solutions and proved that α-amino acids, with larger
alkyl chain in the surfactant solutions, developed stronger
hydrophobic-hydrophobic and hydrophilic hydrophilic
interactions with surfactants. These interactions were noted
to cause certain forces between the alkyl part of the amino
acids and surfactants, and the polar part of the amino acids
and surfactants, respectively. These molecular forces caused
an additional internal pressure resul-ting in shrinkage or
contraction among the acid-surfactant complexes with an
increase in ρ0. It may thus be concluded that amino acid-
306 M. Singh
Fig. 1. The apparent molal volume (Vφ
0) of amino acids in surfactant solutions at different temperatures (CPC = cetyl
pyridinium chloride; CPB = cetyl pyridinium bromide).
Appare
nt
mola
l volu
me (
Vφ
0)
Temperature (K)
4750
4650
4550
4450
4350
4250
4150
4050
3950
3850
3750
3650
3550
3450
3350
3250
3150
3050
2950
2850
2750
2650
2550
2450
2350
2250
2150
2050
1950
1850
1750
1650
1550
1450
1350
1250
1150
282.15 283.15 284.15 285.15 286.15 287.15 288.15 289.15 290.15 291.15 292.15 293.15
glycine in 2% CPC
valine in 2% CPC
leucine in 2% CPC
glycine in 2% CPB
valine in 2% CPB
leucine in 2% CPB
surfactant and amino acid-water-surfactant interactions
seem to be integrated. Further, the larger magnitude of ρ0
values of α-amino acids in CPB surfactant with Br- anion
showed that larger-sized anion was effective and gene-rated
higher intermolecular forces. Probably, the induced dipole
of Br- anion, due to its larger size and 4d empty orbital, stron-
ger London or dispersive forces were generated and led to
polar centre on the outer surface of Br- anion.
Apart from the order of ρ0 values, which demarcated the
solute-solvent interactions, the Sd values of the amino acids
in CPC and CPB surfactants were noted as glycine > valine >
leucine at each temperature, which with temperature was
283.15 > 288.15 > 293.15 for each amino acid, respectively.
Such trends of Sd values proved a weaker effect of composi-
tion on amino acid-amino acid interactions with the size of
alkyl chain. Perhaps, the stronger hydrophobic interaction of
the larger-sized alkyl chain dominated over the interaction of
amino acids caused by their polar centres. Thus, it may be
concluded that an over-weighing of hydrophobic interactions
weakened the composition effect on homomolecular interac-
tions. In general, the Sd values of amino acids with CPB were
found to be higher than that of CPC at each temperature and
indicated that the effect of composition on interactions was
greater at the lower temperature, which weakened with tem-
perature for each amino acid. This can be correlated to the fact
that thermal energy too had structure-breaking influence, and
hence the thermal factor became an additive to destabilizing
the water structure and for the establishment of molecular
interactions. Also, Br-, a larger-sized anion in a state of in-
duced dipoles, facilitated a structure-breaking influence of
surfactants on water. A contribution of the -CH2 group to ρ0
of α-amino acids (Table 4) with temperature was: 283.15 >
293.15 > 288.15 K; and for surfactants as 293.15 > 288.15 >
283.15, and 288.15 > 293.15 > 283.15, for CPC and CPB, respec-
tively. This proves that an addition of -CH2 group to α-amino
acids decreased the ρ0 as it was noted that leucine had nega-
tive values as compared with valine, which denoted that more
were the -CH2 groups in the alkyl chain, less was the ρ0 value,
thus a decrease was greater at 288.15 K. But CPC had changed
this behaviour of -CH2 group as the ρ0 values were found to be
positive for the amino acids indicating a stronger hydropho-
bic interaction of the cetyl chain of CPC with that of the -CH2
group of leucine.
The CPB produced higher ρ0 values, which followed the
same trend for the amino acids in aqueous systems except
at 288.15 K, and the ρ0 values of amino acids from aqueous
to aqueous surfactants was found as: leucine > valine >
glycine. But ρ0tr of CPC was greater than of CPB with a
307Interactions of Amino Acids in Aqueous/Cationic Surfactant Systems
Table 4. The apparent molal volume (Vφ
0) and limiting density
(ρ0) values of -CH2 group of amino acids at different tempera-
tures, obtained by deducting the Vφ
0 and ρ0 values of leucine
from valine as:
(-CH2-) = [(CH
3)
2CH
2CH(NH
2)COOH)]-[(CH
3)
2CH-CH(NH
2)
COOH]
283.15 K 288.15 K 293.15 K
Vφ
0ρ
0 Vφ
0ρ
0 Vφ
0ρ
0
-CH2- values evaluated from (leucine - valine) in aqueous systems
173.61 - 0.0016 266.15 - 0.0028 437.05 - 0.0019
Amino acids in aqueous surfactant systems
Aqueous CPC
- 22.50 0.0003 - 248.90 0.0003 - 367.50 0.0009
Aqueous CPB
317.00 - 0.0008 - 264.70 0.0002 - 21.40 -0.0006
Table 3. Transfer volume, Vφ
0tr (10-6m3 mol-1) and transfer limi-
ting density, ρ0 tr (103 kg m-3) of glycine, valine and leucine
from aqueous to aqueous CPC and CPB surfactant solutions
Temp Vφ0tr ρ
0tr Vφ0tr ρ
0tr Vφ0tr ρ
0tr
(K)
Amino acids from aqueous to aqueous CPC surfactant
glycine valine leucine
283.15 4699.88 - 0.00225 3761.54 - 0.0011 3565.43 0.0008
288.15 2928.03 - 0.00276 2494.42 - 0.0019 1979.37 0.0012
293.15 1554.89 - 0.00297 1464.81 - 0.0027 660.26 0.0001
Amino acids from aqueous to aqueous CPB surfactant
glycine valine leucine
283.15 3566.38 - 0.00125 3231.24 0.0006 3374.63 0.0014
288.15 2779.03 - 0.00206 2364.82 - 0.001 1833.97 0.002
293.15 1849.89 - 0.00187 1542.71 - 0.0007 1084.26 0.0006
CPC = cetyl pyridinium chloride; CPB = cetyl pyridinium bromide
Table 2. Limiting density (ρ0) and apparent molal volumes (Vφ
0)
of cetyl pyridinium chloride (CPC) and cetyl pyridinium bro-
mide (CPB) surfactants in aqueous solution systems at diffe-
rent temperatures
Molality Temperature (K)
(mol kg-1) 283.15 288.15 293.15
ρ0 Vφ
0ρ
0 Vφ0
ρ0 Vφ
0
Aqueous CPC
0.0497 1.00564 -53.59 1.00224 141.96 0.99905 301.20
Aqueous CPB
0.0497 1.00623 -106.59 1.00289 108.30 1.00103 183.48
difference of ±2x10-4
/ 103 kg/m3. The ρ0 and the Vφ
0 values,
calculated together from the weights of solutions, were con-
sidered as independent measurements and hence the trend
of Vφ
0 values was considered useful for the investigation.
In general, the Vφ
0 values of glycine and leucine in aqueous
systems were found to increase with temperature, while
valine reported a considerable decrease. For instance, for
glycine, the Vφ
0 value was ±2.04/10-6 m3 mol-1 and for leucine
it was ±100/10-6
m3 mol-1
. However, the order of decrease in
Vφ
0 values for valine from 283.15 to 288.15 K was ±23, and
between 288.15 and 293.15 K was ±5/10-6 m3 mol-1. It con-
firms that in the case of glycine molecule, sandwiching of
its hydrophobic sphere of -CH2 group by the hydrophilic
sphere of polar -COO- and -NH3
+ groups occurred, while
the sandwiching effect for leucine hydrophobic interactions
was weaker because of slightly stronger hydrophobic in-
teractions due to its longer [-CH (CH3)
2] chain. Thus, hy-
drophobic interaction of leucine dominated over those of
glycine.
At each temperature, Vφ
0 values of amino acids for the
aqueous and surfactant systems were found to have a
linear relationship with the composition. From 0.03 and
0.05 mol/kg surfactant, however, the Vφ
0 of amino acids
decreased, whereas for 0.07 mol/kg the values increased.
This trend of values fits to the polynomial relation of Vφ
0
values against the composition of surfactants. Table 1
shows that the Sv´ values (slope constant of m, molality
of surfactants) were positive, ranging from 18 to 83x104/
10-6 m3 kg2 mol-3, which indicate that surfactants devel-
oped micelle of mild nature with amino acids. Not-ably,
the Sv´ values for CPB were found to be higher than for
CPC, thus predicting comparatively dominating micelle
for-mation with the former. The Vφ
0 values decreased con-
tinuously with temperature by almost 1000/10-6m3/mol.
With temperature, a change in the trend of Vφ
0 values
weakened the intermolecular forces due to thermal
energy, which seemed to be least effective for valine as it
had almost a balance between the interactions of polar
groups [-(NH3
+)(COO-)] and its hydrophobic [-CH(CH3)
2]
groups. Such kinds of arrangements of molecular forces
have shown a strengthening of the forces in aqueous
solutions with its composition. Similarly, the magnitude
of Sv value of glycine was positive and increased with
temperature, whereas the values for valine and leucine
were negative, which for valine, however, increased with
temperature while decreased for leucine. The magnitude
of increase of values for glycine was ±0.03, whereas
for valine it was ±55/10-6 m3/mol and for leucine it was
±150/10-6
m3/mol. These trends prove that for glycine,
the temperature enhanced the glycine-glycine and
aqueous-glycine interactions, which were weakened in
the case of valine and leucine (Singh, 2004). Also,
wea-ker valine-valine, aqueous-valine, leucine-leucine,
and aqueous-lencine interactions, as related with
temperature, lead to the the conclusion that valine and
leucine strengthened cage formation of water around their
alkyl chains. The larger Vφ
0 values of valine and leucine
too indicate that a cage formation around the chains was
of structured water and partly hydrated water due to their
polar groups. It seems that both the structured and
hydrated waters have a grip over their hydropho-bic and
hydrophilic parts, respectively, of these amino acids, thus
establishing a better coordination among the caged and
hydrated parts. Such an orientation of water around the
amino acid molecules seems to result in larger values
with valine and leucine, and a further trend of Vφ
0 values
proved that this grip was strengthened at the higher
temperature in the case of leucine, while weakened for
valine, due to the larger size of alkyl chains.
Similarly, amino acids in aqueous surfactants have larger
Vφ
0 values, which were in the range of 1000/10-6m3/mol
for amino acids (Fig. 1), than those in the aqueous medium
only, resul-ting in higher transfer values (Table 3). The
trend of Vφ
0 for the amino acids with temperature in
aqueous CPC was observed as glycine > valine > leucine,
confirming a binding of polar group of glycine with N+ of
pyridine ring of CPC and cetyl chain surroundeding the
glycine. Alternatively, the glycine could also surround the
polar part of the surfactant. Yet another possibility is that
hydrated water may also establish interaction with CPC
and get attached with glycine. Such a complex must be of
larger in size, as the amino acids have larger Vφ
0 values in
aqueous surfactant systems. Surprisingly, with tempera-
ture, the Vφ
0 values of amino acids in aqueous surfactants
marked an observable decrease of the same order for both
the surfactants, i.e., 800/10-6m3/mol. In general, molecules
or the interconnected complexes expanded with tempera-
ture, but with surfactants there was noted a gradual
decrease. This proved that with amino acid-surfactant
interactions, thermal energy played a crucial role for the
stability of interaction and the surfactant molecules
associated with the amino acids. This indicated a thermal
destabilization, thus detaching the surfactant molecules
either from acids or the hydrated water, which after
detachment may form smallersized interaction complexes.
However, this decrease was higher with CPC than with
308 M. Singh
CPB, thus proving that surfactants with smaller anion
Cl- had less stability with the amino acids, or hydrated
water of acids. It can be attributed to an induced dipole
of Br- with a slightly higher binding force than of Cl-.
Surfactants with Cl-, therefore, do not apply stronger
intermolecular force in the aqueous systems.
The composition of surfactants was noted to produce
negative Sv values for amino acids, which further increased
with temperature for both the surfactants. But Sv values
of amino acids for CPB systems were found lower than
those of CPC. The values were of the order of _
65x103/
10-6 m3 kg mol -2, which indicated that composition of
surfactants from 0.03 to 0.07 mol/kg established
structure-making effect on water. It may be noted from
Table 1, that glycine among amino acids had stronger
structure making effect in surfactant solutions at each
temperature. However, an increase in Sv values with
temperature proved that the cage effect decreased, with
the thermal contents disrupting the cage formation/
geometry/ dyna-mics, and an influence was found in the
order of glycine > valine > leucine in surfactants. It
matched well with the structure of acids as there was
least stability of the cage model of water with glycine.
Surfactants, however, seemed to cement cage formation,
which however did not remain stable with temperature.
The Vφ
0tr values (Table 3) of amino acids indicated a greater
effect at lower temperature, with a greater effect at 283.15
and 288.15 for CPC than for CPB. At 293.15, CPB caused
greater decrease in Sv values proving stronger caging of
water aro-und amino acids, or the CPB molecule at the
higher temperature. The ρ0 and V
φ
0 of amino acids, as
affected by temperature in the aqueous medinium, were
glycine > leucine > valine, and the aqueous surfactants as
valine > glycine > leucine (Table 5). This proved that a
greater effect of temperature, on glycine in aqueous
systems, may be due to a greater attraction between polar
groups. Additionally, surfactants decreased and increased
ρ0 and V
φ
0 of glycine and valine, respectively, as compared
with the aqueous systems, indicating a dominance of
hydrophobic interactions with valine over hydrophilic
interactions due to the presence of polar groups. The Sd(T)
for aqueous systems of acids was found in the order of :
valine > leucine > glycine, whereas for CPC and CPB
systems it was glycine = leucine > valine and glycine =
valine = leucine, res-pectively. This points to the fact that
temperature sensitized valine as it had polar and hydro-
phobic groups just adjacent to each other, thus leucine
was the next to glycine in respect of the temperature
effect on intermoleculer interactions.
The amino acids in aqueous systems, in relation with
temperature, showed Vφ
0 as leucine > valine > glycine,
and Sv(T) as glycine > valine > leucine. V
φ
0 in CPC and
CPB were glycine > leucine > valine, and leucine > gly-
cine > valine, respectively. However, Sv(T) was valine >
leucine > glycine in CPC, and glycine > valine > leucine
in CPB (Table 5), Thus proving a compact linkage of
leucine at the absolute zero degree temperature.
Conclusion
The ρ and Vφ, and regressed data for a series of selected
amino acids with an increase of -CH2 group in their alkyl
chains were noted to enhance the hydrophobic and weak-
Table 5. The limiting density (ρ0) and apparent molal volume
(Vφ
0) data with slope constants for binary and ternary systems
obtained by regressing against temperature
ρ0 (T) Vφ
0 (T)
ρ0(T) S
d(T) Vφ
0(T) Sv(T) S
v(T)
Aqueous glycine
1.0217 - 8.00x10-5 13.87 0.10
Aqueous valine
1.0076 - 3.00x10-5 976.55 - 2.85
Aqueous leucine
1.0142 - 6.00x10-5 153313.00 - 1084.70 1.92
Aqueous CPC
1.0122 0.0007 - 402.00 35.48
Aqueous CPB
1.0112 - 0.0005 - 955.54 112.84 -2.79
Glycine in aqueous CPC
1.0361 - 0.0001 85456.00 - 286.13
Valine in aqueous CPC
1.0460 - 0.0002 63603.00 - 211.51
Leucine in aqueous CPC
1.0313 - 0.0001 72478.00 - 243.09
Glycine in aqueous CPB
1.0350 - 0.0001 47425.00 - 155.14
Valine in aqueous CPB
1.0412 - 0.0001 47387.00 - 155.84
Leucine in aqueous CPB
1.0341 - 0.0001 56879.00 - 188.79
ρ0(T) = 103 kg m-3T-1; S
d = 103 kg2 m-3 mol-1 K-1; Vφ
0(T) = 10-6 m3
mol-1 T-1; Sv = 10-6 m3 kg mol-1K-1; S
v´(T) = 10-6 m3 kg2 mol-1 K-1;
CPC = cetyl pyridinium chloride; CPB = cetyl pyridinium
bromide
309Interactions of Amino Acids in Aqueous/Cationic Surfactant Systems
ening of hydrophilic interactions of polar groups of the
amino acids. A weak dependence of composition of amino
acids on their structure-breaking behaviour was noted in
aqueous and aqueous surfactant solutions. The surfactants
were observed to produce mild micelle formation with amino
acids resulting in exceptionally higher Vφ
0 values for amino
acids, with higher values for CPB. Additionally, the CPB
was seen as an effec-tive structure-breaker than CPC, while
amino acids also showed comparatively stronger structure-
breaking effect of CPB than of CPC. An induced dipole of
Br- was accountable for stronger interactions of CPC.
Acknowledgement
The author is highly thankful to the University Grants Com-
mission, Govervment of India, New Delhi, for financial sup-
port, Mr. Anil Kumar for fruitful association, and Dr. Nabeel
Siddiqui for encouragement.
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Liquid-Liquid Equilibrium Data of Ternary Systems of Water, Acetic Acid
and Alkanols (1-Butanol, 1-Pentanol and 1-Hexanol)
M. A. Rahman, M. S. Rahman, M. N. Nabi and M. A. K. Azad*
Department of Applied Chemistry and Chemical Technology, University of Rajshahi, Rajshahi-6205, Bangladesh
(received August 17, 2004; revised March 8, 2005; accepted March 22, 2005)
Pak. J. Sci. Ind. Res. 2005 48(5) 312-317
Abstract. The liquid-liquid equilibrium data are presented for phase behaviour of ternary system of water, acetic acid and
alkanols (1-butanol, 1-pentanol and 1-hexanol) at the temperature of 30±0.1 °C. From the data, binodal curves, tie lines,
plait points and equilibrium distribution diagram were determined experimentally, and the distribution coefficients and
separation factors were computed, with a view to examine the suitability of alcoholic solvents to extract acetic acid from its
aqueous solution. It has been found that the solubility of acetic acid increased with the increasing number of carbon atoms
in the chain of the alcohol used as the solvent, giving higher values of distribution coefficients and the separation factor.
Keywords: liquid-liquid equilibrium, tie line, liquid-liquid extraction, phase behaviour, distribution coefficient, acetic acid
extraction, binodal curve, equilibrium distribution
*Author for correspondence; E-mail: [email protected]
Introduction
Liquid-liquid extraction has established itself as a unique unit
operation during the last few decades. Nowadays, it finds
application in a number of commercial processes, such as sepa-
ration of organic isomers, isolation of antibiotics, extraction of
metals, separation of trace elements, and removal of pollu-
tants for pollution control. Since the system is based on solu-
bility, it has distinct advantage over distillation in which the
application of heat is required, so that heat sensitive materials
and compounds not differing appreciably in their boiling
points can be easily separated.
The extraction of organic acids from aqueous solutions resul-
ting from fermentation processes, and from spent or recycle
solutions, is industrially important. Many solvents have been
previously tried to improve such extractions using liquid-
liquid extraction (Acre et al., 1995; Briones et al., 1994; Dramur
and Tatli, 1993). Within the scope of this procedure, several
alcohols (Fahim et al., 1997; Kirk and Othmer, 1992) and aceta-
tes (Colombo et al., 1999; Correa et al., 1989) have been used
as solvents to extract acetic acid from aqueous solutions.
Only few of these, however, were noted to have distribution
coefficients greater than one.
The present work was undertaken to investigate fundamen-
tals of the process of extraction of acetic acid, using aliphatic
alcohols as solvents, by studying the liquid-liquid phase
equilibria (LLE) of ternary systems comprising of water, ace-
tic acid and alkanols (1-butanol, 1- pentanol and 1-hexanol).
The LLE of theses systems was studied by determining their
binodal curves and equilibrium distribution diagram experi-
mentally, and computing from these, the tie lines, plait points,
distribution coefficients and separation factors of the respec-
tive systems.
Materials and Methods
Materials. Acetic acid glacial (E. Merck, Germany, 99.5%), 1-
butanol (E. Merck, India, 99%), 1-pentanol (BDH, England,
98%) and 1-hexanol (BDH, England, 98%) were used without
further purification. Distilled water was used throughout this
work.
Solubility data. The solubility data for the three systems,
namely, water + acetic + 1-butanol (WAB), water + acetic acid
+ 1-pentanol (WAP), and water + acetic acid + 1-hexanol
(WAH) were determined by the cloud point method (turbidity
method) (Feki et al., 1994; Correa et al., 1989). Ten ml water was
measured in a 125 ml closed Erlenmeyer flask and a solvent
(alcohol) was added from a burette and agitated till the solu-
tion started to appear turbid. The amount of solvent added
was recorded as the maximum solubility of the solvent in water
and gave the first point on the base line of the binodal curve
(mutual solubility curve) plotted on a triangular diagram. The
appearance of turbidity indicated the beginning of the forma-
tion of the second phase, i.e., the solvent layer. Further addi-
tion of a small amount of the solvent gave a heterogeneous
mixture. Acetic acid was then added from a burette until the
first appearance of distinctly clear homogeneity. This gave
another point of the binodal curve. The procedure was repea-
ted to construct the binodal curve from the aqueous side. The
same procedure was repeated, but by starting with an initially
measured quantity of the solvent (alcohol) and the addition of
312
water and acetic acid to construct the binodal curve from the
solvent side. Each point on the binodal curve indicated a
homogeneous mixture with known compositions, which
could be read from the binodal curve (mutual solubility curve).
Equilibrium data. The equilibrium data for the three systems
were obtained experimentally at 30±0.1 °C in a thermostati-
cally controlled waterbath (SB-3, Grant Instruments Ltd.,
England). Twenty ml each of water and the solvents were
taken in four different 250 ml closed Erlenmeyer flasks and
then various amounts of acetic acid were added. The flasks
were vigorously shaken on an electric shaker (Model-1866,
Jencons Scientific Ltd., England) for 3 h and the Erlenmeyer
flasks were then immersed in a thermostatically controlled
waterbath and allowed to settle for 3 h. It was ensured that
longer stirring and settling times did not exhibit any change
in the phase composition. After the settling period was over,
the samples were pipetted out first from the upper phase and
then from the lower phase. Concentrations of acetic acid in
each phase were determined by potentiometric titration using
OSK-8022 potentiometric automatic titrator, (Ogawa Seiki Co.,
Ltd., Japan) with 0.1 N NaOH. Replicate titrations of a stan-
dard mixture gave an error of 0.21%. Water contents of each
phase were measured by OSK 8023 Karl Fischer tritrator
(Ogawa Seiki Co., Ltd., Japan) using the Karl Fischer reagent.
In this case, the replicate titrations of a standard sample gave
an error of 0.27%. Concentrations of alcohols in each phase
were computed by difference.
Results and Discussion
The solubility data (composition defining the binodal curves)
for water + acetic acid + 1-butanol (WAB); water + acetic acid
+ 1-pentanol (WAP); and water + acetic acid + 1-hexanol
(WAH) systems, obtained experimentally, are given respec-
tively in Tables 1, 2, and 3. These results were plotted as tri-
angular diagrams to give ternary diagrams (binodal curve) of
the above three systems, as shown respectively in Fig. 1, 2,
and 3. A close examination of the curves in these figures revea-
led that the heterogeneous region involving the 1-hexanol
system was greater than that of the 1-pentanol and 1-butanol
systems. Binary mixtures in both the ends of the ternary dia-
gram, at the base line, revealed that mutual solubility of water
and solvent (alcohols) was greater in the organic-rich phase
than in the aqueous-rich phase. The mutual solubility in both
the phases decreased as the number of carbon in the alcohol
molecule, as =CH2 increased. This may be attributed to the
fact that the polarity of alcohols decreases in the order of
1-butanol > 1-pentanol > 1-hexanol (Katayama et al., 1998).
The experimental equilibrium data indicating the composi-
tions of the coexisting phases are presented in Table 4. The
Table 1. Solubility data of the water + acetic acid + 1-butanol
system at 30±0.1 °C
Weight (%)
water 1-butanol acetic acid
Water-rich phase 96.1 3.9 0
86.6 7.0 6.4
79.0 1.06 10.4
72.4 14.7 12.9
67.0 19.0 14.0
62.9 23.0 14.1
58.9 26.2 14.9
55.6 29.3 15.1
50.4 34.5 15.1
46.8 37.9 15.2
1-Butanol-rich phase 19.8 80.2 0
21.5 75.6 2.9
26.5 65.1 8.4
30.5 57.5 11.9
34.2 52.3 13.5
37.4 48.2 14.4
40.5 44.8 14.7
42.9 41.9 15.2
45.3 39.4 15.3
Plait point 54.2 30.7 15.1
corresponding equilibrium distribution diagram, based on
these data was drawn (Fig. 4), while the separation factor for
different systems was also drawn from the obtained solubility
data (Fig. 5). The distribution coefficient and separation fac-
tors for the respective systems were computed from the equi-
librium data. The distribution coefficient is defined as the ratio
of concentration of the solute (acetic acid) in the extract phase
(organic phase ) to that in the raffinate phase (aqueous phase),
while the separation factor is defined as the ratio of the distri-
bution coefficient of the solute (acetic acid) to that of water.
Both the distribution coefficient and separation factor are the
measures of the ability of the solvent to separate the solute
(acetic acid) from water. Greater values of the distribution
coefficient are desirable, since less solvent is then required
for the extraction. A separation factor greater than one would
indicate that the solute is preferentially more soluble in the
solvent (alcohol) than in water to facilitate its extraction. Val-
ues of distribution coeffecients and separation factors, for
the respective systems, are incorporated in Table 4 indica-
ting that all the solvents studied were suitable for separating
acetic acid from its aqueous solution. It may be noted from
Table 4 that the distribution coefficients were always greater
than unity. The previously reported distribution coefficient
of 1.613 at 26.7 °C for water + acetic acid + 1-butanol system
(Perry et al., 1984) is comparable with the present results. For
313Liquid-Liquid Equilibrium Data of Ternary Systems
in terms of electron donar-acceptor concepts, or on the basis
of Lewis acidity and Lewis basicity. They argued that ethanol
and water have both electron donating and electron accep-
ting capabilities, but ethanol has a slightly larger electron donar
number and lower electron acceptor number than water.
Katayama et al. (1998) for their systems, however, interpreted
their results in terms of polarities and dielectric constants of
the solvents. In the present study, as the carbon chain in the
alcohols increased the presence of more methylene groups
(=CH2 groups), the alcohol molecules become increasingly
more electron donars and less electron acceptors. The hetero-
geneous region (binodal region) in the triangular diagram, the
distribution coefficient, and the separation factor were, there-
fore, relatively large showing the order of 1-butanol < 1-pen-
tanol < 1-hexanol. Similar results were obtained by Maeda
et al. (1997) for water + fatty acids + acetone (or ethanol) sys-
tems, showing that as the carbon number of fatty acids incre-
ased the binodal region of the two liquid phases became larger.
Direct separation of the observed tie lines is difficult to make
from a triangular diagram (Feki et al., 1994; Hand, 1930).
Therefore, Hand’s plots for each system, based on 1-butanol,
1-pentanol, or 1-hexanol were, respectively, drawn in Fig. 5,
6, and 7, so that interpolation could be done and the plait
point compostions for these systems could be obtained gra-
phically from these plots in accordance with the procedure of
Perry et al. (1984). The plait point compositions for different
systems were computed as (Tables 1-3): water + accetic acid +
1-butanol (54.2, 15.1, 30.7 by %, wt); water + acetic acid + 1-
pentanol (41.4, 29.6, 29 by %, wt); and water + acetic acid + 1-
hexanol (36.7, 36.3, 27.0, by %, wt).
water + acetic acid + mesityl oxide system, distribution coeffi-
cient of almost the same magnitude was obtained by Hegazi
and Salem (1983). The separation factors in the present stu-
dies were found to be of fairly high values, so that 1-butanol,
1-pentanol and 1-hexanol may be considered as good solvents.
A comparison of the results on the use of 1-butanol, 1-penta-
nol and 1-hexanol, as the solvents, showed that the hetero-
geneous region in the triangular diagram increased in the
order of 1-butanol < 1-pentanol < 1-hexanol. Munson and
King (1984) interpreted the solvent capacity and selectivity
Fig. 1. Binodal curve for the water + acetic acid + 1-buta-
nol system at 30±0.1 °C.
1-Butanol (weight, %)
Wate
r (w
eig
ht, %
)
Ace
tic a
cid (
weig
ht, %
)80
60
40
40
20
20 60 80
80
60
40
20p
Fig. 3. Binodal curve for the water + acetic acid + 1-hexanol
system at 30±0.1 °C.
1-Hexanol (weight, %)
Wate
r (w
eig
ht, %
)
Ace
tic a
cid (
weig
ht, %
)
80
60
40
40
20
20 60 80
80
60
40
20
p
314 M. A. Rahman et al.
Fig. 2. Binodal curve for the water + acetic acid + 1-pen-
tanol system at 30±0.1 °C.
1-Pentanol (weight, %)
Wate
r (w
eig
ht, %
)
Ace
tic a
cid (
weig
ht, %
)
80
60
40
40
20
20 60 80
80
60
40
20
p
Table 2. Solubility data of the water + acetic acid + 1-pentanol
system at 30±0.1 °C
Weight (%)
water 1-pentanol acetic acid
Water-rich phase 97.6 2.4 0
82.7 3.4 13.9
70.7 5.7 24.3
61.2 9.9 28.9
56.9 13.8 29.3
53.2 17.2 29.6
49.9 20.2 29.9
47.0 23.0 30.0
44.8 25.4 29.8
42.7 27.5 29.8
40.7 29.6 29.739.0 31.5 29.5
1-Pentanol-rich phase 8.0 92.0 0
10.3 83.2 6.5
16.3 65.8 17.9
20.8 56.0 23.2
24.5 49.7 25.8
27.9 45.2 26.9
30.7 41.4 27.9
33.2 38.4 28.4
35.4 35.8 28.837.1 33.4 29.5
Plait point 41.4 29.0 29.6
Table 3. Solubility data of the water + acetic acid + 1-hexanol
system at 30±0.1 °C
Weight (%)
water 1-hexanol acetic acid
Water-rich phase 100 0 070.9 1.7 27.464.1 3.1 32.859.3 5.3 35.454.1 9.2 36.750.6 12.3 37.044.8 18.2 37.040.3 23.1 36.636.7 27.0 36.3
1-Hexanol-rich phase 5.8 94.2 09.5 79.9 10.613.5 64.8 21.717.8 53.4 28.822.0 44.7 33.324.0 42.0 34.026.0 39.0 35.028.3 36.0 35.731.7 32.0 36.334.0 29.7 36.3
Plait point 36.7 27.0 36.3
Conclusion
The liquid-liquid phase equilibria of water + acetic acid + 1-
butanol, or 1-pentanol, or 1-hexanol systems were measured
at a temperature of 30±0.1 °C. The binodal curves, tie lines,
distribution coefficients and separation factors were deter-
315Liquid-Liquid Equilibrium Data of Ternary Systems
Fig. 4. Distribution coefficients for the extraction of acetic
acid from water by different solvents (1-butanol,
1-pentanol, 1-hexanol) at 30±0.1 °C.
1.2
1.1
1.0
0.9
0.8
0.7
5 10 15 20 25 30 35
1-butanol
1-pentanol
1-hexanol
Dis
trib
utio
n c
oe
ffic
ien
t (K
D)
Acetic acid concentration in the feed (weight, %)
Fig. 5. Separation factors for the extraction of acetic acid
from water by different solvents (1-butanol, 1-
pentanol, 1-hexanol) at 30±0.1 °C.
10
8
6
4
2
05 10 15 20 25 30 35
1-butanol
1-pentanol
1-hexanol
Separa
tion f
acto
r (α
)
Acetic acid concentration in the feed (weight, %)
Fig. 6. Hand-type ternary diagram for the water + acetic
acid + 1-butanol system.
0.5
-0.5
-1.0
-1.5
( )
( )
0.0
-1.5 -1.0 -0.5
p
p( )
binodal curve
tie lines
plait point
-2.0
Log (
acetic a
cid
/ 1
-buta
nol)
Log (acetic acid / water)
( )
( )1.0
1.5
0.0
-0.5
-1.0
-1.5-2.0 -1.5 -1.0 0.0
p
p( )
binodal curve
tie lines
plait point
-0.5
Log (acetic acid / water)
Log (
acetic a
cid
/ 1
-penta
nol)
Fig. 7. Hand-type ternary diagram for the water + acetic
acid + 1-pentanol system.
Table 4. Comparison of coexisting phase in acetic acid + water + 1-butanol, or 1-pentanol, or 1-hexanol systems at 30±0.1 °C
Compositions of Compositions of Compositions of Distribution Separation
initials mixtures organic pahse aqueous phase coefficients factor
(weight, %) (weight, %) (weight, %) (KD
a) (αb)
water 1-butanol acetic water 1-butanol acetic water 1-butanol acetic
acid acid acid
52.2 42.3 5.5 24.0 70.1 5.9 89.0 5.9 5.1 1.157 4.28
50.8 41.2 8.0 27.0 64.4 8.6 85.6 7.2 7.2 1.194 3.79
49.5 40.1 10.4 29.8 59.0 11.2 81.5 9.2 9.3 1.204 3.29
48.2 39.1 12.7 33.3 54.0 12.7 76.0 12.2 11.8 1.076 2.46
water 1-pentanol acetic water 1-pentanol acetic water 1-pentanol acetic
acid acid acid
52.2 42.3 5.5 10.0 84.7 5.3 91.5 3.0 5.5 0.964 8.84
48.2 39.1 12.7 13.0 74.9 12.1 84.0 3.5 12.5 0.968 6.24
42.8 34.7 22.5 20.0 57.9 22.1 72.3 5.0 22.7 0.974 3.52
40.1 32.5 27.4 29.4 43.4 27.2 63.6 8.4 28.0 0.971 2.10
water 1-hexanol acetic water 1-hexanol acetic water 1-hexanol acetic
acid acid acid
49.5 40.2 10.3 8.8 82.5 8.7 84.3 7.0 12.0 0.725 7.18
44.8 36.5 18.7 11.8 71.0 17.2 78.5 1.0 20.5 0.839 5.59
41.0 33.3 25.7 14.8 61.0 24.2 70.7 1.9 27.4 0.883 4.22
37.8 30.7 31.5 19.5 50.0 30.5 63.0 3.7 33.3 0.916 2.95
KD
a = expressed as weight per cent acetic acid in organic phase/weight per cent acetic acid in aqueous phase, at equilibrium
316 M. A. Rahman et al.
mined. Hand’s method was used to correlate the tie lines and
to calculate coordinates of plait points. The heterogeneous
region of 1-hexanol appeared to be broader than those of 1-
pentanol and 1-butanol systems. The solubility of these sys-
tems increased with the increase in the carbon chain length of
the alcohol used as the solvent, giving high values of the
distribution coefficient and the separation factor. For water +
acetic acid + 1-butanol, water + acetic acid + 1-pentanol, and
water + acetic acid + 1-hexanol systems, the distribution coef-
ficients were found to be in the range of 1.767-2.488, 1.543-
2.989, and 1.801-2.544, respectively, while the separation fac-
tors were found to be in the range of 2.456-4.296, 2.103-8.454,
and 2.961-7.200, respectively.
References
Arce, A.A., Souza, B.P., Vidal, I. 1995. Liquid-liquid equilibria
of the ternary mixtures: water + methyl ethyl ketone and
water + propanoic acid + methyl propyl ketone. J. Chem.
Engg. Data 40: 225-229.
Briones, J.A., Mullins, J.C., Thies, M.C. 1994. Liquid-liquid
equilbria for the oleic acid + β-sitisterol + water system at
elevated temperatures and pressure. Ind. Engg. Chem.
Res. 33: 151-156.
Colombo, A., Battilana, P., Ragaini, V.L., Bianchi, C.L. 1999.
Liquid-liquid equilibria of the ternary systems water +
acetic acid + ethyl acetate and water + acetic acid + iso-
phorone (3,5,5-trimethyl-2-cyclohexen-1-one). J. Chem.
Engg. Data 44: 35-39.
Correa, J.M., Blanco, A., Arce, A. 1989. Liquid-liquid equilibria
of the system water + acetic acid + methyl isopropyl
ketone between 25 and 55 °C. J. Chem. Engg. Data 34:
415-419.
Dramur, U., Tatli, B. 1993. Liquid-liquid equilibria of water +
acetic acid + phthalic esters (dimethyl phthalate and
diethyl phthalate) ternaries. J. Chem. Engg. Data 38: 23-25.
Fahim, M.A., Al-Muhtaseb, S.A., Al-Nashef, I.M. 1997. Liq-
uid-liquid equilibria of the ternary systems water + acetic
acid + 1-hexanol. J. Chem. Engg. Data 42: 183-186.
Feki, M., Fourati, M., Chabouni, M.M., Ayedi, H.F. 1994. Puri-
fication of wet process phosphoric acid by solvent extrac-
tion liquid-liquid equilibrium at 25 and 40 °C of the system
water + phosphoric acid + methyliso butylketone. Canad.
J. Chem. Engg. 72: 939-944.
Hand, D.B. 1930. The distribution of a consolute liquid bet-
ween two immiscible liquids. J. Physiol. Chem. 34: 1961-
2000.
Hegazi, M.F., Salem, A.B. 1983. Ternary data for the acetic acid
+ water + mesityl oxide system. J. Chem. Technol. Biotech-
nol. 33A: 145-150.
Katayama, H., Hayakawa, T., Kobayashi, T. 1998. Liquid-liquid
equilibria of three ternary systems: 2-propanone + glyce-
rol + acetic acid, 2-butanone + glycerol + ethanol, and 2-
butanone + glycerol + 2-propanol in the range of 283.15
to 303.15 K. Fluid Phase Equilibria 144: 157-167.
Kirk, R.E., Othmer, D.F. 1992. Encyclopaedia of Chemical Tech-
nology, vol. 1, p. 121, 4th edition, Wiley-Interscience, Inc.,
New York, USA.
Maeda, K., Yamada, S., Hirota, S. 1997. Binodal curve of two
liquid phases and solid-liquid equilibrium for water + fatty
acids + acetone systems. Fluid Phase Equilibria 130:
281-294.
Munson, C.L., King, C.J. 1984. Factors influencing solvent
selection for extraction of ethanol from aqueous solu-
tion. Ind. Engg. Chem. Process Des. Dev. 23: 109-115.
Perry, R.H., Green, D.W., Maloney, J.O. 1984. Perry’s Chemi-
cal Engineers’ Handbook, pp. 15-5, 15-10, 6th edition,
McGraw-Hill International Editions, New York, USA.
317Liquid-Liquid Equilibrium Data of Ternary Systems
( )
( )
1.5
1.0
0.5
0.0
-0.5
-1.0-1.5 -1.0 -0.5 0.0
p
p( )
binodal curve
tie lines
plait point
Fig. 8. Hand-type ternary diagram for the water + acetic
acid + 1-hexanol system.
Log (acetic acid / water)
Log (
acetic a
cid
/ 1
-hexanol)
Synthesis and Antimicrobial Activity of Some Heterocycles: Part-V
M. M. H. Bhuiyan, Khandker M. M. Rahman*, M. K. Hossain and M. A. RahimDepartment of Chemistry, University of Chittagong-4331, Bangladesh
(received November 21, 2003; revised July 20, 2005; accepted July 23, 2005)
Pak. J. Sci. Ind. Res. 2005 48(5) 318-321
Abstract. Ortho-aminonitrile (1) was prepared from ethoxymethylenemalononitrile. 4-Allylamino-1-methyl-6-
methylthiopyrazolo[3,4-d]pyrimidine (4) was prepared by an initial treatment of compound (1) with carbon disulfide in
pyridine followed by methylation with methyl iodide and subsequent reaction with allylamine in acetonitrile. Ortho-
aminoester (5) was prepared from ethyl (ethoxymethylene) cyanoacetate. Reaction of compound (5) with formamide
yielded compound (6), which was then tosylated. All compounds were screened for their antibacterial and antifungal
activities.
Keywords: ethoxymethylenemalononitrile, pyrazolo[3,4-d]pyrimidine, antimicrobial activity, synthesis of heterocycles
*Author for correspondence
Introduction
Substituted heterocyclic compounds offer a high degree of
structural diversity, which have proven to be broadly useful
as therapeutic agents (Thompson and Ellman, 1996). Fused
heterocyclic systems containing pyrazole ring are ranked
among the most versatile bioactive compounds, possessing
diverse biological activities, such as fungicidal (Sasse et al.,
1986), herbicidal (Ohyama et al., 1986), virucidal (Zikan et al.,
1986),and insecticidal (Hasan et al., 1994). The pyrazolo[3,4-d]
pyrimidines have been described as biologically active agents
(Elmaati, 2002). The most widely used pyrazolo[3,4-d] pyrim-
idines, allopurinol and oxyallopurinol, are established inhibi-
tors of xanthine oxidase and thus interfere in the biosynthesis
of uric acid, the causative agent of gout. This group of com-
pounds also exhibits antineoplastic activity (Hansch et al.,
1990a). Antitumor and antiviral compounds have been syn-
thesized based on the antibiotic formycin, a nucleoside of
pyrazolo[3,4-d]pyrimidene (Hansch et al., 1990b). Potent anti-
inflammatory and analgesic activities have been reported in a
number of 5-arylpyrazolo[3,4-d]pyrimidines (Machon and
Witkiewiz 1985; Shishoo et al., 1999). Prompted by these
observations, and as a continuation of the ongoing programme
on fused heterocycles (Rahman et al., 2003; Chowdhury et al.,
2001; 2000; Chowdhury and Bhuiyan, 2000; 1997), the synthe-
sis and antimicrobial activity of some new pyrazolo[3,4-d]
pyrimidine derivatives is reported here.
Materials and Methods
Melting points were determined in open capillary tubes and
are reported as uncorrected values. 1H- and 13C-NMR spec-
tra were recorded on a Bruker AC 200 spectrometer, using
DMSO-d6/CDCl3 as the solvents and TMS as an internal
standard (chemical shifts in δ, ppm). TLC was carried out on
silica gel-G plates and spots were located by iodine vapour.
All evaporations were conducted under reduced pressure at
the waterbath temperature below 50 °C. Various steps
involved in the synthesis of seven heterocyclic compounds
(1-7) are shown in Fig. 7 as scheme 1 (compounds 1-4) and
scheme 2 (compounds 5-7).
Synthesis of 5-amino-3-cyano-1-methylpyraxole (1). To a
solution of ethoxymethylenemalononitrile (10.85 g, 88.9 ml) in
absolute ethanol (35 ml) was added methylhydraxine (3.6 g,
57.5 mmol) in absolute ethanol (30 ml). Slight heat was pro-
duced during the addition. The resulting solution was then
refluxed with continuous stirring for 1 h, after which the sol-
vent was evaporated under reduced pressure. The obtained
solid was washed with ether and recrystallized from water to
give compound (1), as shining white plates; yield: 7.6 g (70%),
m. p. = 222-223 °C; 1H-NMR (CDCl3): δ 7.50 (s, 1H, 3-H), 6.51 (s,
2H, NH2), 3.51 (s, 3H, CH3); 13C-NMR (CDCl3): δ 151.4, 139.7,
115.2, 72.1, 34.5.
Synthesis of 1-methylpyraxolo[3,4-d]pyrimidine-4, 6(5H,
7H)-dithion (2). A mixture of compound (1) (1 g, 8.196 mmol)
and carbon disulfide (3.53 ml, 40.69 mmol) in pyridine (5 ml)
was refluxed for 10 h. The progress of the reaction was
monitored by TLC (n-hexane : ethyl acetate, 1:1 v/v). After
completion of the reaction, the mixture was cooled to room
temperature and ethanol (30 ml) was added to the mixture.
The orange coloured solid thus obtained was then collected
by filtration, washed with ether (10 ml) and recrystallized
from ethanol to yield compound (2); yield: 1.23 g (75.9%),
m.p. > 250 °C. Without further analysis, the compound was
taken to the next step synthesis.
318
Synthesis of 1-methyl-4,6-dimethylthiopyrazolo[3,4-
d]pyrimidine (3). To the solution of compound (2) (0.25 g, 1.26
mmol) in aqueous sodium hydroxide (10%, 1.2 ml) was
dropwise added methyl iodide (0.25 g, 1.26 mmol) and stirred
at room temperature for 20 h. The yellowish coloured solid
was collected by filtration, washed with distilled water and
recrystallized from ethanol to furnish compound (3); yield:
0.20 g (71.4%), m. p. = 103-105 °C; 1H-NMR (CDCl3); δ 7.81 (s,
1H, CH), 4.0 (s, 3H, CH3), 2.65 (s, 3H, SCH3); 13C-NMR (CDCl3):
δ 168.48, 164.66, 151.76, 131.24, 109.24, 33.61, 14.22, 11.70.
Synthesis of 4-allylamino-1-methyl-6-methylthiopyrazolo
[3,4-d]pyrimidine (4). A mixture of compound (3) (0.15 g, 0.66
mmol) and allylamine (1.02 ml, 13.5 mmol) in acetonitrile (3 ml)
was heated in a stainless steel vessel at 120-130 °C for 30 h.
The mixture was concentrated in vacuo to 1 ml and was then
allowed to set in a refrigerator for 6 h. The obtained solid was
collected by filtration and washed with acetonitrile to yield
compound (4); yield: 0.082 g (52.9%), m.p. = 86-88 °C; 1H-
NMR (CDCl3): δ 7.73 (s, 1H, CH), 6.94-6.82 (m, 1H, CH), 5.30-
5.19 (m, 2H, CH2), 4.23 (s, 2H, CH2), 3.93 (s, 3H, CH3), 2.58 (s,
3H, SCH3), 1.22 (s 1H, NH); 13C-NMR (CDCl3): δ 172.12, 151.10,
117.44, 114.56, 106.22, 98.64, 82.82, 33.69, 29.67, 14.14. Analyti-
cal calculated values for C10H13N5S (235.31): C 51.04, H 5.57, N
29.76; experimentally found values: C 51.25, H 5.53, N 29.70%.
Synthesis of ethyl-5-amino-1-methylpyrazole-4-carboxylate
(5). The compound (5) was prepared from ethyl (ethoxy-
methylene)cyanoacetate by reacting with methyl hydrazine
using the same method as was used for the preparation of
compound 1 as shining white plates; yield: 70% yield, m.p. =98-100 °C; 1H-NMR (CDCl3): δ 7.60 (s, 1H, 3H), 5.00 (s, 2H,
NH2), 4.15 (q, 2H, OCH2), 3.60 (s, 3H, NCH3), 1.35 (t, 3H, CH3);
13C-NMR (CDCl3): δ 164.30, 149.30, 138.90, 95.80, 59.30, 33.90,
14.30.
Synthesis of 1-methylpyrazolo[3,4-d]pyrimidine-4 (5H)-one
(6). A mixture of ortho-amino ester compound (5) (1 g, 5.92
mmol) and formamide (4 ml) was refluxed for 2 h at 180 °C.
After cooling, the mixture was poured onto crushed ice and
stirred for 1h. The precipitate was collected by filtration and
recystallized from ethanol to give compound (6) as white crys-
tals: yield; 0.68 g (68.8%), m.p. > 250 °C; 1H-NMR (CDCl3): δ
8.05 (s, 1H, CH), 8.00 (s, 1H, CH), 4.48 (s, 1H, NH), 3.88 (s, 3H,
CH3); 13 C-NMR (CDCl3): δ 157.27, 151.78, 147.78, 134.05, 105.60,
34.01.
Synthesis of 1-methyl-4-oxo-pyrazolo[3,4-d]pyrimidine-5-p-
toluene sulfonate ester (7). A solution of compound (6) (0.25
g, 1.67 mmol) and p-toluene sulfonyl chloride (0.32 g, 1.67
mmol) in ether (6 ml) was stirred at 30 °C for 6 h. The progress
of the reaction was monitored by TLC [n-hexane : ethyl
acetate, 1:3 v/v). After completion of the reaction, the solvent
was evaporated to dryness and the resulting solid was recrys-
tallized from ethanol to give compound (7) as white crystals;
yield: 0.43 g (84.3%), m.p. = 220-222 °C; 1H-NMR (CDCl3): δ
8.11 (d, J = 8.0 Hz, 2H, Ar-H), 8.00 (d, J = 8.0 Hz, 2H, Ar-H), 7.77
(s, 1H, CH), 7.47 (s, 1H, CH), 3.73 (s, 3H, CH3), 2.27 (s, 3H, CH3);13C-NMR (CDCl3): δ 157.27, 151.79, 147.8, 145.3, 137.8, 134.05,
128.60, 125.50, 105.67, 67.40, 34.02.
Antibacterial and antifungal screenings. All the synthesized
compounds (1-7) were screened for their antibacterial activity
against the gram-positive bacteria Bacillus cereus, B.
megaterium, Staphylococcus aureus, and the gram-negative
bacteria Shigella dysenteriae, Salmonella typhi, Escherichia
coli (Table 1). The antifungal activity was tested against the
fungi Macrophomina phaseolina, Fusarium equiseti, Alter-
naria alternata and Colletotricum corchori (Table 2). For the
detection of antibacterial activities, the disc diffusion method
was followed (Bauer et al., 1966). Poisoned-food technique
was used to assess the antifungal activities (Grover and
Fig. 1. Schemes 1 and 2 for the synthesis of some heterocy-
clic compounds; reagents for various steps and conditions:
CH3NHNH2, EtOH, reflux, 1 h; (b) CS2, pyridine, reflux, 10 h;
(c) CH3I, 10% NaOH, stirred at room temperature, 20 h; (d)
allylamine, CH3CN, 12-130 °C, 30 h; (e) HCONH2, 180 °C, 2 h;
(f) p-TSCl, ether, 30 °C, 6 h; compounds synthesized: 1: 5-
amino-3-cyano-1-methylpyrazole; 2: 1-methylpyrazole[3, 4-d]
pyrimidine-4, 6(5H, 7H)-dithione; 3: 1-methyl-4, 6-
dimethylthiopyrazolo[3, 4-d]pyrimidine; 4: 4-allylamino-1-
methyl-6-methylthiopyrazolo[3, 4-d]pyrimidine; 5: ethyl-5-
amino-1-methylpyrazole-4-carboxylate; 6: 1-methyl-
pyrazolo[3, 4-d]pyrimidine-4(5H)-one; 7: 1-methyl-4-
oxo-pyrazolo[3, 4-d]pyrimidine-5-p-toluene sulfonate ester;
p-TSCl = p-toluene sulfonyl chloride.
319Synthesis of Some Antimicrobial Heterocycles
EtO CN
H CN
CN
NN
CH3
NH2
a
1
NN N
N
S
H
S
HCH32
b
cSCH3
SCH3
NN N
CH3
N
3
H.N
N N N
N
SCH3
d
4
EtO
H CN
CO Et2
a
CO Et2
NN
CH3
CH3
NH2
e
5
NN N
CH3
N.H
O
6
Scheme 1
Scheme 2
f NN N
N TS
CH3
O
7
Moore,1962). A commercial antibacterial Ampicillin and an
antifungal Nystatin were also tested under similar conditions
for comparison. Nutrient agar (NA) and potato dextrose agar
(PDA) were used as the basal media for the culture of the
tested bacteria and fungi, respectively. Dimethyl formamide
(DMF) was used, as a solvent, to prepare 1% solution of the
compounds. Proper control was maintained with DMF.
Results and Discussion
Ortho-aminonitrile, 5-amino-3-cyano-1-methylpyrazol (1), was
prepared from ethoxyemthylenemalononitrile as described in
scheme 1 (Fig. 1) (Chowdhury and Bhuiyan, 1997). The struc-
ture of compound (1) was confirmed by 1H-NMR spectrum. It
showed a one-proton singlet at δ 7.50 for 3-H, a two-proton
singlet at δ 6.51 for NH2 and a three-proton 3.51 for N-CH3
group. The cyclized compound (1) was further proved by 13C-
NMR spectrum. The resonances displayed at δ 151.4 for C-5,
139.7 for C-3, 72.1 for C-4 carbon atoms and down field shift at
δ 34.5 for N-CH3 carbon atom, respectively. Treatment of com-
pound (1) with carbon disulfide in pyridine under reflux af-
forded 1-methylpyrazolo[3,4-d]pyrimidine-4,6(5H, 7H)-dithione
(2) in good yield. Compound (2) was then methylated with
methyl iodide in aqueous sodium hydroxide to afford 1-
methyl-4,6-dimethylthiopyrazolo[3,4-d]pyrimidine (3) with
71.4% yield, m.p. 103-105 °C. The formation of compound (3)
was established by its 1H-NMR spectrum, which displayed
two three-proton singlets at δ 2.65 and 2.59 corresponding to
two SCH3 groups. This structure was also supported by 13
C-
NMR spectrum, which displayed two peaks at 14.22 and 11.70
for two SCH2 groups in the molecule.
A substitution reaction of compound (3) with allylamine in
acetonitrile at 120-130 °C afforded 4-allylamino-1-methyl-6-
methylthio-pyrazolo[3,4-d] pyrimidine (4). The disappearance
of one SCH3 peak in the 1H-NMR spectrum of compound (4)
and appearance of allylic two-proton multiplet at δ 5.30-5.90
and one-proton multiplet at δ 6.94-6.82 confirmed the intro-
duction of allylic group in the molecule. The peaks were also
in good agreement with 13C-NMR spectrum. The microanalyti-
cal data of the compound (4) for C, H, N were in accordance
with the calculted values.
Ortho-aminoester, ethyl-5-amino-1-methylpyrazole-4-carboxy-
late (5), was prepared from ethyl (ethoxymethylene)
cyanoacetate following the same method as was used for the
preparation of compound (1) as shining white plates in 70%
yield, m. p. 98-100 °C, as shown in scheme 2 (Fig.1). The 1H-
NMR spectrum of compound (5) showed a one-proton singlet
at δ 7.60 for 3-H, a two-proton singlet at δ 5.00 for NH2, and a
two-proton quartet at δ 4.15 for OCH2 and a three-proton trip-
Table 2. Fungicidal screening of some heterocyclic compounds (1-7)
Inhibition of mycelial growth by 100 µg dry wt of different compounds/ml PDA (%)
Fungal comp comp comp comp comp comp comp Nystatin
species 1 2 3 4 5 6 7
Macrophomina phaseolina 35.00 46.00 23.00 95.10 30.30 22.00 69.00 71.78
Alternaria alternata 13.00 29.07 51.50 32.34 43.43 49.50 34.00 51.55
Fusarium equiseti 40.10 32.00 43.30 90.20 21.00 19.10 29.16 44.50
Colletotrichum corchori 17.00 48.78 32.00 24.44 49.11 22.22 29.00 40.51
Table 1. Antibacterial screening of some heterocyclic compounds (1-7)
Dia of the zone of inhibition by 100 µg dry wt of different compounds/disc (mm)
Bactrial comp comp comp cmp comp comp comp Ampicilin
species 1 2 3 4 5 6 7 25 µg/disc
Bacillus cereus 9 15 9 21 14 9 -- 21
B. megaterium 6 9 5 7 -- -- 7 20
Staphylococcus aureus 10 9 11 8 -- -- -- 19
Shigella dysenteriae 10 11 12 8 -- -- -- 30
Salmonella typhi 9 9 13 10 -- -- 10 24
Escherichia coli 6 6 9 20 8 7 -- 12
1: 5-amino-3-cyano-1-methylpyrazole; 2: 1-methylpyrazole[3, 4-d]pyrimidine-4, 6(5H, 7H)-dithione; 3: 1-methyl-4, 6-dimethylthiopyrazolo[3,
4-d]pyrimidine; 4: 4-allylamino-1-methyl-6-methylthiopyrazolo[3, 4-d]pyrimidine; 5: ethyl 5-amino-1-methylpyrazole-4-carboxylate; 6: 1-
methylpyrazolo[3, 4-d]pyrimidine-4-(5H)-one; 7: 1-methyl-4-oxo-pyrazolo[3, 4-d]pyrimidine-5-p-toluene sulfonate ester
320 M. M. H. Bhuiyan et al.
let at δ 1.35 for CH3, which were coming from ethyl ester. The13C-NMR spectrum of (5) was also consistent with the
structure.
Compound (5) in formamide under reflux furnished 1-
methylpyrazolo[3,4-d]-4(5H)- one (6) in 68.8% yield. In its 1H-
NMR spectrum the disappearance of NH2 peak at δ 5.00 and
appearance of one-proton singlet at δ 8.00 for H-6 confirmed
the formation of pyramidine ring. The 13C-NMR spectrum
displayed the presence of six carbons corresponding to its
molecular formula C6H6N4O. The structure of compound (6)
was futher confirmed by its conversion to tosylate derivative
(7) with p-toluene sulfonyl chloride.
Most of the compounds showed moderate to significant anti-
bacterial and antifungal activities (Tables 1 and 2). Generally,
allylamino pyrimidine derivative exhibited higher activities.
Compound (4) showed the highest antibacterial activity
against B. cereus and E. coli. Compounds (3, 4, 5, 6) showed
high antifungal activities against A. alternata; M. phaseolina
and F. equiseti; C. corchori; and A. alternata, respectively.
However, none of the synthesized compounds showed better
antifungal and antibactrial activities than the standards Nys-
tatin and Ampilicilin, respectively, used during the present
investigations for the purpose of comparison.
Acknowledgement
Authors wish to thank Mr. M. S. Rahman, Department of
Microbiology, Chittagong University, Bangladesh, for
assistance in the determination of antimicrobial activities of
the synthesized heterocyclic compounds.
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Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Truck, M. 1966.
Antibiotic susceptibility testing by a standardized single
disc method. Am. J. Clin. Pathol. 45: 493-496.
Chowdhury, A.Z.M.S., Bhuiyan, M.M.H. 1997. Isothiocyanato
mediated synthesis of fused pyrimidine: synthesis of
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321Synthesis of Some Antimicrobial Heterocycles
Introduction
Industry requires large supplies of water for their processes.
Only a small fraction of it is incorporated in their products
and some is lost by evaporation, while the rest of it is released
as the wastewater containing sludge. This sludge contains
organic pollutants as the major constituent and inorganic salts
as dissolved solids. If this sludge is released into the water
bodies untreated, it severely affects the quality of water. This
sludge may contain toxic metals that directly affect the aquatic
life, or it may contain plant nutrients, such as nitrates and phos-
phates that may stimulate the growth of aquatic weeds, or it
may have a high demand for dissolved oxygen thus resulting
in anaerobic conditions. Under anaerobic conditions, H2S gas
is generated, which produces offensive odours. In order to
protect the environment from the undesirable toxic materials,
therefore, the wastewater is required to be suitably treated
before its discharge to natural water streams. Many of the steps
taken to treat the wastewater result in further concentration
of the dissolved pollutants in the sludge (Priestly, 1991). The
sludge, as a consequence, becomes unstable, putrescine and
pathogenic. Sludge must, therefore, be treated before dispo-
sal or for reuse in order to alleviate pollution problems and
thus not become a burden on the environment.
Pharmaceutical sludge contains biodegradable organic mat-
ter. Bacteria may be used to bring about its degradation, so as
to stabilize the waste for ultimate disposal or reuse. However,
non-biodegradable substances remain unaffected during the
process of bacterial biodegradation. That is why a combina-
tion of biological and physicochemical methods was investi-
gated in the present study, during which the wastewater slu-
dge from the pharmaceutical industry was characterized and
treated by integration of the two procedures. For this pur-
pose, the physicochemical methods of coagulation and floc-
culation were used in conjunction with the aerobic biological
method. This approach was considered adequate to produce
a well-treated effluent and sludge, having better efficiency
for reuse than was achievable with either method when used
singly.
Materials and Methods
Industrial sludge used. The source of sludge used in the
present study was obtained from a pharmaceutical factory
located in Ikeja, Lagos. The factory produces a variety of drugs,
including, Trosyl cream, Tetramycine, Ergonovine, Uvacin,
Combantrin, Obron, Oxytetracyclin, Diflucan and Diphenydra-
mine. The average wastewater generated per day by this fac-
tory was 3.1x104 litres at optimum production. The wastewa-
ter from which the sludge was obtained originated from a com-
bination of various processes, including syrup preparation,
malt preparation, production of pastilizers, and from tablet
rejection. The effluent was heterogeneous in nature and
included water used for washing equipment, from braken
vessels containing the pharmaceutical preparations, rejected
tablets that were crushed and washed down the drain, starch*Author for correspondence; E-mail: [email protected]
Pak. J. Sci. Ind. Res. 2005 48(5) 322-328
Combined Aerobic and Physicochemical Treatment of
Pharmaceutical Industry Sludge
I. O. Asiaa* and C. M. A. Ademoroti
b
aDepartment of Chemistry, Ambrose Alli University, Ekpoma, Nigeria
bUniversity of Benin, Benin City, Nigeria
(received September 9, 2003; revised July 8, 2005; accepted July 23, 2005)
Abstract. Composite samples of sludge obtained from a pharmaceutical factory were analysed for their pollution cha-
racteristics. The samples were then treated by integrated aerobic biological and physicochemical methods. The analysis
revealed that the BOD and COD of the sludge liquor were high, as well as were the levels of solids concentration, nit-
rogen, phosphorus and bacterial count. These showed that sludge from this industry had a high pollution potential, and
therefore needed treatment before disposal or reuse in other applications. Percentage solids reduction achieved were in
the range of 26.1 to 29% of total soluble solids, 26.1 to 33% of suspended solids, and 43 to 52% of volatile solids. BOD
and COD reductions were in the range of 96.1 to 98.2%, and 96.8 to 98.4%, respectively. Ammonia nitrogen reductions in
this sludge were about 85.2 to 93.3%. Total nitrogen and phosphorus were also found to be appreciably reduced by the
combined aerobic and physicochemical treatment methods.
Keywords: aerobic biodegradation, physicochemical degradation, pollution, wastewater treatment, euprophication,
pharmaceutical sludge
322
washed from the equipment and starch moulds, a wide range
of chemicals arising from different processing units, oils,
emulsions, and a variety of ether extractable materials. All
these found their way into the effluent tank, which was ulti-
mately emptied into the drain. Wastewater flowchart of the
pharmaceutical factory is as shown in Fig. 1.
Sampling of sludge. Samples of sludge were obtained from
the sedimentation tank of the industry. The samples were sto-
red in a refrigerator to prevent biodegradation. Stock solu-
tions of alum, iron (III) chloride, hydrated lime, and polyacry-
lamide partially hydrolysed with soda, used as coagulants,
were prepared fresh at the time of the test. For coagulation
studies, jar tests were conducted. The objective of the jar test
was to determine the optimum dose and the pH value at which
a coagulant should be introduced to the sludge.
Combined aerobic and physicochemical treatment proce-
dure. The sludge samples were first treated using the aerobic
biological systems as described below, but without dewater-
ing and disinfection. The resultant sludge was then treated by
the physicochemical method as described earlier (Asia and
Ademoroti, 2002; Ademoroti, 1982). The flow chart for the
combined aerobic and physicochemical treatment is shown
in Fig. 2.
Aerobic treatment procedure. Two litres of the sludge was
placed in a four litre plastic container, which served as the
digester (Fig. 3). Natural air served as the aeration system.
Five ml of phosphate solution was added as the buffer. One
ml each of magnesium sulphate, calcium chloride, and iron
(III) chloride solutions were added as the nutrient source for
the bacterial population in the sludge. One ml of 50% hydro-
gen peroxide was also added so as to keep the dissolved sul-
Chemicals from
production units
Washing of process
equipment
Starch washed from
equipment and mould
Breakage of vessels
containing prepared drugs
Rejected products crushed
and washed down the drain
Equalization tank
(wastewater and sludge)Drain
Fig. 1. Wastewater flowchart of the pharmaceutical factory
located at Ikeja Lagos, Nigeria, which was investi-
gated for the treatment of the generated sludge.
Supernatant
layer
Digested sludge Digested sludge out
Supernatant
Loose cover
Stirrer
Fig. 3. Laboratory apparatus (digester) for conducting
aerobic digestion studies on the sludge generated
by a pharmaceutical factory.
phide below 0.6 mg/l and to eliminate odour. The plastic con-
tainer was covered with loose plastic cap so as to allow air to
enter the digester system. The sludge was stirred at an interval
of 6 h and was allowed to digest the sludge for 45 days (Asia,
2000; Ademoroti, 1983). The digestion was deemed to have
been completed, as the pH at this stage was noted to have
stabilized. The digested sludge was then analysed to deter-
mine the compositional changes that had occurred. The sludge
was then treated with bleaching powder (calcium oxochlo-
ride) until the level of residual chlorine was between 0.2 and
0.7 mg/l (Henry and Heinke, 1989). This was done to ensure
323Treatment of Pharmaceutical Industry Sludge
Fig. 2. Flowchart for combined aerobic and chemical treat-
ment of pharmaceutical sludge.
Collection
Primary settling
Aerobic treatment
Chemical treatment
Sedimentation tank
Dewatering by
sand bed
Effluent for pH
correction and
chlorination
Sludge for
sun-drying and
incineration
Wastewater and sludge
S1, S2 and S3 = different sampling points
S1
S2
S3
obtaining the maximum level of bacterial kill before dewate-
ring. Dewatering was done by sand bed filtration, using tef-
lon as the filter medium. The wet sludge was pasteurized for
30 min at 100 °C, followed by sun-drying for three days.
Analytical procedures. All samples were analyzed, as per
the standard methods for wastewater and effluent analysis
(Ademoroti, 1996; APHA, 1985). Where analysis was not
immediately possible, the digested sludge samples were pre-
served to inhibit biodegradation. All the reagents used for
the analysis were of analytical grade obtained from BDH
Chemicals Limited, Poole, England.
Results and Discussion
Characterization of the sludge under study. The results of
the characterization carried out on the fresh sludge obtained
from a pharmaceutical industry are shown in Tables 1 and 2.
These results showed that the pH value for fresh pharmaceu-
tical sludge was 6.7, indicating that fresh pharmaceutical
sludge was slightly acidic. The average turbidity of 980 NTU
showed that the colloidal matter in the sludge was high and
by implication the sludge contained high concentration of
solids. The total solids, suspended solids and the volatile
solids were 14,031 mg/kg, 1,231 mg/kg and 10,800 mg/kg,
respectively. The BOD5, COD and the total bacterial count of
the fresh sludge were 452 mg/l, 1446 mg/l and 3.8 x 106 CFU,
respectively. These values are quite high when compared
with the WHO standards. These indicate a strong pollution
potential, and therefore call for treatment of the sludge before
disposal, or for any further application. The results also
showed that the ratio of COD : BOD was 3.20, indicating that
the sludge was capable of undergoing about 50-90% sub-
strate biodegradation (Quano et al., 1978).
As is evident from these results, most of the nitrogen present
in the sludge was more in the form of ammonium-nitrogen.
Consequently, if these sludge samples, containing such high
concentrations of ammonium-nitrogen are discharged to the
environment, depletion of oxygen in the receiving water re-
sources may occur, as the ammonia is oxidized to nitrates by
some groups of aerobic bacteria. Also, nitrates and phosphates,
derived from the sludge are inorganic nutrients, which pro-
mote plant and algal growth. Although the amounts neces-
sary to trigger algal blooms are not well established, yet con-
centrations as low as 0.01 mg/1 of phosphorus and 0.1 mg/1
of nitrates may be sufficient for eutrophication when other
elements are in excess (Henry and Heinke, 1989). In addition
to having a detrimental aesthetic effect on lakes (odour and
appearance), algae can be toxic to cattle, spoil the taste for
use as potable water, block filtration units, and increase chemi-
cal requirements in the water treatment procedures (Henry
and Heinke, 1989).
324 I. O. Asia and C. M. A. Ademoroti
Table 2. Characteristics of fresh settled sludge from a phar-
maceutical factory
Sludge liquor Units Range of values Mean
characteristics
Settleable solids mg/kg 840-1580 1231
Moisture % 95-98 97
Volatile solids mg/kg 8199-12010 10800
Total solids mg/kg 10900-14390 14031
Ash mg/kg 16.5-24.7 23.0
Total nitrogen mg/kg 51.44-80.9 66.80
Phosphorous mg/kg 10.1-14.9 14.67
Potassium mg/kg 1.80-4.2 3.03
Oil and grease mg/kg 3.2-6.7 5.0
Iron mg/kg 0.11-0.17 0.13
Calcium mg/kg 50.1-80.4 68.1
Magnesium mg/kg 41.7-57.6 49.5
Manganese mg/kg nil nil
Copper mg/kg 0.22-0.32 0.28
Cadmium mg/kg nil nil
Chromium mg/kg nil nil
Lead mg/kg 0.9-1.5 1.2
Zinc mg/kg 1.01-1.31 1.2
Table 1. Characteristics of fresh sludge liquor from a pharma-
ceutical factory
Sludge liquor Units Range of values Mean
characteristics
pH 5.21-7.20 6.70
Temperature °C 27-29.5 28.0
Conductivity Scm-1 90-130 120
Specific gravity 1.01-1.03 1.02
Turbidity NTU 740-1145 980
Dissolved oxygen mg/1 1.9-2.8 2.3
BOD5
mg/1 410-480 452
COD mg/1 1010-1640 1446
Total alkalinity mg/1 490-555 530
Bicarbonate alkalinity mg/1 38.4-45.3 42
Ammonia-nitrogen mg/1 41.4-66.0 50.7
Nitrate-nitrogen mg/1 23.3-40.1 38.2
Organic-nitrogen mg/1 10.1-14.9 12.5
Chloride mg/1 127-159 134.2
Sulphate mg/1 117-159 134.2
ABS mg/1 13.2-24.3 21
Total bacterial count cfu/100ml 3.1-4.1 x 106 3.8 x 106
cfu = colony forming units
On the basis of an earlier study (Tables 3, 4; Fig. 4), Asia
(2000) had determined 5 g/l of alum, 4 g/l of iron (III) chlo-
ride, 15.5 g/l of lime and 400 mg/l of polyelectrolyte as the
optimum coagulant/flocculant doses for the treatment of
pharmaceutical industry sludge.
The reduction in solids contents. The amount of solids pre-
sent in the raw and treated sludge are shown in Fig. 5. It is
evident from these observations that the pharmaceutical
industry sludge treated by combined aerobic and physicoche-
mical methods had undergone the total solids reductions of
26.1-29%, suspended solids of 26.1-33%, and volatile solids
of 43-52%.
Oxygen demand values. The results of the study on oxygen-
demand of raw and treated sludge are shown in Fig. 6. It may
Table 4. Results of a pharmaceutical sludge liquor analysis,
after treatment with different doses of lime and polyelectro-
lyte
Lime Polyelectrolyte
dosage pH COD dosage pH COD
(g/l) reduction (mg/l) reduction
(%) (%)
7.5 6.7 - - 6.7 -
9.5 8.1 49.9 100 6.2 41.9
11.5 9.4 53.8 200 5.9 64.7
13.5 10.3 64.7 300 5.8 70.7
15.5 11.1 72.4 400 5.6 77
17.5 11.8 70.3 500 5.6 66.8
19.5 12.2 71.6 600 5.4 66.8
21.5 12.6 69.9 700 5.3 63.3
23.5 12.8 68.2 800 4.9 59.4
25.5 12.8 64 900 4.9 56.4
1000 4.8 53.4
Table 3. Results of a pharmaceutical sludge liquor analysis,
after treatment with different doses of alum and iron (III)
chloride
Alum Iron (III) chloride
dosage pH COD dosage pH COD
(g/l) reduction (g/l) reduction
(%) (%)
- 6.7 - - 6.7 -
2 5.9 48.1 2 6.2 37.9
2.5 5.7 51.5 2.5 5.9 54.1
3 5.4 55.4 3 5.7 64
3.5 5.1 59.2 3.5 5.1 71.6
4 4.8 67.5 4 4.9 79
4.5 4.7 68.7 4.5 4.7 72.5
5 4.7 69 5 4.4 69.6
5.5 4.4 53.7 5.5 4.3 47.4
Fig. 4. The treatment of pharmaceutical sludge using vari-ous methods: (a) alum treatment; (b) iron (iii) chlo-ride treatment; (c) lime treatment; polyelectrolytetreatment; optimum values for (a), (b), (c), (d), res-pectively, were 5 g/l, 4 g/l, 15.5 g/l, 400 mg/l.
325Treatment of Pharmaceutical Industry Sludge
80
70
60
50
40
30
20
10
02 2.5 3 3.5 4 4.5 5 5.5
(a)
CO
D r
eduction (
%)
Alum doses (g/l)
2 2.5 3 3.5 4 4.5 5 5.5
80
70
60
50
40
30
20
10
0
(b)90
CO
D r
eduction (
%)
Iron (III) chloride doses (g/l)
CO
D r
eduction (
%)
Polyelectrolyte doses (mg/l)
80
70
60
50
40
30
20
10
0
(d)90
100 200 300 400 500 600 700 800 900 1000
CO
D r
eduction (
%)
Lime doses (g/l)
80
70
60
50
40
30
20
10
07.5 9.5 11.5 13.5 15.5 17.5 21.5
(C)
19.5 23.5
(c)
326 1. 0. Asia and C. M. A. Adetnoroti
be noted from these observations that the combined biologi- cal and physicochemical treatment methods had proved to be more efficient than either the biological, or the physicochemi- cal method alone in terms of BOD and COD reduction. About 96.1-98.2% BOD and 96.8-98.4% COD reductions were achieved by the combination of these treatment methods.
Reduction in nitrogen. Nitrogen concentrations in the com- bined aerobic and physicochemically treated sludge is shown in Fig. 7. These results show that there was considerable
reduction in the nitrogen contents of the treated sludge. The highest reductions were noticed in the aerobic lime-treated sludge. Ammonia nitrogen reductions in this sludge were about 85.2-93.3%. This may be due to the nitrification pro- cess during the digestion stage in which some ofthe ammonia present was converted to nitrates, while more ammonia was further reduced by the physicochemical treatment processes. Total nitrogen was also found to be reduced in all the sludge samples treated by the combined aerobic and physicoche- mical methods. Reductions were in the range of 64.8-86.7%.
Raw Aerobic AIPC-Alum AIPC-FeCI, NPC-Lime NPC- Polyelectrolyte
Treatment methods
Fig. 5. Solids contents of raw and variously treated sludge from a pharmaceutical factory; TSS = total suspended solids, TS = total solids, VS = volatile solids, AIPC = aerobic/physicochemical combined treatments.
Raw Aerobic NPC-Alum NPC-FeCI, AtPC-Lime NPC- Polyelectrolyte
Treatment methods
Fig. 6. BOD and COD of raw and variously treated sludge from a pharmaceutical factory; BOD = biological oxygen demand, COD = chemical oxygen demand, AIPC = aerobiclphysicochemical combined treatments.
Treatment of Pharmaceutical Industry Sludge 327
0
Raw Aerobic NPC-Alum NPC-F~CI, NPC-L~me AIPC- Polyelectrolyte
Treatment methods
Fig. 7. Nitrogen concentration of raw and variously treated sludge from a pharmaceutical factory; AIPC = aerobiclphysico- chemical combined treatments.
Raw
25
Aerobic AIPC-Alum NPC-FeCI, NPC-Lime NPC-Polyelectrolyte
Treatment methods
I Liquor Cake
Fig. 8. Phosphorus concentration of raw and variously treated sludge from a pharmaceutical factory; A/PC = aerobiclpharma- ceutical combined treatments.
The highest reductions were also found in the lime-treated Conclusion This that lime was a better The present study has revealed that the combined aerobic and
coagulant, if the nitrogen removal was the focus of treatment physicochemical methods are efficient for the treatment of from the aerobically digested sludge. pharmaceutical industry sludge. The choice of an aerobic Reduction in phosphorus concentration. The results on the method over an anaerobic system lies in the fact that pharma- reduction of phosphorus concentrations are shown in Fig. 8, ceutical industries produce a large volume of water-contain- which revealed that the combined aerobic and physicochemi- ing sludge coupled with high values of BOD and COD, and cal methods can be used to reduce phosphorus from the sludge high concentration of volatile solids. Keeping in view the liquor. The phosphorus was adsorbed by the sludge solids economics, time and efficiency, an aerobic treatment system which could in turn be used to condition soil, for the purposes is recommended for the treatment of pharmaceutical sludge. of agricultural applications. This is due to the presence of aerobic organisms with a high
respiratory rate, which acclimatize and treat the sludge in ashort period of time. In addition, in the combination methods,while the aerobic biological system converts almost all theammonia present in the sludge to nitrates, the physicochemi-cal method removes the nitrates from the solution by denitri-fication.
ReferencesAdemoroti, C.M.A. 1982. Recent developments in research
in wastewater renovation in Nigeria. J. Nig. Soc. Engrs.17: 31-36.
Ademoroti, C.M.A. 1983. Trickling filter-activated sludgeplant performance in Ibadan. Effluent Water TreatmentJournal 23: 415-419.
Ademoroti, C.M.A. 1996. Standard Methods for Water andEffluents Analysis, pp. 44-65, Foludex Press Ltd., Ibadan,Nigeria.
APHA. 1985. Standard Methods for the Examination ofWater and Wastewater, 16th edition, American Public
Health Association, American Water Works Association,Water Pollution Control Federation, Washington D.C.,USA.
Asia, I.O. 2000. Studies on Industrial Sludge Treatment Op-tions. Ph.D. Thesis, pp. 1-89, University of Benin, BeninCity, Nigeria.
Asia, I.O., Ademoroti, C.M.A. 2002. The application of physi-cochemical methods in the treatment of aluminium extru-sion sludge. Afric. J. Sci. 3: 609-623.
Henry, J.G., Heinke, G. 1989. Environmental Science andEngineering, Prentice Hall, Eaglewood Cliffs, New Jer-sey, USA.
Priestly, A.T. 1991. Report on Sewage Sludge Treatment andDisposal; Environmental Programs and Research Needsfrom an Australian Perspective, pp. 1-44, CSIRO, Divi-sion of Chemicals and Polymers, Sydney, Australia.
Quano, E.A.R., Lohani, B.N., Thanh, N.C. 1978. Water Pol-lution Control in Developing Countries, Asian Instituteof Technology, Bangkok, Thailand.
328 I. O. Asia and C. M. A. Ademoroti
Essential Oil Composition of Green Peel of the Inter-Varietal Mandarin
Hybrid, Kinnow Orange
Shahid Mahmud*, Amran Waheed, Tanzeela Nazir and Razia KhanumPCSIR Laboratories Complex, Shahrah-e-Jalaludding Roomi, Lahore – 54600, Pakistan
(received October 17, 2003; revised May 25, 2005; accepted May 25, 2005)
Pak. J. Sci. Ind. Res. 2005 48(5) 329-333
Abstract. The essential oil of green peel of large-sized Kinnow fruits was obtained by steam distillation, which recorded a yield
of 0.34%. Kinow is a hybrid of inter-varietal cross of the mendarin orange cultivars, King x Mediterranean. The oil was
analyzed by GC and MS procedures. Among a variety of the oil constituents, 24 compounds were identified by GC, which
were further analyzed for their chemical nature through GC-MS. The major proportion of the indentified constituents
comprised of 6-methyl-5-heptene-2-one (15.33%), carvone (13.8%), cis-carveol (10.04%) and thujanol (4.55%). Rest of the
twenty identified compounds occurred in minor amounts, comprised of 35.84% of the total oil. Limonene, usually the major
component of the citrus oils (35-85%), was present in rather low quantities (2.76%) in the Kinnow orange green peel oil.
Keywords: essential oils, limonene, Kinnow orange, Rutaceae, green orange peels, Citrus reticulata var. Kinnow
*Author for correspondence
Introduction
Pakistan is rich in the production of citrus fruits, especially
orange, the Kinnow orange, grapefruit and lemon. Citrus fruits
and their by-products are used in the production of bever-
ages, confectionery, ice-creams, flavours and pharmaceuticals.
Essential oils are the major by-products obtained from citrus
fruit peels, which find wide applications in food, flavour and
pharmaceutical industries for the manufacture of a variety of
valuable products (Mori, 2002; Lehner, 2000; Vargas-Arispuro
et al., 1998). Extensive research work has been done on the
essential oils of various citrus species. These studies relate
with the improvement of technologies involved in the produc-
tion and consumption of citrus oils. Limonene, a monoterpene
hydrocarbon, being the major constituent of citrus essential
oils, has been separated using a process called deterpenation
(Boelens and Jimenez, 1989; Ferrer and Matthews, 1987; Timelli,
1987; Owsusu-Yaw et al., 1986; Uchida et al., 1984). Using this
process, terpeneless citrus oils are obtained, which are rich in
oxygenated fractions comprising of aldehydes, alcohols and
esters (Sugisawa et al., 1989; Yamamoto et al., 1989).
The present study reports the essential oil composition,
obtained from green peels of Citrus reticulata var. Kinnow,
which is a hybrid produced by the inter-varietal cross of
two mandarin cultivars of C. reticulata, namely, King x Medi-
terranean (Nordby and Nagy, 1975). Constituents of the Kinnow
orange green peel oil were examined, and a comparison was
made with those of mature Kinnow orange peel oil reported
earlier (Haque, 1989). Detection and identification of the green
peel essential oils was carried out by GC-MS. Gas chromato-
gram provided the information of constituents present in the
essential oils, while mass spectra of the compounds aided in
the confirmation of chemical nature of these constituents.
Materials and Methods
The peel material for essential oils extraction. The full-sized
fruits of Kinnow orange fruit having green peel were obtained.
Immature fruits were selected for the essential oil extraction.
Due to the presence of chlorophyll, the fruit was green in
colour and thus capable of photosynthetic fixation of CO2,
similar to the process occurring in leaves (Kefford and Chan-
dler, 1970; Jhon and Sunday, 1965). When fruit ripens, the
chlorophyll contents change to carotenoids, transforming the
colour of the fruit from green to yellow. Green Kinnow orange
fruit of full size was collected, and 4 kg peel was shredded.
Hydrodistillation of fresh and finely divided green peels was
carried out to obtain the green peel essential oil fraction
(Gunther, 1948). The oil was extracted with ether : hexane (1: 4),
the solvent mixture was dried over anhydrous Na2SO4, and
removed under vacuum distillation. Vacuum distillation of the
solvent extract gave essential oil fraction as the residue. The
oil obtained was yellow in colour.
Gas chromatography-mass spectrometry. Jeol model JMS-
AX505H mass spectrometer in combination with Hewlett
Packard 5890 gas chromatograph was used for the GC-MS
analysis. Oil sample was injected into a 25 m x 0.22 mm WCOT
BP5 (5% phenyl, 95% dimethylsiloxane) fused silica column,
using helium as the career gas with the split ratio of 1:100; EI
positive mode, electron energy 70 ev, ionization current 300
µA, ionization source temperature 250 °C, interface tempera-
ture 230 °C, column temperature programmed at 60 °C for 4
329
min, with a 6 °C/min rise at 220 °C. Data acquisition and pro-
cessing were performed by JEOL JMA-DA 5000 system. Vari-
ous components were identified by their retention time and
peak enhancement with standard samples in the gas chro-
matographic mode and MS library search from the derived
fragmentation pattern of the various components of the es-
sential oil.
Results and Discussion
Yield of the essential oil was 0.34%, which was comparatively
lower than the peel oil obtained from matured fruits (0.41%).
The physical characteristics were determined by following
standard methods (Gunther, 1948), which included refractive
index, specific gravity, and acid and ester values (Table 1).
These are comparable to the values reported for other citrus
oils (Sattar et al., 1986).
In order to determine the composition of the essential oils
obtained from green Kinnow orange peels, gas chromato-
graphic analysis was carried out. All EI mass spectra were
scanned at 70 ev. The gas chromatogram of the Kinnow orange
green peel oil is shown in Fig. 1. Each peak in the chromatogram
was assigned to a specific compound according to the reten-
tion time. A total of 62 peaks were detected. Twenty four of
which were identified, which constituted 77.64% of the essen-
tial oils analysed (Table 2).
The identified compounds were further classified in to two
fractions, namely, the oxygenated fraction and the hydrocar-
bon fraction. The oxygenated fraction comprised of octanal,
nonanal, hexadecanoic acid, cis-carvyl acetate, limonene
oxide, citral, decanal, trans-carveol, carvone, undecenal,
carvone oxide, thujanol, ascaridol, farnasol, 6-methyl-5-
heptene-2-one, 3,7-dimethyl-1,7-octadiene-3,6-diol, p-
mentha-2,8-dien-1-ol, 2,3-bornandiol, 3-(2-oxopropyl)
cycloheptanone, and p-mentha-1(7),8-dien-2-ol, while
limonene, ledane, 6-hexadecene-4-yne (E), and 12-methyl-1,
5,9,11-tridecatetraene were found as hydrocarbon fractions
of the essential oils (Table 2).
Limonene, which is a major monoterpene hydrocarbon of cit-
rus oils (Shaw, 1979) occurring to the extent of (50-90%), was
found only up to 2.76 %. The hydrocarbons portion of the oil
was 4.34% and oxygenated hydrocarbons fraction was 68.8%
in the total oil fraction.
The twenty four compounds given in Table 2 were identified
and confirmed by mass spectral studies. The mass spectral
data of these components is given in Table 3. Mass spectra
were found more informative to confirm the structure by show-
ing the molecular weight of each compound. Extensive mass
cracking in the EI mode, analysis of base peak and stable
fragments further aided in the confirmation of structure of
each compound.
The essential oils of Kinnow orange peel were earlier studied
by Haque (1989), reporting twenty three components.
Limonene in that study was the major component (93.7%),
while β-myrecene, ocimene, citral and decanal were present in
minor amounts. The mature orange peel oil analysis has also
been reported by Shaw et al. (1979), which revealed that
limonene was present in large amounts (80-90%). The percent-
Table 1. Physical characteristics of essential oils fraction of
Kinnow orange green peel
Colour light yellow
Smell pleasant
Yield 0.34%
Specific gravity 0.8403
Refractive index 1.4127
Acid value 3.16
Aldehyde value 0.84
Table 2. Composition of the essential oil fraction of Kinnow
orange green peels
Peak No. Name of Compound (%)
1 Octanal 0.193
2 d-Limonene 2.762
3 Nonanal 0.550
4 Hexadecanoic acid 3.531
5 cis-Carvyl acetate 1.55
6 Limonene oxide 0.60
7 Citral 0.53
8 Decanal 1.03
9 trans-carveol 10.04
10 Carvone 13.80
11 Undecenal 0.84
12 Carvone oxide 2.05
13 Thujanol 4.55
14 Ascaridol 2.19
15 Farnasol 1.26
16 6-Hexadecene-4-yne (E) 1.3
17 6-Methyl-5-heptene-2-one 15.33
18 12-Methyl (1,5,9,11-tridecatetraene) 3.30
19 Ledane 1.60
20 3,7-Dimethyl-1,7-octadiene-3,6-diol 3.271
21 p-Mentha-2,8-dien-1-ol 3.245
22 2,3-Bornandiol 0.47
23 3-(2-Oxopropylcycloheptanone) 1.08
24 p-Mentha-1(7),8-dien-2-ol 2.66
330 S. Mahmud et al.
Table 3. Mass spectral data of components of the essential oil fraction of Kinnow orange green peel
Components Molecular formula, Mass spectral data
Molecular weight
Octanal C8H
16O, 128 M+ very weak, 111 (4%), 95 (10%), 69 (22%), 56 (53%), 43 (100%), 41 (75%), 29 (68%)
d-Limonene C10
H16
, 136 M+ 20%, 121 (18%), 93 (50%), 79 (22%), 68 (100%), 67 (45%), 65 (9%), 53 (22%), 41 (25%)
Nonanal C9H
18O, 142 M+ 2%, 124 (5%), 109 (1%), 95 (20%), 81 (25%), 70 (45%), 57 (100%), 41 (98%), 29 (80%)
Hexadecanoic acid C16
H32
O2, 256 M+ 3%, 125 (5%), 112 (35%), 98 (100%), 84 (30%), 69 (25%), 55 (98%), 41 (75%)
cis-Carvyl acetate C12
H18
O2, 194 M+ very weak, 152 (45%), 135 (5%), 134 (30%), 119 (85%), 84 (80%), 67 (20%), 55 (25%),
43 (98%), 28 (100%)
Limonene oxide C10
H16
O, 152 M+ 10%, 137 (20%), 119 (15%), 108 (22%), 93 (40%), 81 (60%), 67 (65%), 55 (45%),
43 (100%), 41 (85%)
Citral C10
H16
O, 152 M+ 10%, 137 (12%), 123 (10%), 109 (15%), 94 (20%), 83 (13%), 69 (100%), 41 (80%),
29 (10%)
Decanal C10
H18
O, 154 M+ very weak, 147 (2%), 138 (9%), 128 (5%), 112 (25%), 95 (20%), 82 (30%), 57 (65%),
43 (98%), 41 (100%)
trans-Carveol C10
H16
O, 152 M+ 28, 134 (20%), 119 (35%), 109 (100%), 93 (22%), 91 (40%), 84 (75%), 41 (52%)
Carvone C10
H14
O, 150 M+ 20, 135 (10%), 109 (12%), 108 (65%), 93 (40%), 82 (100%), 79 (14%), 54 (50%)
Undecenal C11
H22
O, 170 M+ very weak, 109 (12%), 110 (5%), 95 (20%), 81 (45%), 79 (80%), 67 (65%), 55 (70%),
41 (100%), 29 (40%)
Carvone oxide C10
H14
O2, 166 M+ 20, 123 (35%), 109 (25%), 91 (15%), 81 (50%), 67 (30%), 43 (100%)
Thujanol C10
H18
O, 154 M+ very weak, 136 (15%), 121 (40%), 95 (70%), 81 (65%), 79 (50%), 67 (55%), 55 (90%),
43 (100%), 41 (88%)
Ascaridol C10
H16
O2, 168 M+ very weak, 152 (5%), 136 (15%), 121 (10%), 109 (70%), 79 (68%), 67 (30%), 55 (20%),
43 (100%)
Farnasol C15
H26
O, 222 M+ 5, 179 (3%), 161 (4%), 136 (8%), 121 (6%), 107 (12%), 93 (20%), 81 (22%), 69 (100%),
41 (50%)
6-Hexadecene C16
H28
, 220 M+ 8, 177 (5%), 163 (5%), 149 (9%), 135 (10%), 121 (15%), 107 (20%), 93 (32%), 79 (100%),
-4-yne (E) 65 (12%), 51 (10%), 43 (35%), 41 (45%)
6-Methyl-5- C8H
14O, 126 M+ 18, 111 (15%), 108 (50%), 93 (12%), 69 (40%), 55 (45%), 43 (100%), 41 (50%)
heptene-2-one
12-Methyl-1,5, C14
H22
, 190 M+ 10, 175 (4%), 147 (10%), 133 (12%), 121 (28%), 107 (35%), 93 (100%), 79 (70%), 65 (12%),
9,11-tridecatetraene 55 (50%), 41 (90%)
Ledane C15
H26
, 206 M+ 8, 163 (20%), 135 (15%), 121 (25%), 107 (38%), 93 (40%), 79 (30%), 67 (45%), 55 (40%),
41 (100%)
3,7-Dimethyl-1,7- C10
H18
O2, 170 M+ very weak, 152 (5%), 137 (10%), 119 (15%), 109 (12%), 91 (10%), 82 (60%), 71 (95%),
octadiene-3,6-diol 67 (100%), 55 (60%), 43 (90%), 41 (70%)
p-Mentha-2,8 C10
H16
O, 152 M+ 5, 138 (10%), 137 (55%), 109 (72%), 95 (30%), 79 (75%), 67 (35%), 43 (100%)
-dien-1-ol
2,3-Bornandiol C10
H18
O2, 170 M+ very weak, 152 (15%), 134 (10%), 121 (12%), 111 (30%), 95 (100%), 81 (40%), 69 (42%),
55 (38%), 43 (60%), 41 (55%)
3-(2-Oxopropyl) C10
H16
O2, 168 M+ 10, 150 (3%), 126 (2%), 111 (68%), 97 (12%), 83 (25%), 67 (13%), 55 (20%), 43 (100%)
cycloheptanone
p-Mentha-1(7), C10
H16
O, 152 M+ 10, 137 (15%), 134 (98%), 119 (55%), 109 (100%), 91 (50%), 67 (55%), 55 (60%), 41 (80%)
8-dien-2-ol
331Essential Oil Composition of Kinnow Orange Peel
ages of terpene hydrocarbons and oxygenated fractions in
the mature peel oils of citrus species has been reported to be
85-93% and 5-7%, respectively (Sattar et al., 1986; Kefford
and Chandler, 1970). From the present studies it is concluded
that in the essential oil of green peels, the percentage of
terpene hydrocarbon (2.76 %), limonene, was low, while the
percentage of oxygenated compounds (68.8 %) was very high,
as compared to the essential oils extracted from peels of
mature Kinnow orange (Haque, 1989), mature orange (Shaw
et al., 1979), and mature citrus fruits (Kefford and Chandler,
1970). It was observed that among the total essential oil
composition, 6-methyl-5-heptene-2-one (15.33%), carvone
(13.8%), cis-carveol (10.04%), and thujanol (4.55%) were found
as the major components, with higher percentages. Aldeyhdic
and alcoholic contents of the essential oils of Kinnow orange
green peel were 3.143% and 26.42%, respectively.
The results obtained from the analysis of green peel essential
oil fraction indicate that the biogenetic pathway leading to
the synthesis of terpenes is incomplete in the immature state.
With the maturation of the fruit, some of the intermediate mol-
ecules, such as 6-methyl-5-heptene-2-one, 3,7-dimethyl-1,7-
octadiene-3,6-diol, 3-(2-oxopropyl) cycloheptanone, 3,7-dim-
ethyl-1,7-octadiene-3,6-diol, 6-hexadecene-4-yne (E), and 12-
methyl-1,5,9,11-tridecatetraene and others are transformed
through biosynthetic routes to the monoterpenes normally
found in the essential oil of mature Kinnow orange and other
citrus fruits (Kefford and Chandler, 1970).
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peak numbers and the compounds listed in Table 2.
6.405
6.685 12.159
1
3
2
9.229
12.871
13.489
4
5
6
15.812
9
7
16.209
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19
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24
18
13
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100
SD12(N)17.271 18.371 22.724
20.658
23.763
25.572
27.217
28.723
%
010.000 15.000 20.000 25.000 30.000 35.000 40.000
rt
8
10
332 S. Mahmud et al.
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333Essential Oil Composition of Kinnow Orange Peel
Introduction
Malaria is widely distributed, manifesting itself in different
populations in different ways due to differences in the vari-
ous disease determining factors in different localities, such as
the strain of parasite, vector, prevalent environment, and the
humans themselves. The disease burden in Africa rests pre-
dominantly on young children and pregnant women, whereas
in Asia, adults are affected as much as are the children. In
Africa, the disease is characterized by death as the most sig-
nificant end-result, whereas in other parts of the world, physi-
cal debility and loss of economic productivity are the pre-
dominant end-results (Fansidar Monograph, 1998; McComark
and Morgan, 1987; Muto et al., 1971). Several reports have
been published on the successful treatment of acute attacks
of falciparum malaria in adults with sulfadoxine pyrimethamine
combination since the middle sixties todate, both in chloro-
quine sensitive and chloroquine resistant areas. The earlier
reports were mainly focused on efficacy, tolerance and the
dose determining studies (Walker et al., 1993; Weidekamn et
al., 1987; Walter et al., 1986; Waxman and Herbert, 1969).
Fansidar, an antimalaria drug, is composed of two active in-
gredients: sulfadoxine (N´-(5,6-dimethoxy-4-pyrimidinyl)-
sulfanilamide and pyrimethamine (2,4-diamino-5-(p-
chlorophenyl)-6-ethyl pyrimidine) in a ratio 20 : 1 (sulfadoxine
500 mg and pyrimethamine 25 mg). It is available in tablets,
syrup and ampoule (Fansidar Monograph, 1998). The mode
of action is based on the reciprocal potentiation of its two
Pak. J. Sci. Ind. Res. 2005 48(5) 334-337
Biochemical Changes Induced in Some Rabbit Tissues on theAdministration of an Antimalaria Drug, Fansidar
O. I. Oloyede* and M. F. Asaolu
Department of Biochemistry, University of Ado-Ekiti, Ekiti State, Nigeria
(received January 7, 2004; revised July 4, 2005; accepted July 23, 2005)
Abstract. The effect of Fansidar (40 mg/kg), a widely used antimalaria drug, was investigated on enzyme activities and
some other biochemical constituents in some selected rabbit tissues. The enzymes assayed were alanine transaminase,
aspartate transaminase and alkaline phosphatase. Total protein and glucose contents in the tissues were also deter-
mined. The results obtained showed a decrease in the activities of alanine transaminase and aspartate transaminase in
liver and heart when the drug was administered. This indicates tissue damage, which was complicated with an increase
in the activities of these enzymes in the blood due to cell leakage. There was a significant elevation of alkaline
phosphatase activity in liver, heart and blood, on the third day of drug administration, which continued upto the
seventh day only in the heart. This shows that prolonged usage of the antimalaria drug, Fansidar, may lead to cell
destruction and degradation.
Keywords: antimalaria drug, Fansidar, alanine transaminase, aspartate transaminase, tissue damage, alkaline phosphatase
components. The antimalaria action is accomplished by se-
quential blockade of the two enzymes involved in the bio-
synthesis of folic acid within the parasites. Sulfadoxine is a
structural analogue of para-aminobenzoic acid (PABA), and
it competitively inhibits the enzyme, dihydropleroate syn-
thetase, which is responsible for the incorporation of PABA
into dihydrofolic acid and decrease in the amount of meta-
bolically active tetrahydrofolic acid, a cofactor for the syn-
thesis of purines, thymidines and DNA. Pyrimethamine binds
to, and reversibly inhibits, the protozoal enzyme dihydro-
folate reductase, selectively blocking the conversion of
dihydrofolic acid to its functional form, tetrahydrofolic acid.
When pyrimethamine is administered concurrently with
sulfadoxine, synergism occurs, which is attributed to the
inhibition of tetrahydrofolate production at two sequential
steps in its biosynthesis (Fansidar Monograph, 1998).
The main objective of the present was to investigate the
effect of Fansidar on transaminase activities, so as to ensure
a prompt cure of malaria without any undue adverse effects.
Materials and Methods
The antimalaria drug. Fansidar tablets were obtained from
SwissPharma, Nigeria Limited (formerly Roche), Lagos State,
Nigeria. All other reagents used were of analytical grade and
were prepared in double glass-distilled water.
Animal groupings. Eight rabbits, both sexes, weighing
256 to 550 g, were obtained from the Animal Unit of the
Anatomy and Physiology Department, University of Ibadan,*Author for correspondence
334
Ibadan, Nigeria. The animals were divided into four groups of
two rabbits each. Rabbits in group one served as the control.
They were fed with pellets and water ad libitum.
Drug administration. Fansidar tablets (8 g), liquified in 100
ml distilled water, were orally administered to the rabbits at a
dose of 40 mg/kg body weight. Rabbits in group two were
given only one dose, those in group three were administered
two doses at 24 h intervals, while those in group four were
administered three doses at 24 h intervals and were kept alive
for four days. The animals were dissected on the respective
completion of one, two, and three dose regimens after two,
three and four days of the administration of the respective
drug doses to collect the tissue samples. The control group
was given distilled water, instead of the drug.
Collection of tissues. The rabbits were bled, while under ana-
esthesia, into a clean, dry beaker and serum was prepared as
described by Akanji and Ngaha (1989). The blood was kept
frozen at -20 °C, until required. Animals in each group were
killed 24 h after the completion of each dose regimen. They
were killed while still under anaesthesia by cervical disloca-
tion and were quickly dissected. Liver and heart were removed
into ice-cold 0.25 M sucrose solution. The tissues were
decapsulated and washed free of blood, before weighing. Each
organ tissue was cut very thin with a clean sterile blade and
then homogenized in ice-cold 0.25 M sucrose solution
(1 : 5 w/v). The homogenates were kept frozen at -20 °C over-
night, before use for enzyme analysis. This was to ensure the
maximum release of the enzymes located in the cell organelles
(Akanji and Ngaha, 1989; Ngaha, 1984).
Enzyme and protein determinations. Alkaline phosphatase,
alanine transaminase and aspartate transaminase activities
were determined, using appropriate buffer systems. Alkaline
phosphatase activity was determined by measuring the p-
nitrophenol liberated from p-nitrophenyl phosphate at 400 nm
(Wright et al., 1972). Protein content of the tissues were deter-
mined by the biuret method (Plummer, 1978). Spectrophoto-
metric method was used to determine alanine and aspartate
transaminases (Kings, 1960). Alanine transaminase was
measured by monitoring the concentration of pyruvate
hydrazone formed with 2,4-dinitrophenylhydrazine at 546 nm.
Aspartate transaminase was determined by monitoring
the concentration of oxaloacetate hydrazone formed with
2,4-dinitro-phenylhydrazine at 546 nm.
Results and Discussion
Table 1 illustrates the changes in the activities of aspartat
transaminase, alanine transaminase, and alkaline phos-
phatase in some selected rabbit tissues, following the daily
administration of Fansidar. There were significant changes
in the liver and heart tissues (p < 0.05) throughout the dura-
tion of drug administration, as compared with the control
values. Aspartate transaminase activity reduced in the liver
after the administration of the first dose (p < 0.05). The re-
duction continued until the third day of the drug administra-
tion. The activity of this enzyme in the liver recovered by the
seventh day, however showing a small increase in the aspar-
tate transaminase activity as compared with the control val-
ues. The alanine transaminase activity, on the other hand,
was noted to reduce throughout the study period. Alkaline
phosphatase activity increased on the third and seventh
day, as compared with the control value. In the heart tissue,
activities of all the enzymes under investigation reduced (p
< 0.05) after the first dose of drug administration, but later
increased appreciably on the third day. The increase in ala-
nine transaminase and alkaline phosphatase activities in the
heart tissue lasted throughout the duration of the drug
administration period.
Table 1. Effect of Fansidar (40 mg/kg animal body weight) on some enzyme activities* in the rabbit tissues
Tissues Days Group Aspartate Alanine Alkaline
after injection transaminase transaminase phosphatase
Liver 0 1(control) 142.00+2.83 217.10+2.41 23.40+14.14
1 2 (one-dose) 62.20+1.00 28.60+0.89 1988+16.97
3 3 (two-dose) 11.10+0.28 28.60+1.67 3748.80+16.97
7 4 (three-dose) 62.00+2.12 17.10+1.34 221.5+4.95
Heart 0 1 (control) 144.45+7.85 100.00+1.41 3976.00+33.94
1 2 (one-dose) 79.98+0.03 34.29+1.71 170.40+13.58
3 3 (two-dose) 106.65+0.64 51.42+0.82 5026.80+32.81
7 4 (three-dose) 66.66+0.48 120.00+13.44 7071.60+26.02
*enzyme activities are expressed as specific activity (unit per litre); + = standard deviation; statistical significance was tested using student’s
t-test, compared with control value p < 0.05
335Biochemical Changes Induced by Antimalaria Drug
The level of enzyme activities in the serum are shown in
Table 2. There was an increase in the activity of all the
enzymes under investigation on the third day. Aspartate
transaminase and alkaline phosphatase activities reduced
(p < 0.05) on the seventh day, whereas alanine transaminase
activity was noted to increase, till the termination of the
drug administration period.
The pattern of variations in protein and glucose contents
in the rabbit tissues is shown in Table 3. Decrease in
protein and glucose contents occurred in the liver, which
lasted for the duration of drug administration period. A
decrease in protein contents was observed in the heart,
immediately after the administration of the first dose as
opposed to an increase in the glucose contents. The heart
tissue showed a continuous increase in the protein con-
tents until the termination of the experiment. Oral admin-
istrations of 40 mg/kg animal body weight of Fansidar
were tolerated by the rabbits and adverse drug reaction
was not observed.
The results of the present study indicate that Fansidar
administration (40 mg/kg) resulted in a significant reduc-
tion in the activities of aspartate transaminase, alanine tran-
saminase and alkaline phosphatase in the rabbit liver and
heart. The reduction in the activities of these enzymes in
the liver and heart may be attributed to the loss of mem-
brane components in the tissues, resulting in the release of
biochemicals including the enzymes, in to the extracellular
environment. This may be due to destruction of the lyso-
somal membrane of the liver and heart by excess dosage of
Fansidar (i.e., on the 3rd day), which leads to the loss of
enzymes from these tissues to the extra-cellular environ-
ment, i.e., in to the blood, thus increasing the level of these
biochemicals in the serum.
Elevated alkaline phosphatase activity observed in the heart
on the seventh day, viz., on termination of the experiment,
may have resulted from increased synthesis of plasma mem-
brane proteins during the repair of the damages caused by
the drug molecules (Wright et al., 1972; Brain and Kay,
1927). It may also be due to the increase in the functional
activity of the organs (Brain and Kay, 1927). The lack of
further significant increase in aspartate transaminase activ-
ity in the heart, with increasing number of doses, may be
due to a reduction in the effect of the drug, or as a sign of
cell recovery (Ngaha, 1984).
In conclusion, the results obtained from this study have
revealed that enzyme activities decreased with increased
dose of Fansidar in the tissues studied, as opposed to an
increase in the blood (especially on the 3rd day). On the
basis of these observations, it may be suggested that
prolonged usage and high dosage of the drug (Fansidar)
can lead to damage of organs such as liver and heart.
Table 2. Effect of Fansidar (40 mg/kg aninmal body weight) on some blood serum enzyme activities* in rabbits administerad the
antimalaria drug
Days Group Aspartate Alanine Alkaline
after injection transaminase transaminase phosphatase
0 1 (control) 20.00+0.74 17.10+0.42 153.00+1.00
1 2 (one-dose) 13.30+0.28 5.70+0.28 70.40+2.97
3 3 (two-dose) 62.20+0.51 22.90+0.42 454.40+8.49
7 4 (three-dose) 24.40+0.14 32.28+1.02 284.00+5.66
*enzyme activities are expresed as specific activity (unit per litre); + = standard deviation; statistical significance was tested using student’s
t-test, compared with control value p < 0.05
Table 3. Effect of Fansidar (40 mg/kg animal body weight) on
protein and glucose contents of rabbit tissues
Tissues Days Group Protein Glucose
after (g/l) (mg/100 ml)
injection
Liver 0 1(control) 239.00+1.00 403.36+4.75
1 2(one-dose) 45.50+1.00 365.77+8.16
3 3(two-dose) 32.80+3.96 232.43+10.71
7 4(three-dose) 13.20+1.98 194.59+7.65
Heart 0 1(control) 119.50+0.71 1144.14+5.66
1 2(one-dose) 49.20+4.40 1702.70+3.82
3 3(two-dose) 92.70+3.82 567.57+10.71
7 4(three-dose) 219.45+0.78 464.86+21.41
Serum 0 1(control) 51.90+2.55 209.01+1.40
1 2(one-dose) 38.20+0.28 335.14+6.87
3 3(two-dose) 38.20+0.57 219.82+0.25
7 4(three-dose) 50.50+0.71 227.47+1.41
+ = standard deviation; statistical significance was tested using
student’s t-test, compared with control value p < 0.05
336 O. I. Oloyede and M. F. Asaolu
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337Biochemical Changes Induced by Antimalaria Drug
Introduction
Interpecific variations in the fecundity of fishes are important
attributes of the inland water fisheries resources. Bagenal and
Braum (1978) defined fecundity as the number of ripe ova in
the female prior to the next spawning period, determined by
counting all mature eggs in the ovary in relation to the length,
body weight and ovary weight. Interspecific variations in the
fecundity of fishes is also critical in the fisheries biology due
to the relevance of these parameters in various applications,
such as the assessment of population capacity and spatio-
temporal regimes in the egg production capacity (Omoregie
et al., 1998; King, 1996). The knowledge of fecundity variance
among different families of fishes is of significance in the
investigations related to reproduction, life history, investment
in fisheries, and various applied aspects of fisheries biology
and pisciculture (King, 1997). Fecundity assessment of fishes
has been further useful in race identification, progeny
survival studies, stock evaluation, aquaculture-based induced
spawning, and egg incubation (Coastes, 1988; Mareus, 1982;
Bagenal, 1978). As a result, fecundity assessment of several
fishes has been the subject of a number of biological and
ecological studies (Pullin and Lowe-McConnell, 1982; Balarin,
1979; Fryer and Iles, 1972; Burchard, 1967).
King (1998) reported, in a study on the weight-fecundity rela-
tionship of Nigerian fish populations, that as the maximum
body weight of the fish increased, the number of eggs pro-
duced per gram increased. This trend may be linked to the
biomechanism where a fish continues to grow after the fecun-
dity has been stabilized, so that the fecundity for a given size
appears to decline, albeit remaining constant (Bagenal, 1978).
This phenomenon, best known in individual species, was also
observed in multispecific populations. Little is known about
interspecific fecundity variance of different families of fishes
in numerous small rivers and streams in Nigeria (Komolafe and
Arawomo, 1998; King, 1996). Besides the work of Roff (1986),
there appears to be no previous investigation, with a holistic
approach aimed at determining the fecundity variance with
regards to different fish families in a given small body of water.
Most of such studies in Nigeria have centered on families in-
habiting large rivers, lakes, lagoons and reservoirs (Fawole
and Adewoye, 1998; Mgbenekan and Eyo, 1992; Adebisi, 1987;
Nwadiaro, 1987; Nwadiaro and Okorie, 1986; Fagade, 1979;
1978; Akintunde and Imevbore, 1979).
Pak. J. Sci. Ind. Res. 2005 48(5) 338-344
Interspecific Variations in the Fecundity of Some Dominant Fish
Populations in Ikpoba River, Nigeria
L. I. N. Ezemonye* and F. A. OsiezagheDepartment of Zoology, University of Benin, Benin City, Edo State, Nigeria
(received February 4, 2003; revised August 8, 2005; accepted August 18, 2005)
*Author for correspondence; E-mail: [email protected]
Abstract. Interspecific variability and trends in the fecundity of five dominant fish species, namely, Auchanoglanis
occidentalis (Bagridae), Brycinus longipinnis (Characidae), Tilipia mariae (Cichlidae), Malapterurus electricus (Malap-
teruridae), and Xenomystus nigri (Notopteridae) from Ikpoba river were studied, vis-à-vis certain morphological attri-
butes. Estimates of the “b” value (regression coefficient; exponent of length-fecundity relationship) ranged between 0.301
in T. mariae and 3.265 in A. occidentalis with a mean of 1.850. The maximum size of the fish populations examined did not
significantly influence the relative magnitude of “b” (regression coefficient). The parameter β of the linear length-fecundity
(LF) relationship of the form F= β are also presented. Estimates of β (slope of regression coefficient) ranged from min 2.18
in X. nigri to max 142 in M. electricus. LF data in this study suggested that absolute fecundity of the fish populations was
dependent on the cube of their length, and hence body volume. There was a positive allometric functional relationship
between the mean total body weight, mean body condition and mean absolute fecundity (p < 0.05). The mean absolute
fecundity varied considerably among the families (coefficient of variation, cv = 74.04%). The decreasing order of variance
of the mean for absolute fecundity was M. electricus > T. mariae > B. longipinnis > A. occidentalis > X. nigri. The hierarchy
of mean absolute fecundity was B. longipinnis > M. electricus > T. mariae > A. occidentalis > X. nigri. There was no
significant relationship between the mean absolute fecundity and mean total length (p > 0.05). Interspecific divergence in
fecundity and morphometric attributes of these species (cv = 18.8-89.20%) appeared to enhance reproduction isolation or
partitioning. This explains, in part, the reason for sustainable coexistence of these fish species within the same habitat in
Ikpoba river.
Keywords: interspecific variation, fish fecundity, Ikpoba river, length-fecundity relationship, inland fishery
338
Earlier studies on the fishes of Ikpoba river have focused on
heavy metal pollution (Eghaboh, 1998; Oguzie, 1996; Fufeyin,
1994), and length-weight relationship (LWR) of fish popula-
tion (Ezemonye and Oshiokpekhai, 1999). No work has been
done on the dynamics of fecundity variations of the fish popu-
lations of this river. The present work aims at contributing
information on the fecundity dynamics of some dominant and
economically important fishes found in Ikpoba river, Benin
City, Southern Nigeria.
Materials and Methods
The fish samples were collected from four stations of Ikpoba
river (Fig. 1) during November 2000 and October 2001. The
fishing was carried out using traditional fishing gear and traps
consisting of cast nets, gill nets, basket traps, and hook and
line. Each specimen was identified by reference to the taxo-
nomic works of Leveque et al. (1992), Teugels et al. (1992),
Fischer and Bianchi (1984), and Reed et al. (1967). Routine
body measurements of the total length (TL), the distance from
the anterior part of the snout to the flexure line of the caudal
penduncle; standard length (SL), the distance from the ante-
rior part of the snout to the posterior end of the caudal fin;
and the total weight (TW) were determined.
For the dominant and commercially important fish species the
parameters, “a” (regression constant) and “b” (regression coef-
ficient) of the allometric length-fecundity relationship (LFR)
of the form, as described by Bagenal (1978), was estimated as
below:
F = aLb
(1)
this was transformed as:
Log F = Log a + b Log L (2)
Using LF data pairs and least squares linear regression, the
linear LF functions of the form
F = a + βL (3)
were determined
where:
L = length
F = fecundity
LF = length-fecundity
a = regression constant
b = regression coefficient (intercept on Y-axis)
β = slope of regression constant
Reproductive studies were carried out on the dominant gravid
fish samples. They were sexed after dissection to reveal the
gonads. Absolute fecundity, the total number of eggs found
in a ripe female before spawning, as defined by Bagenal and
Braum (1978), was determined by the direct count of all eggs
after air-drying at 27-29 °C ambient temperature for 6 h (King,
1996). The determination of interspecific variation of egg
production capacities was facilitated by the calculation of
relative fecundity (weight-specific fecundity), i.e., the count
of number of eggs per g total body weight (Bagenal, 1978).
Standard fecundity (length-specific fecundity) was expressed
as the number of eggs per cm total length (King, 1991; Nikolsky,
1963). The general well-being of the fish samples, defined as
the condition factor (K), was computed as:
K = wt 100 (TL)-3
where:
wt = total body weight (g)
TL = total body length (cm)
Determination of the trend in interspecifc variations in fish
morphometry (length, weight) and fecundity were statistically
examined by coefficient of variations (cv) in accordance with
Lowentin (1966), and the regression analysis using untrans-
formed or double-log-transformed (common logarithm) data
sets as described by King (1996).
Results and Discussion
A total of 247 fish specimens belonging to 13 families and 17
species were collected and studied. Out of these 64 fish
339Interspecific Variations in the Fecundity of Fishes
1
2
3
4
Isianor
Ugbowo
University of Benin
Ekosodinr
Omoregie
NIGERIA
Area ofstudyN
Fig. 1. Map of Benin City showing Ikpoba river and the
sampling points (1, 2, 3, 4).
Legends
Ikpoba river
Benin moat
Major roads
Sampling points
specimens (25.9% of the total catch) were sexually active (ripe)
females. The number of specimens of each of these species
examined is given in Table 1. The dominant and gravid females
examined belonged to five species, each a member of different
family.
Morphometric measurements. A wide range of variations in
the mean length and weight were observed within each spe-
cies. These ranged from min total body length of 8.05 cm in
Brycinus longipinnis (Characidae) to a max of 19.36 cm in
Malapterurus electricus (Malapteruridae). The weight varied
from 20.82 g in Xenomystus nigri (Notopteridae) to 106.27 g
in Malapterurus electricus (Table 2).
The coefficient of variations for mean total length and weight
were 18.82% and 82.42%, respectively (Table 3). Mean total
body weight increased with mean total body length: degree
of freedom (df) = 4; correlation coefficient (r) = 0.8537; p < 0.05.
The length-fecundity relationship (LFR) data of the fish popu-
lations studied, along with the available information on fish
population specific results, ancillary statistics such as sample
size or the number of individuals (n), length ranges, fecundity
ranges, and their means are summarized in Table 2.
All the recorded length fecundity (LF) correlations were not
significant at p = 0.05. Estimates of “b” ranged from 0.30 in
Tilipia mariae to 3.265 in Auchanoglanis occidentalis (i.e.,
10-fold variation), with the mean “b” value of 1.849 (standard
deviation = 1.36). The exponential variability in values of “b”
showed that the fish population in Ikpoba river had coeffi-
cient of variation (cv) = 73.62%, exhibiting high heteroge-
neous profile. A regression of the fish maximum length (Lmax)
versus length exponent in “b” was not significant (r = 0.424,
df = 8, p = 0.05).
Across the five fish populations, for which Lmax - β data pairs
were calculated, a significant inverse relationship existed bet-
ween Lmax and “b” (r = 0.570, df = 4, p = 0.05). Values of β
(Table 1) were heterogeneous (cv = 90.24%). These varied
from βmin (minimum slope) of 2.18 (X. nigri) to βmax (maximum
slope) of 142.88 (M. electricus), i.e., 65-fold variation, with a
mean β of 29.0, which shows that the egg production capacity,
in terms of increase in the egg number per cm, increased with
body length.
Body condition. The body condition factor determined in this
study was the morphological expression of the overall well-
being of the fishes (King, 1991). It is an index of fitness of the
fish populations and operates on the assumption that heavier
the fish for a given length, the better condition (K) it has, and
vice versa (Froese and Binohlan, 2002; Bolger and Connolly,
1989). Mean body condition varied remarkably between the
species (cv = 59.06%). It ranked lowest in X. nigri and the
highest in T. mariae (Table 2). It had no significant relation-
ship between mean total body length, mean total body weight,
and mean absolute fecundity at p > 0.5.
Fecundity. The fecundity values of the fish populations exa-
mined are shown in Table 2. The mean absolute fecundity
varied remarkably among the species (cv = 74.04%). The de-
creasing order of mean absolute fecundity was M. electricus
(Malapteruridae) > T. mariae (Cichlidae) > B. longipinnis
(Bagridae) > X. nigri (Notopteridae). There was a significant
positive relationship between mean absolute fecundity and
mean total body weight (df = 4, r = 0.8345, p > 05). There was
340 L. I. N. Ezemonye and F. A. Osiezaghe
Table 1. Length-fecundity relationships and related statistics for fish populations of Ikpoba river, Benin City, Edo State, Nigeria
Fish species/ Fecundity Length-fecundityFamily parameters
Number Length range min max mean b β r(n) (cm) (cm) (cm) (cm)
Auchanoglanis occidentalis 8 Tl: 12.2-15.7 58.89 515.90 189.30 3.265 26.66 0.331(Family: Bagridae) Sl: 11.4-13.2
Brycinus longipinnis 31 Tl: 7.9-9.8 77.19 986.70 477.59 1.733 93.33 0.104(Family: Characidae) Sl: 7.0-9.5
Tilipia mariae 9 Tl: 12.2-16.7 484.30 1191.30 758.72 0.301 9.375 0.080(Family: Cichlidae) Sl: 10.2-13.7
Malapterurus electricus 11 Tl: 13.5-22.6 177.60 2822.60 914.97 3.194 142.88 0.601(Family: Malapteruridae) Sl: 11.0-19.8
Xenomystus nigri 5 Tl: 11.7-18.4 49.00 139.00 77.88 0.756 2.18 0.303(Family: Notopteridae) Sl: 11.2-17.7
b = regression coefficient (intercept on Y-axis); β = slope of regression coefficient; r = correlation coefficient; Tl = total body length; Sl =standard length
no significant relationship between the mean absolute fecun-
dity and mean total body length. However, with the exclusion
of X. nigri and A. occidentalis, mean absolute fecundity incre-
ased with mean total length from 477 eggs in B. longipinnis
(8.05 cm) to 914 eggs in M. electricus (19.36 cm). These were
not statistically significant, which may be due to the small
amount of data involved in the computations. For fish of the
Ikpoba river, a summary of the degrees of interspecific varia-
tions in the species, as measured by coefficient of variation,
regarding morphometric and fecundity parameter, is presen-
ted in Table 3. Only the body length had values less than
20%, the variation in all other parameters exceeded 60%.
The ability of a fish population to survive to sexual maturity
and contribute to the gene pool is a measure of its fitness.
Collectively, these surviving individuals show remarkable
interspecific variations in total body length and weight of
fecund females. This variation often determines the level of
survival of the entire population. To protect a fish resource,
with management regime in the face of unguided exploitations,
it is important to relate fecundity variations to niche prefe-
rence and survival. In this study, there were noticeable inter-
specific variations in the total body length, weight and fecun-
dity of the sexually (ripe) active females examined. This may
impose corresponding differences in resource exploitation
pattern and reproductive habitat preference. Related studies
have shown that body length and weight are correlates of
reproductive investment, sexual maturity and egg production
capacity (Fawole and Adewoye, 1998; King, 1991; Bagenal
and Braum, 1978; Lagler et al., 1977; Fryer and Iles, 1972).
Dajoz (1977) and Wootton (1973) have reported that varia-
tions in body size in fish may result in differences in the choice
of diet quality and quantity, which in turn may influence their
reproductive attributes (maturity size, fecundity, egg size and
variability of the eggs). It does imply that the observed inter-
specific divergence in the size of sexually matured females of
the fish population in Ikpoba river could enhance reproduc-
tive isolation since reproductive investment and fecundity
attributes have been noted to be size dependent.
The interpopulation variability of exponential LFR value of
“b” in this study was lower than those reported by King (1997)
for some Nigerian fish populations, which were moderately
homogeneous. The “b” values of 0.301-3.265 fall below the
341Interspecific Variations in the Fecundity of Fishes
Table 2. The means and ranges (in parenthesis) of total length (TL) and total weight (TW), condition factor (K), and fecundity-
related observations of fish populations of Ikpoba river, Nigeria
Parameters Auchanoglanis occidentalis Brycinus longipinnis Tilipia mariae Malapterurus electricus Xenomystus nigri
(Family: Bagridae) (Family: Characidae) (Family: Cichlidae) (Family: Malapteruridae) (Family: Notopteridae)
TL (cm) 14.11 8.05 14.94 19.36 15.14
(12.2-15.7) (7.9-9.8) (12.2-16.7) (13.5-22.6) (11.7-18.4)
TW(g) 33.43 9.72 77.82 106.27 20.82
(22.74-45.03) (7.30-12.92) (40.40-94.22) (29.28-180.60) (10.73-28.05)
Condition 1.1900 0.6358 2.3337 1.4645 0.5999
factor (K)
Fecundity (range) 58.89-515.90 77.19-986.70 484.30-1191.30 138.10-2822.60 49.00-139.00
Absolute 189 477 758 914 77
fecundity (mean)
Relative 5 49 9 8 3
fecundity
Standard 13 41 50 47 5
fecundity
Number of 25 63 32 25 6
specimens
examined
Number of 8 31 9 11 5
specimens with
ripe ovaries
range (1.56-5.77) reported by King (1997). These values also
fall outside the values of “b” (2.3 - 5.3) reported by Bagenal
(1978) for most fish. Similarly, Kock and Kellermann (1991)
recorded “b” = 1.088-6.03 for Antarctic notothenioid fish. The
mean of the length exponent “b” is not significantly different,
showing that the “Cube Law” (Bagenal, 1978) can be applied
to most of the fish studied. The observation is similar to that
reported earlier for some selected Nigerian fish populations
by King (1997). The implication, therefore, is that the body
volume is an important morphometric determinant for fecun-
dity dynamics in the fish studied.
The result of the regression of fish maximum size (Lmax) exa-
mined versus length exponent in “b” showed that the maxi-
mum size of fish had no significant influence on the relative
magnitude of the length exponent of the LFR. The inverse
relationship exhibited between Lmax and β suggested that as
the maximum body length of the fish increased, the number of
egg production also increased. This could be attributed to
biomechanism, whereby fish continues to grow even after the
fecundity has stabilized, so that the fecundity for a given size
appears to decline albeit remaining constant (Bagenal, 1978).
There was a positive allometric functional relationship between
the total body weight, condition factor and absolute fecun-
dity. Earlier, Victor and Akpocha (1992) had reported an inverse
relationship between the condition factor and length of Para-
channa obscura, which was similar to the observations in the
present study for the dominant fish populations in Ikpoba
river, thus suggesting that habitat conditions were unfavor-
able for the larger length groups.
There was no significant relationship between the absolute
fecundity and total body length in the present study. Fish
species of the same length or weight had variable fecundities.
Bagenal (1957) had earlier reported a wide variation in fecun-
dity among individuals of the same fish species, with regard
to size and age. The absence of any interspecific increase in
absolute fecundity with total length of cichlids was similar to
the observation of Nwadiaro (1987) for cichlids of the Somb-
rerio river. A comparison of the absolute fecundity values for
some fish populations (T. mariae and M. electricus) in this
study with other related studies showed that the values
reported by King (1998; 1996), Nwadiaro (1987), and Camara
(1984), for T. mariae were higher than the values reported in
the present study for the same species. However, the values
reported in this study were higher than the reports of Adebisi
(1987) for the five species. Fecundity values as reported by
King (1998) for M. electricus were higher than those reported
in this study, but the values from this study were higher than
the observations of Okon (1994) for the same species in Ikpoba
river. The relative fecundity data also followed the same pat-
tern as the absolute fecundity. From the comparison and data
available in this study, it would appear that a wide range of
egg production occurred in different fish populations of the
species stu-died. Several studies have reported that interpopu-
lation disparity in egg production capacity is attributable to
several fecundity related factors such as size of specimen,
growth dynamics, and food resource conditions (King, 1998;
1996; Alvarez-Lajonchere, 1982; Bagenal and Braum, 1978;
Bagenal, 1978; Dajoz, 1977; Wootton, 1973). Bagenal (1969;
1966) further suggested that variations in fecundity may be
due to differential in the abundance of food.
The assumption of the domineering role of nutritional resour-
ces in regulating variations in fecundity (Leveque, 1997; Dajoz,
1977; Wootton, 1973), where fish in “good” body condition
would be expected to have larger egg output than those in
“poor” body condition (Baltz and Moyle, 1982), did not apply
in this study, suggesting that other factors were more critical.
Undeniably, fish ecological and fishery management experts
have established a strong relationship between morphologi-
cal and ecological similarities (Watson and Balon, 1984). It
was assumed that the morphological similarity between spe-
cies should lead to similar pattern of the same resource-exploi-
tation and habitat preference. This suggests that resource use
competition should be higher in morphologically similar spe-
cies and linked to the behavioural pattern.
The significant level of divergence in morphological and
reproduction character observed in this study appears to
enhance minimization of intense resource competition and
stable coexistence.
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Mean absolute fecundity 89.20%
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344 L. I. N. Ezemonye and F. A. Osiezaghe
Numerical Taxonomy of Two New Mite Species of the Genus
Caloglyphus Berlese (Acaridae) from Pakistan
Muhammad Sarwar*a, Muhammad Ashfaq b and Shamshad Akbar b
aNuclear Institute of Agriculture, Tando Jam - 70060, Sindh, PakistanbUniversity of Agriculture, Faisalabad - 38040, Pakistan
(received April 30, 2004; revised May 7, 2005, accepted August 18, 2005)
Pak. J. Sci. Ind. Res. 2005 48(5) 345-353
Abstract. During the mite sampling of the genus Caloglyphus, the taxonomical analysis of diagnostic features of the
hypopial stage confirmed two new taxa named as C. agrios and C. hadros. When compared with the already known
worldwide species within this genus, these species showed sufficient dissimilarity to be classified as separate taxa. The
characteristic features differentiating these new species from the other Caloglyphus species are the shapes of gnathosoma,
propodosoma, hysterosoma, apodemes, coxal fields, suctorial shield, and chaetotaxy and solenidiotaxy of legs. The present
study reports the historical review of the genus, completed with the information on already known species, their descrip-
tion, illustration of main characters, geographical distribution, host range, remarks on the new species, matrixes showing
comparisons, and the percentage of similarity along with a key for their identification based on hypopial characters for the
species known from Pakistan.
Keywords: new mite species, Caloglyphus, mite taxonomy, Caloglyphus agrios, Caloglyphus hadros, Acaridae, hypopus
*Author for correspondence; E-mail: [email protected]
Introduction
Mites are the widely distributed tiny arthropods, that can be
found in granaries, threshing floors, stacks of hay and straw,
dead organic matter, soil, and plant residues. Family Acaridae
contains some damage-causing mite species, which are com-
monly encountered in granaries. The mite-attacked grains lose
nutrients and the ability to germinate, due to the mite-feeding
on the germ. Crushed bodies of the storage mites cause colou-
ration in flour that reduces the product value. Under normal
conditions, these mites develop according to the following
pattern: egg, larva, nymph-I, nymph-II, and adult. Some spe-
cies may produce hypopus under unfavourable conditions.
Hypopus is a diapause form that can be carried by rodents or
insects to other storing places. This stage offers maximum
conservation attributes and provides stable taxonomic cha-
racters. Therefore, taxonomy of the stored commodity mites
is largely based on the characters of this stage.
The genus Caloglyphus has been recorded from many coun-
tries of the world and is a key pest among the mite fauna in
some of these areas. The present bibliography attempts to
compile the world literature on Caloglyphus, which is likely
to prove a useful tool to researchers involved in all aspects
of managing this serious pest. Berlese (1923) first described
the genus Caloglyphus, designating Caloglyphus berlesei
Michael, 1903, as its type species. Some species of the genus
Caloglyphus have been reported from several regions of the
world. Zakhvatkin (1941) made a comprehensive review of this
genus: described four new species, and redescribed six spe-
cies with improved descriptions. Nesbitt (1949; 1944), and
Samsinak (1966), respectively, added four and one new spe-
cies to this genus. Mahunka (1979; 1978; 1974; 1973) described
five new species. Hughes (1976) made a good addition to the
knowledge of this genus. Tseng and Hsieh (1976) redescribed
one species with improved description. Samsinak (1980) revi-
sed the tribe Caloglyphini, reestablished the genus Calo-
glyphus and described one new species. Channabasavanna
et al. (1981), Rao et al. (1982), and Ashfaq and Chaudhri (1983),
respectively, added one, one, and four new species. Samsinak
(1988) mentioned one new species to the tribe Caloglyphini.
Zou and Wang (1989), Sevastyanov and Radi (1991), Sher
et al. (1991), Klimov (1996), and Eraky (1999) added one, three,
two, one, and one new species, respectively. Klimov (2000)
analyzed acarid mites of the tribe Caloglyphini with the
narrative of a fresh species. Klimov and Oconnor (2003)
published phylogeny, historical ecology and systematics of
various mites, together with full descriptions of each taxon,
keys and natal informations. Sarwar and Ashfaq (2004)
identified and described two new species from Pakistan.
Materials and Methods
For conducting the present study, representative localities in
various ecological zones of Pakistan were surveyed to explore
new taxa and distribution range of mites of the genus
Caloglyphus. All the collected samples of different stored
commodities were brought to the laboratory to process them
further by employing Berlese’s Funnel Method (Berlese, 1923).
345
Standard and improved techniques were tested and utilized
for collection, sorting, storing, mounting and describing the
taxa. All the drawings were made on graph paper using an
ocular grid. Measurements of body length, width and other
body parts were made with the help of an ocular micrometer.
The abbreviations used in figures are: dorsocentral seta 1, 2, 3,
4 (d1, d2, d3, d4), internal vertical seta (vi), external vertical
seta (ve), internal scapular seta (sci), external scapular seta
(sce), subscapular seta (scs), internal humeral seta (hi), exter-
nal humeral seta (he), anterior lateral seta (la), posterior lateral
seta, 1, 2 (lp1, lp2), external sacral seta (sae), internal sacral
seta (sai), apodeme 1, 2, 3, 4 (ap1, ap2, ap3, ap4), metasternal
seta (mts), sternum 1, 2 (st1, st2), ventral humeral seta (hv),
genital disc 1, 2, 3 (gdi1, gdi2, gdi3), genital seta (pr), coxal
disc I, II (di1, di2), seta on femur (vF), seta on tarsus (e),
solenidion on tarsus (w1), seta on genu (ó), setae on tibia
(hT), and milimicron (mµ).
The similarity between species based the presence or abse-
nce of 25 characters quoted from the entire body was expres-
sed (Table 1). A complete comparison of the presently repor-
ted new species (including differences and similarities), with
eight other species of the genus was also furnished (Table 2).
Numerical classification was performed using resemblance
function and average linkage clustering strategy by following
the techniques devised by Sokal and Sneath (1963) and phe-
netic relationships with the species already reported in the
literature were examined to help build a background for the
genus Caloglyphus.
Results and Discussion
1. Caloglyphus agrios, new species, hypopus (Fig. 1): des-
cription
Dorsum. Body 300 mµ long, 243 mµ wide, divided into pro-
podosomal and hysterosomal shields. Propodosomal shield
with rostral projection antero-medially, 90 mµ long, 215 mµ
wide, dotted medially, broken striations antero-laterally; setae
vi, ve, sci, sce and scs each 1 pair, simple, scs broad medially,
setae respectively measuring 25 mµ, minute (very small), 6 mµ,
18 mµ and 25 mµ in length; sci-sci 36 mµ, sce-sce 85 mµ and
sci-sce 25 mµ apart; setae sci and sce forming semi-circular
line. Hysterosomal shield 233 mµ long, 243 mµ wide, smooth
medially, lateral margins of hysterosomal shield turn towards
venter, encircled by transverse, broken striations anteriorly,
longitudinal, broken striations latero-posteriorly, a row of
transverse striations latero-posteriorly. Hysterosomal shield
with 11 pairs of setae, simple, 3 pairs of visible pores. Setae d1
= d2 = d3 = d4 = 8 mµ; hi 5 mµ, he 6 mµ; la 6 mµ; lp1 = lp2 =
5 mµ; sae 25 mµ; sai 8 mµ long; d1-d1 72 mµ, d2 - d2 55 mµ,
d3 - d3 66 mµ, d4 - d4 68 mµ; d1 - d2 37 mµ, d2 - d3 68 mµ,
d3 - d4 53 mµ, and la - la 155 mµ apart. Hysterosomal shield
anterior margin overlapping propodosomal shield posterior
margin by 23 mµ with transverse, broken striations (Fig. 1a).
Venter. Gnathosoma fused pedipalpi, 2-segmented, with para-
llel lateral margins, straight at base, 32 mµ long (basal seg-
ment 21 mµ, distal segment 11 mµ), arista 1 pair, 35 mµ long,
bifurcated anteriorly, 2 pairs of small setae. Apodeme 1 (ap1)
Y-shaped, continuing with sternum 1 (st1). Sternum 1 (st1)
free, 41 mµ long; apodeme 2 (ap2) free; apodeme 3 (ap3) free;
apodeme 4 (ap4) not meeting medially, making broad roun-
ded tip. Sternum 2 (st2) double-lined, 24 mµ long, meeting
apodeme 4 (ap4) and apodeme 5 (ap5). Metasternal seta (mts)
1 pair, simple, 7 mµ long, each seta in encircled area of apodeme
4 (ap4) and apodeme 5 (ap5). Seta hv 1 pair, 10 mµ long. Coxal
fields I and II open, III and IV closed. Ventral shield separated
from genital shield. Genital shield dotted, genital slit elongated
with 2 pairs of genital suckers and a pair of genital seta (pr)
messed to genital disc (gdi3). Coxal discs (di1 and di2) present,
conoids. Genital disc (gdi3) kidney-shaped, without radial
striations. Suctorial shield 66 mµ long, 78 mµ wide, dotted,
concave antero-medially, rounded posteriorly; posteriorly
and laterally with broad sclerotized piece with bifurcated,
pointed ends; anterior suckers 1 pair, rounded with radial stria-
tions. Anal suckers 1 pair, rounded, anal suckers equal to ante-
rior suckers. One pair of lateral and 1 pair of posterior suckers,
Fig. 1a. Dorsal side of Caloglyphus agrios, hypopus, new
species.
346 M. Sarwar et al.
cooids; 1 pair of clear areas and 1 pair of dotted areas, repre-
senting the vestigial suckers towards periphery, 1 pair of suc-
kers below the suctorial shield. Suctorial shield separated
towards periphery, 1 pair of suckers below the suctorial shield.
Suctorial shield separated from posterior body end by 27 mµ,
a distance smaller than suctorial shield length (Fig. 1b).
Legs. Strong and stout, I-IV, respectively, measuring 125 mµ,
110 mµ, 93 mµ, and 93 mµ in length (trochanter base to tarsus
tip). Setae and solenidia on legs I-IV segments: coxae 0-0-0-0,
trochanters 1-1-1-0, femora 1-1-0-0, genua 3-3-0-1, tibiae 3-3-2-
2, tarsi 12-9-7-7. Tarsi I and II, 46 mµ and 36 mµ long, respec-
tively. Seta vF on femora I and II, 37 mµ and 54 mµ long,
respectively, absent on femora III and IV. Seta e on tarsi I-IV,
measuring 28 mµ, 19 mµ, 18 mµ and 18 mµ in length, respec-
tively. Seta mG on genua I and II; hT on tibiae I and II, each
lancet-like, measuring 14 mµ, 10 mµ, 28 mµ and 12 mµ in
length, respectively. Seta ó on genu I, a simple seta, on genu
II, a solenidion, 30 mµ and 17 mµ long, respectively. Tarsi I
and II, each with a solenidion, w1 26 mµ and 28 mµ long, res-
pectively. Tarsi I and II 46 mµ and 36 mµ long, respectively,
while III and IV short and stout. Dorsal seta ö on tibiae I and
II, 68 mµ and 41 mµ long, respectively. Seta ba 25 mµ long.
Tarsi I-IV provided with setae: 5 leaf-like + 1 spoon-shaped;
4 leaf-like + 1 spoon-shaped; 3 leaf-like + 1 club-shaped; 3
leaf-like + 1 club-shaped, respectively. Seta d on leg IV, tarsus
66 mµ long (Fig. 1b).
Material examined. Holotype, hypopus, type material was
collected from Jhelum, Pakistan, from groundnut (Arachis
hypogea L.) on 19.9.1994 (Sarwar) and deposited in Acarology
Research Laboratory, Department of Agricultural Entomology,
University of Agriculture, Faisalabad, Pakistan.
Comparative notes. After going through the key, and kee-
ping in view the other species described so far, this new spe-
cies was found to be closely related to C. trigonellum Sher,
Ashfaq and Parvez, however, the following characters sepa-
rated the two.
a . Hysterosomal shield dotted in C. trigonellum, but smooth
in the new species (C. agrios).
b . Gnathosoma not parallels laterally in C. trigonellum, but
parallel in the new species.
c . Sternum 1 (st1) bifid posteriorly in C. trigonellum, but
not bifid in the new species.
d . Apodemes 4 (ap4) meeting medially in C. trigonellum,
but not meeting in the new species.
e . Paragenital seta (pr) antero-medial to genital disc (gdi3)
in C. trigonellum, but messed in the new species.
This new species is also comparable with C. tshernyshevi
Zakhvatkin on the basis of the following points.
a . Basal joint of gnathosoma twice as long as wide in C.
tshernyshevi, but not so in the new species (C. agrios).
b . Propodosomal shield 4-times shorter than hysterosomal
shield in C. tshernyshevi, but 3-times shorter in the new
species.
c . Leg I tarsus with 4 leaf-like setae in C. tshernyshevi, but
with 5 leaf-like setae in the new species.
d . The coxal, genital, posterior and lateral suckers are less
fleshy in C. tshemyshevi, but not so in the new species.
e . The external seta of genu I is thin and hair-like in C. tsher-
nyshevi, but not so in the new species.
2. Caloglyphus hadros, new species, hypopus (Fig. 2): des-
cription
Dorsum. Body 283 mµ long, 218 mµ wide, divided into
propodosomal and hysterosomal shields. Propodosomal
shield with rostral projection antero-medially, 78 mµ long, 196
mµ wide, antero-lateral parts with broken striations, dotted
medially; setae vi, ve, sci, sce and scs, each 1 pair, simple,
measuring 21 mµ, 6 mµ, 10 mµ, 19 mµ and 28 mµ in length,
respectively; sci-sci 33 mµ, sce-sce 85 mµ and sci-sce 25 mµ
apart; setae sci and sce forming a semi-circular line. Hystero-
somal shield 228 mµ long, 218 mµ wide, smooth, dotted and
striated antero-laterally, a row of minute striations latero-
posteriorly. Hysterosomal shield lateral margins turn towards
the venter. Hysterosomal shield with 11 pairs of setae, simple,
3 pairs of visible pores. Setae d1 = d2 = d3 = d4 = 4 mµ; hi 7 mµ,
Fig. 1b. Ventral side of Caloglyphus agrios, hypopus, new
species.
347New Mite Species of the Genus Caloglyphus
he 6 mµ; la 5 mµ, lp1 = lp2 = 8 mµ; sae 32 mµ, sai 10 mµ long;
d1-d1 66 mµ, d2 - d2 45 mµ, d3 - d3 62 mµ, d4 - d4 58 mµ; d1 -
d2 34 mµ, d2 - d3 63 mµ, d3 - d4 55 mµ, and la - la 138 mµ
apart. Hysterosomal shield anterior margin overlapping
propodosomal shield posterior margin by 23 mµ, with trans-
verse, broken straitions and dots (Fig. 2a).
Venter. Gnathosoma fused pedipalpi, parallel lateral margins,
broad and notched posteriorly, bifurcated anteriorly, 2-seg-
mented, 29 mµ long (basal part 17 mµ, distal part 12 mµ), 1 pair
of arista, 33 mµ long, 2 paris of small setae. Apodeme 1 (ap1)
Y-shaped, continuing with sternum 1 (st1), sclerotized. Ster-
num 1 (st1) free, 43 mµ long. Apodeme 2 (ap2) free, curved at
tip. Apodeme 3 (ap3) meeting apodeme 4 (ap4). Apodemes 4
(ap4) not meeting medially. Apodeme 5 (ap5) and apodeme 4
(ap4) meeting anteriorly making broad, rounded tip, not mee-
ting with the same structure from other side. Sternum 2 (st2)
double-lined, 35 mµ long. Metasternal seta (mts) 1 pair, 6 mµ
long, each seta in encircled area of apodeme 4 (ap4) and
apodeme 5 (ap5). Seta hv 8 mµ long, 1 pair. Coxal fields I and II
open, III and IV closed; I, II and III smooth, IV dotted. Ventral
shield separated from genital shield. Genital shield, as shown
in Fig. 2b, dotted, genital slit elongated with 2 pairs of genital
suckers and 1 pair of paragenital seta (pr), messed to genital
disc (gdi3). Coxal discs di1 and di2 present; di1 and di2
conoids. Genital disc (gdi3) kidney-shaped. Suctorial shield
60 mµ long, 70 mµ wide, dotted, anterior margin concave me-
dially, rounded posteriorly, a sclerotized pointed bifurcated
piece arising from latero-posterior margin on either side. Ante-
rior suckers 1 pair, rounded; anal suckers 1 pair, rounded with-
out radial striations, 2 dots in between; anal suckers equal to
anterior suckers, 1 pair lateral and 1 pair of posterior conoids;
2 pairs of peripheral suckers; 1 pair of suckers below suctorial
shield. Suctorial shield separated from posterior end of the
body by 30 mµ, a distance smaller than suctorial shield
length (Fig. 2b).
Legs. Strong and stout, I-IV measuring 118 mµ, 105 mµ, 80 mµ
and 80 mµ in length, respectively (trochanter base to tarsus
tip). Setae and solenidia on legs I-IV segments: coxae 0-0-0-0,
trochanters 1-1-1-0, femora 1-1-1-0, genua 4-3-0-1, tibiae 3-3-2-
2, tarsi 14-10-7-8. Tarsi I and II 45 mµ and 35 mµ long, respec-
tively. Seta vF of femora I, II and III 32 mµ, 40 mµ and 25 mµ
long, respectively, absent on femur IV. Seta e on tarsi I-IV
measuring 25 mµ, 20 mµ, 15 mµ, and 15 mµ in length, respec-
tively. Seta mG on genua I and II spine-like; hT on tibiae I and
II, each lancet-like, 12 mµ, 10 mµ, 17 mµ and 14 mµ long, res-
pectively. Seta ó on genu I, a simple seta, 29 mµ long, on genu
II 14 mµ long, a solenidion. Tarsi I and II, each with a soleni-
dion w1 25 mµ and 27 mµ long, respectively. Tarsi III and IV
short and stout. Seta ö on tibiae I and II 70 mµ and 38 mµ
long, respectively. Seta ba, on tarsus, I 26 mµ long. Tarsi I-IV
provided with setae: 1 spoon-shaped + 4 leaf-like; 1 spoon-
shaped + 5 leaf-like; 1 spoon-shaped + 3 leaf-like; 1 spoon-
Fig. 2a. Dorsal side of Caloglyphus hadros, hypopus, new
species.
Fig. 2b. Ventral side of Caloglyphus hadros, hypopus, new
species.
348 M. Sarwar et al.
shaped + 4 leaf-like; 1 spoon-shaped + 5 leaf-like; 1 spoon-
shaped + 3 leaf-like; 1 spoon-shaped + 4 leaf-like, respectively.
Seta d on leg IV, tarsus 50 mµ long (Fig. 2b).
Material examined. Holotype, hypopus, type material was
collected from Mirpur, Azad Jamu and Kashmir (Pakistan), from
gram (Cicer arientinum L.) on 15.10.1994 (Sarwar) and depo-
sited in Acarology Research Laboratory, Department of Agri-
cultural Entomology, University of Agriculture, Faisalabad,
Pakistan.
Comparative notes. The following points differentiate this
new species from C. agrios, another new species described
above.
a . Gnathosoma not notched in C. agrios, but notched pos-
teriorly in this new species (C. hadros).
b . Apodeme 3 (ap3) not meeting apodeme 4 (ap4) in C.
agrios, but meeting in this new species.
c . Tarsi I-IV with 5-4-3-3 leaf-like setae, respectively, in C. agrios,
but with 4-5-3-4 leaf-like setae, respectively, in this new
species.
d . Seta vF on femur III absent in C. agrios, but present in
this new species.
In its characteristics, this new species is also nearer to C.
merisma Ashfaq and Chaudhri, but the following characters
differ in the two species.
a . Setae sci and sce of equal size in C. merisma, but not
equal in the new species (C. hadros).
b . Coxal field III open in C. merisma, but closed in the new
species.
c . Apodemes 4 (ap4) meeting medially in C. merisma, but
not meeting in the new species.
d . Genital disc (dgi3) simple in C. merisma, but kidney-shaped
in the new species.
e . Suctorial shield anal suckers not equal to anterior suckers
in C. merisma, but equal in size in the new species.
f . Seta e on tarsi III and IV lancet-like in C. merisma, but
spoon-shaped in the new species.
Key to Pakistan species of the genus Caloglyphus (Hypopodes)
1. Sternum 2 (st2) present ........................................................ 2
Sternum 2 (st2) absent ......................................................... 8
2. Apodeme 3 (ap3) meeting apodeme 4 (ap4) .................... 6
Apodeme 3 (ap3) not meeting apodeme 4 (ap4) ............. 3
3. Palposoma lateral, margins parallel .................................... 4
Palposoma lateral, margins not parallel ............................. 5
4. Sternum 1 (st1) bifid posteriorly;
paragenital seta (pr) bifid
C. multaniensis Ashfaq
and Chaudhri
Sternum 1 (st1) not bifid posteriorly;
paragenital seta (pr) not bifid
C. agrios nov. sp.
5. Setae sci and sce forming straight line;
coxal discs (di1, di2) not conoids
C. opacatus Ashfaq and
Chaudhri
Setae sci and sce not forming straight line;
coxal discs (di1, di2) conoids
C. trigonellum Sher,
Ashfaq and Parvez
6. Gnathosoma notched posteriorly....................................... 7
Gnathosoma not notched posteriorly
C. faisalabadiensis Sher,
Ashfaq and Parvez
7. Setae sci and sce of equal size;
apodemes 4 (ap4) meeting medially
C. merisma Ashfaq and
Chaudhri
Setae sci and sce not of equal size;
apodemes 4 (ap4) not meeting medially
C. hadros nov. sp.
8. Palposoma extended beyond the body;
apodemes 4 (ap4), meeting medially
C. morosus Ashfaq and
Chaudhri
Palposoma not extended beyond the body;
apodemes 4 (ap4), not meeting medially .......................... 9
9. Coxal field III open;
genital disc (dgi3) and suctorial shield
with radial striations
C. clemens Sarwar and
Ashfaq
Coxal field III closed;
genital disc; (dgi3) and suctorial shield
without radial striations
C. cingentis Sarwar and
Ashfaq
Surveys of the genus Caloglyphus undertaken previously in
Pakistan represented 8 species. Currently, two new species
have been collected and described. The new species herein
reported are ninth and tenth of this genus reported from Pakis-
tan and Azad Kashmir. A comparison of characters of the ten
species is presented in Table 1, and the matrix of the percent-
age of similarities between these species is shown in Table 2.
The phenogram (Fig. 3) identifies two subdivisions having
sister groups, showing different relationships at different
levels of linkages with one another. A brief discussion per-
taining to the 10 species reported from Pakistan follows
below.
349New Mite Species of the Genus Caloglyphus
The present expression, in the first cluster, shows the highest
shared phenetic affinity of 92% between the new species
agrios and hadros, which are linked in a pair-like format. Both
the species have been collected from adjoining sub-moun-
taineous areas of similar ecological habitats. The second pair
in this cluster depicts an affinity of 80% between the species
faisalabadiensis and trigonellum, as these two species are
common to the same habitat, having identical hosts. The affi-
nity of these species could thus be attributed to be due to the
same ecological zones that they occupy. These two pairs are
linked to each other at 76% level of the phenetic affinity. The
species multaniensis and opacatus show an affinity of 68%
and 60.5%, respectively, with the groups of agrios-hadros,
and faisalabadiensis-trigonellum species, thus completing
the cluster. Since these species are associated with diverse
ecological zones, their affinity among themselves may be due
to the sharing of common genetic characters, rather than eco-
logical relatedness. So, the characters used for the separation
of species within the genus appear to be of consistent occur-
rence.
The second cluster is a combination of four species in which
the phenetic affinity of 88% is clearly evident between the
cingentis-clemens pair, which are the dwellers of varied eco-
logical areas. To this pair, species merisma and morosus join
at 66% and 59% levels of phenetic affinities, respectively. All
the species of this second cluster, though the dwellers of
discrete localities, show affinity among themselves and their
affinity may be due to the consistent characters and instincts
which embody the genus. This infers that similarity could be
an attribute of genetics. This second cluster is bridged to the
first cluster at 59% level of shared affinity, thus forming one
of the largest phenogram in the present study from hetero-
geneous habitats.
Conclusion
The specimens accommodated in the genus Caloglyphus have
a wide range of distribution in Pakistan, as they have been
collected from discrete and diverse ecological habitats. This
indicates that the species have an ability to adapt to diverse
ecological habitats, and can thus be presumed to have a wider
100
90
80
70
60
50
40
30
20
10
0
Perc
enta
ge o
f sim
ilarity
Taxa
Fig. 3. Phenogram of species of the mite genus Caloglyphus Berlese (Acaridae).
ag
rio
s
ha
dro
s
fais
ala
ba
die
nsis
trig
on
ellu
m
mu
lta
nie
nsis
op
aca
tus
cin
ge
ntis
cle
me
ns
me
rism
a
mo
rosu
s
350 M. Sarwar et al.
Table 1. Comparison of characters among the species of the mite genus Caloglyphus Berlese (Acaridae)
Characters multani- opacatus merisma morosus faisalaba- trigonel- cingentis clemens agrios hadros
ensis diensis lum
Propodosomal setae (sci, sce) - - + - - - - - - -
of equal size
Propodosomal setae (sci, sce) - + - - - - - - - -
forming a straight line
Propodosomal setae (sci, sce) + + - - + + - - - -
posterior in position
Hysterosomal shield, dotted + - - - + - - - + +
Gnathosoma parallel, laterally - - + - - - - - - +
Gnathosoma notched, posteriorly + + - - + + - - - -
Gnathosoma distal fork, - - - + - - + + - -
separated from basal joint
Gnathosoma with 2 pairs of + + - - - + - - + -
small setae
Sternum 1 (st1) bifid, posteriorly - - + + - + - - - -
Sternum 2 (st2) absent + + + - + + + + + +
Apodeme 3 (ap3) not meeting + - + - - + + + + +
apodemes 4 (ap4)
Apodemes 4 (ap4) meeting + + - - + + - - + +
medially
Coxal field III, shut + - - - - - - + - -
Ventral shield separated from - + - + + + - - - -
genital shield
Coxal discs (di1, di2), conoids + - - - - - - - - -
Genital disc (gdi3), kidney-shaped + + - + + + + + + +
Genital disc (gdi3), with radial + - - - + + - - + +
striations all around
Paragenital seta (pr), + - + - + + + + + +
anteromedial to disc (gdi3)
Paragenital seta (pr) bifid + - - + - - - - - -
Suctorial shield rounded + + - - - - + + - -
posteriorly
Suctorial shield anal suckers equal + + + + + + + + + +
to anterior suckers
Suctorial shield with lateral and + + + + + + + - + +
posterior conoids
Seta ó on genu II, a solenidion + + + + + + + - + +
Leg I tarsus with 2 leaf-like setae + - + - + + + + + +
Leg II tarsus with 3 leaf-like - - - + - + + + + +
setae
351New Mite Species of the Genus Caloglyphus
genetic plasticity. Probably, more species of the genus
Caloglyphus have to be included from other geographical
locations of Pakistan with more characters taken to make the
picture of phenogram more meaningful. Finally, more work is
still needed before the species relationships can be consi-
dered stable, certainly with the hope that the inter-relation-
ships between different genera in particular and that between
families of Acari in general would provide a basis for future
phylogenetic work by the addition of further characters and
taxa, which would gain worldwide acceptance by both the
cladistics and classifiers.
References
Ashfaq, M., Chaudhri, W.M. 1983. Four new (hypopi) species
of the genus Caloglyphus Berlese from Pakistan (Aca-
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Berlese, A. 1923. Centuria sesta di Acari nuovi. Redia 15: 237-
262.
Channabasavanna, G.P., Krishna, N.S.R., Ranganath, H.R. 1981.
A new Caloglyphus (Astigmata: Acaridae) from poultry
litter in India with taxonomic comments on the genus. Ind.
J. Acarol. 6: 57-63.
Eraky, S.A. 1999. Five new hypopial nymphs (Acari: Acaridae
and Histiostomatidae) described from different habitats.
Folia Entomol. Hung. 60: 45-56.
Hughes, A.M. 1976. The Mites of Stored Food and Houses,
Tech. Bull. No.9, pp. 1-400, Ministry of Agriculture, Food
and Fisheries, London, UK.
Klimov, P.B. 2000. A review of acarid mites of the tribe Calo-
glyphini (Acaridae: Acariformes) with description of a
new genus and species from Siberia and Russian Far
East. Vestink Zoologii Ukraine 34: 27-35.
Klimov, P.B. 1996. A new species of acarid mite from the genus
Caloglyphus (Acari: Acaridae) from the Russian Far East.
Zool. Zhur. 75: 613-619.
Klimov, P.B., Oconnor, B.M. 2003. Phylogeny, historical eco-
logy and systematics of some mushroom associated
mites of the genus Sancassania (Acari: Acaridae) with
new generic synonymies. Invertebrate Systematics 17:
469-514.
Mahunka, S. 1979. The examination of myrmecophilous Aca-
roidea mites based on the investigations of Dr. C.W.
Retten-meyer (Acari: Acaroidea)-II. Acta Zool. Hung. 25:
311-356.
Mahunka, S. 1978. Schizoglyphidae fam. n. and new taxa of
Acaridae and Anoetidae (Acari: Acarida). Acta Zool.
Hung. 24: 107-131.
Mahunka, S. 1974. Auf insekten lebende Milben (Acari: Acarida:
Tarsonemida) aus Afrika-III. Acta Zool. Hung. 20: 137-154.
Mahunka, S. 1973. Auf insekten lebende Milben (Acari: Acarida:
Tarsonemida) aus Afrika-II. Acta Zool. Hung. 19: 289-337.
Michael, A.D. 1903. British Tyroglyphidae, vol. 2, pp. 1-183,
Royal Society, London, UK.
Nesbitt, H.H.J. 1949. Six Mexican mites of the sub-family Rhi-
zoglyphinae Acarina. Pan-Pacific Entomol. 25: 57-70.
Nesbitt, H.H.J. 1944. Three new mites of the sub-family Rhizo-
glyphinae. Canad. Entomol. 76: 21-27.
Rao, N.S.K., Ranganath, H.R., Channabasavanna, G.P., Krishna,
N.S.R., Rao, N.S.K. 1982. Caloglyphus kamatakaensis
sp. nov. (Acari: Acaridae) from India, with taxonomic com-
ments on the genus Caloglyphus. Ind. J. Acarol. 7: 37-43.
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Sarwar, M., Ashfaq, M. 2004. Two new Caloglyphus Berlese
mites (Astigmata: Acaridae) recorded in Pakistan. Pak. J.
Table 2. Matrix showing percentage of similarity among the species of the mite genus Caloglyphus Berlese (Acaride)
Species multaniensis opacatus merisma morosus faisalabadiensis trigonellum cingentis clemens agrios hadros
multaniensis xx
opacatus 60 xx
merisma 44 44 xx
morosus 32 56 56 xx
faisalabadiensis 68 72 56 52 xx
trigonellum 68 68 56 60 80 xx
cingentis 56 56 72 68 60 64 xx
clemens 52 44 60 56 48 52 88 xx
agrios 72 56 64 52 76 80 76 64 xx
hadros 64 48 72 52 76 72 76 64 92 xx
352 M. Sarwar et al.
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353New Mite Species of the Genus Caloglyphus
Introduction
Development of high yielding wheat cultivars has been one
of the major objectives of wheat breeding programmes
throughout the world. Grain yield is a complex trait, which
is the combined effect of many causal factors or plant
parameters. Knowledge of association among various traits
is important to make improvement in the overall crop yield.
Determination of correlation coefficients between various
characters helps in obtaining the best combinations of
attributes in wheat crop for the highest grain yield returns
per unit area.
Cause and effect relationship, or the path coefficient analysis,
is a handy technique which elaborates the extent of relation-
ship among different plant characters. It is an index to predict
the basis for allocation of weightage to each contributing com-
ponent in deciding upon suitable selection criteria for the
genetic improvement of complex characters, such as yield
(Rehman et al., 1998). Many workers have studied the rela-
tionship between grain yield and various yield contributing
components in wheat under different environmental condi-
tions. Hussain and Khan (1990) and Zaheer et al. (1987)
observed positive and direct effect of the number of grains
per spike and 1000-grain weight on yield.
Bahadur et al. (1993) observed that correlation coefficients
at the genotypic level were higher than phenotypic correla-
tion coefficients and observed that grain yield was highly and
positively associated with tillers per plant, the ear-length, spike-
lets per ear, grains per ear, and 1000-grain weight. Uddin et
al. (1997) evaluated genotypic and phenotypic correlation for
grain yield per plant and other quantitative characters in
21wheat varieties and found that grain yield per plant was
positively and significantly correlated with spikes per plant,
spikelets per spike, and 1000-grain weight. Nabi et al. (1998)
estimated phenotypic and genotypic correlation coefficients
among various morphological characters and reported that
grains per spike had positive and significant genetic and
phenotypic correlation with grain yield. Dhonde et al. (2000)
recorded nine yield-related components in 40 genotypes.
Higher genotypic and phenotypic correlation coefficients were
observed for productive tillers per plant, plant height, grains
per ear and grain yield per plant. Number of grains per ear and
grain weight per ear had the highest direct effect on grain yield.
Shukla et al. (2000) evaluated 25 cross-combinations of
wheat for yield related components and recorded significant
positive association of desirable traits, such as grains per
ear, tillers per plant and 1000-grain weight. The present study
was undertaken to evaluate the relationship amongst
different biometric traits and their direct and indirect effect
on grain yield in bread wheat under rainfed conditions.
Materials and Methods
Twenty-five cross-combinations comprising five parents,
namely, GPW-235, GPW-273, GPW-272, GPW-36 and GPW-
37, ten F-1s, and ten reciprocals were evaluated for establish-
Cause and Effect Relationship for Some Biometric Traits in
Bread Wheat
Nazar Muhammad Cheema*, Muhammad Ashraf Mian, Muhammad Ihsan,
Muhammad Azeem Tariq, Ghulam Rabbani and Abid Mahmood
Barani Agricultural Research Institute, P.O. Box 35, Chakwal, Pakistan
(received July 27, 2004; revised June 6, 2005; accepted August 31, 2005)
Abstract. Genotypic and phenotypic correlations and path coefficient analyses were conducted for grain yield and
some biometric traits in 25 cross-combinations of bread wheat under the rainfed conditions. Significant positive geno-
typic and phenotypic association was observed between grain yield per plant, and the yield components, such as
productive tillers per plant, spike-length, spikelets per spike, grains per spike and 1000-grain weight. The path coeffi-
cient analysis revealed that the number of grains per spike and 1000-grain weight had the maximum direct effect on
grain yield. These traits may be considered as the selection criteria for developing high yielding wheat genotypes for
rainfed areas.
Keywords: bread wheat, biometric traits, grain yield, correlation coefficient, path coefficient analysis, rainfed wheat
crop
*Author for correspondence
Pak. J. Sci. Ind. Res. 2005 48(5) 354-357
354
ing cause and effect relationship in bread wheat under
medium rainfall conditions (annual rainfall 350-500 mm). The
study was carried out at the Barani Agricultural Research
Institute, Chakwal, Pakistan, during the 2002-2003 crop sea-
son. The material was sown using randomized complete block
design with three replications. Each entry comprised a single
row of 3 m length, whereas row to row distance was kept at
30 cm and plant to plant distance was kept at 5 cm. Normal
agronomic practices were carried out during the crop season.
At maturity, ten guarded plants, excluding the border ones,
were randomly selected for recording data for the number of
productive tillers per plant, spike-length (cm) of the main tiller,
spikelets per spike, grains per spike, 1000-grain weight (g)
and grain yield per plant (g).
The data were subjected to analysis of variance following Steel
and Torrie (1980). Genotypic and phenotypic correlation
coefficients were calculated as followed by Al-Jibouri et al.
(1958). Path coefficient analysis was carried out as illustrated
by Singh and Chaudhary (1985).
Results and Discussion
Parents and their cross-combinations exhibited significant dif-
ferences (p < 0.01) for all the traits studied, i.e., productive
tillers per plant, spike-length, spikelets per spike, grains per
spike, 1000-grain weight, and grain yield per plant (Table 1).
Genotypic and phenotypic correlation coefficients (Table 2)
revealed that productive tillers per plant had significant posi-
tive genotypic correlations with spike-length, spikelets per
spike and grain yield per plant, but non-significant positive
genotypic correlations with the number of grains per spike
and 1000-grain weight. This trait showed significant positive
phenotypic correlations with grain yield per plant, but non-
significant positive phenotypic association with spike-length,
number of spikelets per spike, number of grains per spike,
and 1000-grain weight.
Spike-length showed significant positive genotypic, as well
as phenotypic correlations with spikelets per spike, number
of grains per spike, 1000-grain weight, and grain yield per
plant, whereas significant positive genotypic but non-
significant positive phenotypic association was observed
between spike-length and productive tillers per plant. The
number of spikelets per spike had significant positive geno-
typic and phenotypic correlations with spike-length, 1000-
grain weight and grain yield per plant; significant positive
genotypic but non-significant positive phenotypic correla-
tions were noted with the number of productive tillers per
plant; whereas non-significant positive genotypic as well as
phenotypic association was observed between the number
of spikelets per spike and the number of grains per spike.
The number of grains per spike showed significant positive
genotypic, as well as phenotypic correlations with spike-
length and grain yield per plant, but non-significant positive
correlations with the number of productive tillers per plant,
the number of spikelets per spike, and 1000-grain weight.
The 1000-grain weight had significant positive genotypic, as
well as phenotypic correlations with spike-length, the number
of spikelets per spike and grain yield per plant. This trait
showed non-significant positive correlations with the number
of productive tillers per plant and the number of grains per
spike. Significant positive genotypic, as well as phenotypic
association was observed between grain yield per plant and
all the traits studied, i.e., the number of productive tillers per
plant, spike-length, the number of spikelets per spike, the num-
ber of grains per spike, and 1000-grain weight.
Table 1. Mean squares for tillers per plant, spike-length, spikelets per spike, grains per spike, 1000-grain weight, and grain yield
per plant in wheat under rainfed conditions
Source of df Tillers/ Spike- Spikelet/ Grains/ 1000-Grain Grain
variation plant length spike spike weight yield/plant
Genotypes 24 1.298 ** 1.676 ** 3.650 ** 33.178 ** 16.186 ** 3.163 **
Replications 02 0.168 ns
0.161ns
0.587 ns
11.232 * 0.382 ns
0.020 ns
Error 48 0.223 0.192 0.986 3.225 1.249 0.235
Total 74
cv (%) 9.61 3.88 4.86 3.19 2.80 5.83
lsd 5 (%) 0.775 0.731 1.630 2.948 1.835 0.796
lsd 1 (%) 1.034 0.975 2.175 3.933 2.448 1.062
ns = non-significant; *, ** significant at 5% and 1% level of probability, respectively; cv = coefficient of variation; lsd = least significant
difference; df = degree of freedom
355Cause and Effect Relationship of Biometric Traits in Wheat
Table 2. Genotypic (rg) and phenotypic (r
p) correlation coefficients among tillers per plant, spike-length, spikelets per spike,
grains per spike, 1000-grain weight, and grain yield per plant in wheat under rainfed conditions
Biometric traits Tillers/ Spike-length Spikelet Grains/ 1000-Grain
plant per spike spike weight
Spike-length rg
0.505 **
rp
0.227 ns
Spikelets per rg
0.562 ** 0.802
spike rp
0.277 ns
0.504 **
Grains per rg
0.111 ns
0.447 ** 0.262 ns
spike rp
0.093 ns
0.389 ** 0.233 ns
1000-Grain rg
0.277 ns
0.353 * 0.434 ** 0.096 ns
weight rp
0.127 ns
0.296 * 0.361 * 0.109 ns
Grain yield per rg
0.438 ** 0.643 ** 0.631 ** 0.638 ** 0.517 **
plant rp
0.365 * 0.457 ** 0.348 * 0.472 ** 0.366 *
ns=non-significant; *,**=significant at 5% and 1% level of probability, respectively
Table 3. Direct (in parenthesis) and indirect effect of tillers per plant, spike-length, spikelets per spike, grains per spike, and
1000-grain weight, on grain yield per plant in wheat under rainfed conditions
Biometric Tillers/ Spike-length Spikelet Grains/ 1000-Grain Genotypictraits plant per spike spike weight correlation
coefficient
Tillers per plant (0.132) 0.007 0.157 0.057 0.085 0.438
30.14 1.60 35.84 13.01 19.41
Spike-length 0.066 (0.014) 0.224 0.231 0.108 0.643
10.26 2.18 34.84 35.92 16.80
Spikelets per spike 0.074 0.011 (0.279) 0.135 0.132 0.631
11.73 1.74 44.21 21.40 20.92
Grains per spike 0.014 0.006 0.073 (0.517) 0.028 0.638
2.19 0.94 11.44 81.03 4.40
1000-Grain weight 0.036 0.005 0.121 0.049 (0.306) 0.517
6.96 0.97 23.40 9.48 59.19
*figures in the second row of each biometric trait indicate the effect in percentage
Path coefficient analysis (Table 3) revealed that the number
of productive tillers had considerable direct effect (30.14%),
as well as indirect effect via the number of spikelets per spike
(35.84%), on grain yield. Spike-length had negligible direct
effect on grain yield, but considerable indirect effect via the
number of spikelets per spike (34.84%), and the number of
grains per spike (35.92%). The number of spikelets per spike
had substantial direct effect (44.21%) and some indirect ef-
fect through the number of grains per spike (21.40%) and
1000-grain weight (20.92%) on grain yield. The number of
grains per spike and 1000-grain weight exhibited the maxi-
mum direct effect (81.03% and 59.19%, respectively) on
grain yield, and some indirect effect via the number of spike-
lets per spike.
Significant differences were observed for different biometric
traits in the cross-combinations of wheat investigated. All the
traits studied, i.e., productive tillers per plant, spike-length,
number of spikelets per spike, number of grains per spike and
1000-grain weight showed significant positive genotypic and
phenotypic association with grain yield per plant. Similarly,
356 N. M. Cheema et al.
most of the yield components exhibited significant positive
genotypic, as well as phenotypic association amongst
themselves. Positive and significant genotypic and phenotypic
correlations amongst yield and different yield contributing
components have been observed by Dhonde et al. (2000),
Shukla et al. (2000), Nabi et al. (1998), Uddin et al. (1997),
and Bahadur et al. (1993). It was also observed that
genotypic correlation coefficients were higher in all the traits,
as compared to the phenotypic correlation coefficients.
Similar findings were reported by Bahadur et al. (1993).
Dhonde et al. (2000), Hussain and Khan (1990), and Zaheer
et al. (1987) have reported positive direct effect of number of
grains per spike and 1000-grain weight on grain yield. The
present studies revealed that the number of grains per spike
had the highest direct effect on grain yield per plant under
rainfed conditions followed by 1000-grain weight, number of
spikelets per spike and the number of productive tillers per
plant. These plant characters may be used as the selection
criteria for developing high yielding wheat varieties for the
rainfed areas.
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357Cause and Effect Relationship of Biometric Traits in Wheat
Combining Ability Analysis of Seed Cotton Yield and its Components in
Cotton (Gossypium hirsutum)
Muhammad Tehseen Azhar and Asif Ali Khan*Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad-38040, Pakistan
(received November 10, 2003; revised August 10, 2005; accepted August 13, 2005)
Pak. J. Sci. Ind. Res. 2005 48(5) 358-361
Abstract. In order to examine the genetic mechanisms controlling seed cotton yield and its components, four lines of
Gossypium hirsutum L., MNH-554, Delcerro, Coker-304 and Albacala-(71)1190 were crossed in all possible combina-
tions. Combining ability analysis of the data revealed that general combining ability effects were highly significant at p =
0.01, in respect of the number of bolls, seed cotton yield and lint percentage. The general combining ability variances were
greater than specific combining ability variances, which showed the predominance of additive gene effects. Among the four
parents, MNH-554 appeared to be the best general combiner for all the characters studied. Due to the preponderance
effects of additive genes, it seems that single plant selection in segregating generations would be effective for improving the
seed cotton yield and its various components.
Keywords: combining ability, Gossypium hirsutum, seed cotton yield, additive variance
*Author for correspondence; E-mail: [email protected]
Introduction
For the development of genetically promising plant materi-
als through hybridization, selection of the appropriate par-
ent stock is the crucial step in plant improvement strate-
gies. The selection of desirable plant characteristics in the
breeding population can be effective, if information on the
pattern of inheritance of plant yield and its components is
also available. Several biometrical techniques are available,
which can provide the working knowledge for determining
the genetic basis of variation in different plant characters.
These techniques include graphic analysis (Hayman, 1954),
North Carolina designs (Comstock and Robinson, 1952),
triple test cross technique (Kearsey and Jinks, 1968), gen-
eration mean analysis (Hayman, 1958), line x tester analysis
(Kempthorne, 1957), besides several others. All these tech-
niques describe the mode of gene action controlling the
manifestation of characters. None of these techniques, how-
ever, deal with the potential of the parents involved in a
crossing programme. The combining ability analysis
(Griffing, 1956) is a biometrical method, which identifies the
parents having the best combining ability and the effects
of genes governing the inheritance of characters. This
method was, therefore, followed in the present invisti-
gations to collect information on seed cotton yield and its
components.
In some previous genetic studies, the pattern of inheritance
of seed cotton yield and its components normally appeared
to vary, whereas both additive and non-additive gene effects
have been reported in the literature. The work of Shakeel et
al. (2001) and Khorgade et al. (2000) showed that the seed
cotton yield was normally affected by the genes having domi-
nance characteristics. On the contrary, Islam et al. (2001) and
Hassan et al. (2000) have reported the genetic mechanism
under additive gene effects. Similarly, the number of bolls,
which is an important attribute of cotton plant yield, was
reported to be controlled by non-additive gene effects
(Ahmad et al., 2000). Other studies, however, have shown it
to be affected by genes with cumulative effects (Kumareson
et al., 2000; Ajmal et al., 2000). Ginning percentage was also
observed to be under the influence of genes acting addi-
tively and non-additively (Pavasia et al., 1999). The present
study was carried out to remove this confusion by undertak-
ing investigations on these aspects on some of the genetic
materials available in the Department of Plant Breeding and
Genetics, University of Agriculture, Faisalabad, Pakistan. The
observations so made are likely to be useful for continued
genetic improvement in the Gossypium hirsutum L. lines
through the conventional breeding methods.
Materials and Methods
Study material. For the purpose of examining the genetic
basis of yield and its components in G. hirsutum, the experi-
mental materials used in the studies were developed by cross-
ing four parents, namely, MNH-554, Coker-304, Delcerro, and
Albacala-(71)1190 in 4x4 complete diallel fashion.
Experimental design and collection of data. The seeds of the
parents were sown in 30x30 cm earthen pots using the green-
house facility. At the time of flowering, the plants were
358
emasculated and pollinated. All the necessary precaution-
ary measures were taken to avoid alien pollen contamina-
tion of the genetic material at the time of emasculation and
pollination. Maximum number of flowers were pollinated to
produce sufficient quantity of F-1 seeds of each cross. The
seeds obtained from 12 F-1 crosses, and of the parents,
were planted in the field to raise the F-1 generation plants.
Each entry was sown in three replications, following the
randomized complete block design layout. To ensure uni-
form plant population, the seeds were dibbled, which were
then sown in a single row plot, having 10 plants spaced 30
cm within the row and 75 cm between the rows. The data
were taken on the middle six plants, leaving out the two
plants on either end of the row so as to avoid the border
effects. The usual standard cultural and agronomic prac-
tices and plant protection measures were adopted to obtain
healthy plants. At maturity, the number of bolls developed
on the central eight plants, from each of the set in three
replications, was counted. All of these bolls were picked to
record yield of seed cotton, and the data on boll weight and
lint percentage were recorded (Tables 1-3).
Statistical procedures. The 4x4 diallel data were subjected
to ordinary analysis of variance technique in order to de-
termine the genotypic differences. The data were further
analyzed according to Griffing (1956).
Results and Discussion
The simple analysis of variance of seed cotton yield, the
number of bolls produced per plant, and the ginning per-
centage indicated significant differences in these charac-
ters at p = 0.01 (Table 1). Further analysis of the data, fol-
lowing the combining ability technique (Griffing, 1956), re-
vealed highly significant differences for mean squares due
to the general and specific combining ability for seed cot-
ton yield and the number of bolls. For reciprocal effects,
these differences were non-significant for both the charac-
ters at p = 0.05. For lint percentage, the general combining
ability effects were significant at p = 0.01 (Table 2). The
comparison of genetic variances showed that the magni-
tude due to general combining ability was greater than that
resulting from the specific combining ability for seed cot-
ton yield, the number of bolls, and lint percentage (Table 2).
It indicated that breeding for these characters was easy, as
the inheritance of these characters was less complex (Liang
and Walter, 1968).
The four parents were compared for their general combining
ability effects (Table 3). The observations so made revealed
that MNH-554, with the highest general combining ability in-
dex, was the best general combiner for all the characters. The
Table 1. Mean squares from analysis of variance of three plant
characters in 16 families of Gossypium hirsutum L
Source of df Number Seed cotton Lint
variation of bolls yield (g) (%)
Replications 2 0.054 ns 7.595 ns 0.043 ns
Families 15 36.298** 211.739** 2.175**
Error 30 3.676 10.040 0.044
ns = non-significant; ** = differences highly significant; df = degree
of freedom
Table 2. Mean squares from the combining ability analysis of
three plant characters in 16 families of Gossypium hirsutum L
Source of df Number Seed cotton Lint
variation of bolls yield (g) (%)
General com- 3 52.876** 325.803** 3.587**
bining ability (6.220) (39.541) (0.446)
Specific com- 6 3.273* 9.982* 0.018 ns
bining ability (1.260) (4.083) (0.002)
Reciprocal 6 0.536 ns 3.568 ns 0.001ns
(-0.344) (0.111) (-0.007)
Error 30 1.225 3.347 0.015
*, ** and ns show significant, highly significant and non-significant
differences, respectively; values given in paranthesis are the vari-
ances; df = degree of freedom
crosses of variety MNH-554 with other lines expressed specific
combinations for different characteristics as: MNH-554 x Coker-
304 (2.032) and Albacala-(71)1190 x MNH-554 (0.733) for the
number of bolls per plant, MNH-554 x Delcerro (3.256) and
Albacala-(71)1190 x MNH-554 (2.770) for seed cotton yield, and
MNH-554 x Albacala-(71)1190 (0.176) for lint percentage proved
to be the best combinations. The parents having the high gen-
eral combining ability effects are likely to have the potential to
produce breeding materials for the development of some im-
proved cotton lines. The previous studies, similar to the present,
had also shown that some of the crosses which involved at
least one good general combiner, as a parent, had shown supe-
riority (Islam et al., 1998; Azhar and Rana, 1993a). It has been
also reported that parents known to be poor general combiners,
sometimes yield hybrids that rank better than the others (Azhar
and Khan, 2003; Azhar and Rana, 1993b). These types of crosses
were also noted in the present studies, for example, the crosses
Albacala-(71)1190 x Coker-304 (0.985) for the number of bolls,
and Delcerro x Coker-304 (0.575) for the seed cotton yield.
In the present genetic examination, following the approach
of genetic analysis of Griffing (1956), it was not possible to
estimate the heritability of these characters. However, the
359Combining Ability Analysis of Cotton Genotypes
Table 3. Means of the number of bolls per plant, seed cotton yield, and lint percentage in four parents, and estimates of general
combining ability (GCA), specific combining ability (SCA) and reciprocal effects
Parents/ Number of bolls Seed cotton yield (g) Lint (%)
cross-combinations
Parents mean GCA mean GCA mean GCA
values values values
MNH-554 27.467 3.719 76.730 9.458 35.420 0.887
Coker-304 19.867 - 0.627 58.667 - 1.771 33.900 0.071
Delcerro 18.400 - 2.190 52.757 - 3.995 32.370 - 0.697
Albacala-(71) 1190 20.533 0.901 54.453 - 3.691 33.090 - 0.261
cd1 (gi
- gl) 1.085 1.793 0.119
Cross-combinations mean SCA mean SCA mean SCA
values values values
MNH-554 × Coker-304 27.730 2.032 68.570 - 0.287 34.720 - 0.021
(27.950)* (- 0.110) (71.377) (- 0.403) (34.70) (0.010)
MNH-554 × Delcerro 25.000 0.521 70.490 3.256 33.920 - 0.028
(24.533) (0.233) (72.093) (- 0.802) (33.950) (- 0.015)
MNH-554 × Albacala-(71) 1190 26.400 0.133 72.900 1.790 34.600 0.176
(24.933) (0.733) (67.360) (2.770) (34.500) (0.025)
Coker-304 × Delcerro 19.330 - 0.634 57.520 0.138 33.180 0.043
(19.200) (0.065) (56.370) (0.575) (33.200) (- 0.010)
Coker-304 × Albacala-(71) 1190 22.370 0.197 57.690 0.514 33.550 - 0.008
(20.400) (0.985) (57.560) (0.065) (33.600) (- 0.025)
Delcerro × Albacala-(71) 1190 19.850 0.050 53.610 - 1.567 32.800 - 0.006
(19.500) (0.175) (53.030) (0.290) (32.820) (- 0.010)
cd1 (sij
- sik) 1.879 3.105 0.206
cd1 (rij
- rkl) 2.170 3.586 0.238
* the values given in parenthesis are the means of reciprocal crosses and their SCA effects; cd1(g
i
-gl
) = critical difference for general combining
ability; cd1 (s
ij- s
ik) = critical difference for specific combining ability; cd
1 = critical difference for reciproeal effects
preponderance effect of additive genes suggests that esti-
mates of heritability may be high (Falconer and Mackey,
1996). Thus, from the present results it seems possible that
segregating material would be of potential value, and single
plant selection may be effective in improving seed cotton
yield and its components.
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Combining Ability Analysis of Cotton Genotypes 361
Effect of Storage Fungi on the Seed Quality Parameters of DifferentMustard Varieties
Tamoor Khan*, K. Khan and Muhammad Zahoor ul Haq
Food Technology Division, PCSIR Laboratories, Mastung Road, Quetta, Pakistan
(received May 29, 2004; revised November 30, 2004; accepted January 15, 2005)
Pak. J. Sci. Ind. Res. 2005 48(5) 362-365
Abstract. Seven seed storage fungi (Aspergillus flavus, Alternaria brassicae, Helminthosporium brassicae, Penicillium sp,
Pythium sp, Rhizoctonia solani and Fusarium oxysporum) were isolated from four mustard seed varieties (B-raya, Y-raya,
B-M-1 and S-9). A. flavus was the most predominant fungus found on seeds of all the four mustard seed varieties. The
germination percentage of the fungal infected seeds of B-raya decreased significantly, followed by Y-raya, B-M-1 and S-
9 mustard seed varieties, both in the laboratory and the pot-scale studies. It was noted that weight and oil contents of seeds
of all the four varieties decreased in those that were infected by fungi during storage in comparison with the seeds that were
stored in sterilized bottles. The fungal infected seeds also had lower glucosinolate and erucic acid contents than those of
the non-infected seeds.
Keywords: storage fungi, mustard seed, seed quality parameters, glucosinolate in mustard, erucic acid in mustard, stored
seed germination
*Author for correspondence
Introduction
Mustard (Brassica juncea L) is commonly known as “sarson”
in the Indo-Pakistan sub-continent. It is an annual winter crop
and a major oil seed source in Pakistan. It is cultivated in the
temperate regions of the world, in Europe, China, India and
Pakistan (Shafi et al., 1994). In Pakistan, it is cultivated in all
the provinces while the province of Punjab is its major area of
cultivation. The total area under the cultivation of mustard in
Pakistan is 33950 hectares, averaging 859 kg/ha (PARC, 1998).
The average oil and protein content in the mustard seed is 38-
45% and 28%, respectively (Bhatti and Soomro, 1994). Mus-
tard plant, during all the stages of its growth, is attacked by a
number of diseases, such as downy mildew (Peronospora
parasitica), white rust (Albugo candida), powdery mildew
(Erysiphe cichoracearum), blight (Alternaria brassicae), wilt
(Fusarium oxysporum), stem and root rot (Sclerotinia
sclerotiorum), and seed storage rots caused by species of
Rhizoctonia, Aspergillus, Penicillium and Fusarium (Rakesh
et al., 1995). The seed mycoflora associated with abnormal
wrinkled and discoloured mustard seeds include Chaetomium
indicum, Cladosporium fulvum, Penicillium rubrum, Alter-
naria brassicae, Rhizopus arrhizus, Fusarium sp, Alternaria
brassicae (Kumar et al.,1986); Rhizoctonia brassicae,
Pyrenopeziza brassicae, Botrytis cinerea, Fusarium
oxysporum, Macrophomina phaseolina, Pythium butteri,
Urocystis brassicae (Saha and Singh, 1988); and Altenaria
curvularie, Aspergillus sp, Rhizopus sp and Mucor sp (Rani
et al., 1995). Healthy seeds play an important role in increas-
ing plant population, improving seed quality and yield of any
crop species (Hafiz, 1986).
Keeping in view the importance of mustard seed crop in the
production of oil in the country, the present studies were
carried out to evaluate the effect of storage fungi on the seed
quality parameters of different mustard varieties.
Materials and Methods
Collection of mustard seeds. Seed samples of four mustard
varieties, namely, B-M-1, S-9, brown raya (B-raya) and yellow
raya (Y-raya) were obtained from the Oil Seed Section of Agri-
cultural Research Institute, Quetta, Pakistan. 1 kg seeds of
each variety were stored for 6 months at 25 °C and 8-20%
relative humidity in open atmosphere. Furthermore, 1 kg seeds
of each variety were stored for 6 months in sterilized glass
bottles at 25 °C and 8% relative humidity.
Isolation and identification of storage fungi. 200 seeds of
each variety, stored in the open, were thoroughly washed un-
der running tap water for about 20 min. These seeds were
sterilized by rinsing in 0.01% mercuric chloride for 1-2 min and
washing twice with distilled water for about 2-3 min. 25 seeds
of each variety were placed in petri dishes containing steril-
ized potato dextrose agar medium. All plates were kept at room
temperature (25±1 °C) for 12 days in three replications. The
fungi associated with each seed were identified on the basis
of their specific mycelial and spore characteristics using the
genera identification protocol (Agrios, 1978) . The percentage
of infection in the storage fungi was determined using the
362
naria brassicae, Helminthonsporium brassicae, Penicillium
sp, Pythium sp, Rhizoctonia solani and Fusarium oxysporum.
There were similarities and disparities among the mustard
varieties with regard to the frequency of their presence in
different varieties of mustard seed (Table 1). Overall, the most
common fungus was Aspergillus flavus. The percentage of
this fungus was the highest on the seeds of three varieties
(S-9, Y-raya, B-raya), but the second highest in one variety
(B-M-1). The second most common fungal species present on
the three varieties, S-9, Y-raya and B-raya was Alternaria
brassicae. However, Helminthosporium brasicae was the
most common on the seeds of mustard variety, B-M-1, followed
by Aspergillus flavus as the second most common, and
following formula:
number of seeds infected by each fungus species% = x 100 total number of seeds incubated
Effect of storage fungi on the germination of different mus-
tard varieties
a. Laboratory-scale germination studies. Germination stud-
ies in petri plates were undertaken by taking randomly 200
seeds of each variety stored in the open, as well as those
stored in sterilized bottles. Seeds were surface sterilized with
0.01% mercuric chloride, washed twice with distilled water and
then placed in petri plates containing two layers of sterilized
moist filter papers. Observations were recorded after 12 days,
when the plumules of the germinated seeds were about 1.25
cm long. The percentage of germination was determined.
b. Pot-germination studies. Seed germination studies were
carried out in earthen pots (22 cm dia) containing sterilized soil
with 25 seeds of each variety prepared in the same manner as
was done for labortory-scale germination studies. Seeds stored
under both the storage conditions, namely, in the open and in
the sterilized bottles were used for pot germination. The pots
were irrigated whenever needed. The germination percentage
was recorded after 32 days when seedlings were about 4 cm
long.
Effect of storage fungi on the mustard seed quality
a.1000- seed weight. Three replicates of 1000 seeds of each
variety stored under the two sets of conditions were randomly
selected. Seed weights of all the replicates were recorded.
b. Determination of oil content. Oil content was determined
by taking three replicates of 50 g seeds of each variety kept
under the two storage conditions. The seeds were oven-dried
for 1 h at 130 °C, followed by grinding of the samples. 5 g of
each sample was extracted with petroleum ether in Dickey
John analyzer for oil extraction by heating for 2 h. The recov-
ered oil percentage was calculated as:
oil (%) = oil recovered x 100/5
c. Determination of erucic acid and glucosinolates. Erucic
acid and glucosinolates were determined by gas liquid chro-
matography (GLC) in accordance with Smith et al. (1993).
Data analysis. Data were analyzed for the percentage of isolted
fungi, seed germination and chemical composition by analy-
sis of variance (Steel and Torrie, 1984).
Results and Discussion
Isolation and identification of storage fungi. Seven species
of storage fungi were isolated and identified from each of the
four mustard varieties, which were Aspergillus flavus, Alter-
Table 1. Frequency of storage fungi isolated from four mus-
tard varieties stored under conditions of open atmosphere*
Mustard Storage Fungi Percentage of
variety fungi isolated isolated infection
(number) (%)
S-9 Aspergillus flavus 80 40.0
Alternaria brassicae 35 17.5
Helminthosporium brassicae 26 13.0
Penicillum sp 24 12.0
Pythium sp 4 2.0
Rhizoctonia solani 13 6.5
Fusarium oxysporum 18 9.0
B-M-1 Aspergillus flavus 60 30.0
Alternaria brassicae 32 16.0
Helminthosporium brassicae 80 40.0
Penicillum sp 9 4.5
Pythium sp 13 6.5
Rhizoctonia solani 4 2.0
Fusarium oxysporum 2 1.0
Y-raya Aspergillus flavus 87 43.5
Alternaria brassicae 79 39.5
Helminthosporium brassicae 6 3.0
Penicillum sp 14 7.0
Pythium sp 3 1.5
Rhizoctonia solani 5 2.5
Fusarium oxysporum 6 3.0
B-raya Aspergillus flavus 103 51.1
Alternaria brassicae 48 24.0
Helminthosporium brassicae 16 8.0
Penicillum sp 10 5.0
Pythium sp 4 2.0
Rhizoctonia solani 6 3.0
Fusarium oxysporum 13 6.0
*200 seeds of each variety were tested for recording fungal infection
363Effect of Storage Fungi on Mustard Quality
Table 3. Effect of storage fungi on the seed quality of four
mustard varieties in respect of weight and total oil content
when stored in open atmosphere and sterilized bottles
Variety Seed 1000-seed Oil concenration
storage weight (%)
condition (g)
S-9 sterilized bottles 3.38 a* 38.0a
open atmosphere 3.21a 34.51b
Y-raya sterilized bottles 3.38a 38.0a
open atmosphere 2.40bc 32.75c
B-raya sterilized bottles 3.38a 38.0a
open atmosphere 2.23c 30.35bc
B-M-1 sterilized bottles 3.38a 38.0a
open atmosphere 2.46b 33.40c
lsd 0.19 1.13
cv 6.46% 2.90%
*values with different alphabets in each column are significantly
different from each other; lsd: least significant difference at p = 0.05;
cv: coefficient of variance
Table 4. Effect of storage fungi on the fatty acid and
glucosinolate contents of four mustard varieties stored in the
open and in sterilized bottles
Variety Seed storage Glucosinolate Erucic acid
condition content (%) (%)
S -9 sterilized bottles 0.25a* 41.50a
open atmosphere 0.23a 38.75b
Y-raya sterilized bottles 0.25a 41.50a
open atmosphere 0.20ab 31.25d
B-raya sterilized bottles 0.25a 41.50a
open atmosphere 0.17b 29.67e
B-M-1 sterilized bottles 0.25a 41.50a
open atmosphere 0.22a 37.00c
lsd 0.03 0.72
cv (%) 11.53 2.12
*values with different alphabets in each column are significantly
different from each other; lsd: least significant difference at p = 0.05:
cv: coefficient of variance
Alternaria brassicae as the third most common. The experi-
ment thus indicated some degree of resistance in the mustard
gene pool to most of the fungus species, except Aspergillus
flavus, Alternaria brassicae and Helminthosporium brassicae.
Effect of storage fungi on the germination of four mustard
varieties. The results given in Table 2 show that the germina-
tion percentage of seeds of all the four varieties infected by
fungi during storage was significantly decreased as compared
to those stored in sterilized bottles. The overall lowest germi-
nation was noted for B-raya, which ws 15% and 16% under
laboratory conditions and pot germination, respectively. Vari-
eties S-9 and B-M-I were affectd to a lesser extent by storage
fungi, followed by Y-raya and B-raya.
The overall rate of germination percentage of seeds stored in
sterilized bottles of all the four varieties was (28-93%) as com-
pared to the seeds infected during storage (15-53%).
Effect of storage fungi on seed quality of the four mustard
varieties
a. 1000-seed weight. Seed weight was significantly reduced
in variety B-raya followed by Y-raya, B-M-1 and S-9 in the
seeds infected by fungi in comparison with the seeds stored
in sterilized bottles (Table 3).
b. Oil content. The seeds of B-raya colonized with storage
fungi had lower oil-content percentage as compared to Y-raya,
B-M-1 and S-9 mustard seed varieties (Table 3).
Table 2. Effect of storage fungi on the seed germination of
four mustard varieties in laboratory-scale and pot-scale
studies*
Variety Scale Seed storage Number Germination
condition of seeds (%)
germinated
S-9 laboratory sterilized bottles 185 92.5
open atmosphere 107 53.3
pots sterilized bottles 166 83.0
open atmosphere 119 59.5
Y-raya laboratory sterilized bottles 91 45.5
open atmosphere 43 21.5
pots sterilized bottles 92 46.0
open atmosphere 52 26.0
B-raya laboratory sterilized bottles 56 28.0
open atmosphere 30 15.0
pots sterilized bottles 73 36.5
open atmosphere 32 16.0
B-M-1 laboratory sterilized bottles 186 93.0
open atmosphere 101 50.5
pots sterilized bottles 174 87.0
open atmosphere 128 64.0
*200 seeds of each variety were studied for germination
Effect of storage fungi on fatty acid and glucosinolate
contents
a. Fatty acids. The fatty acid (erucic acid) percentage was
significantly reduced in the fungal infected seeds of all the
four mustard varieties, as compared to seeds stored under
sterile conditions (Table 4). The overall erucic acid percent-
age was the lowest in the fungal infected B-raya variety.
b. Glucosinolate. The results given in Table 4 indicate that
glucosinolate content was significantly reduced in the infected
364 T. Khan et al.
seeds of B-raya as compared to those of Y-raya, B-M-1and
S-9 fungal infected varieties.
Conclusion
The undertaken studies revealed that all the seed quality
parameters were severely affected by the seed mycoflora in all
the four mustard varieties stored in open conditions, as com-
pared with seeds stored in sterilized bottles. Fungal infection
during storage also reduced the percentage germination of
the seeds. It was further observed that poor quality seed lose
oil yield and have poor market value. It is, therefore, sug-
gested that healthy seeds should be used for sowing while all
agricultural management practices should be carried out with
the primary aim of reducing the incidence of storage fungi,
thus preventing the erosion of seed quality parameters.
References
Agrios, G.N. 1978. Plant Pathology, pp. 192-243, Academic
Press, New York, USA .
Bhatti, I.M., Soomro, A.H. 1994. Agriculture Inputs and Field
Crop Production in Sindh, pp. 1-127, Agricultural Re-
search, Hyderabad, Sindh, Pakistan.
Hafiz, A. 1986. Plant Diseases, pp. 1-148, Pakistan Agricul-
tural Research Council, Islamabad, Pakistan.
Kumar, K., Srivastava, S.S.I., Singh, J. 1986. Fungi associated
with Brassica juncea seeds, their effect and control. J.
Farm Sci. 17: 38-41.
PARC. 1998. Area Cultivation and Production of Mustard
and Rapeseed in Pakistan, Pakistan Agricultural Research
Council, Islamabad, Pakistan.
Rakesh, K., Gupta, P.P., Gupta, S.L., Kumar, R. 1995. Effect of
microflora on oil quality of seeds of crucifers. J. Food Sci.
Technol. 32: 345-348.
Rani, P., Agarwal, A. 1995. Effect of fungicides on seed myco-
flora and seed germination of mustard. Advances Plant
Sci. 8: 342-344.
Saha, L.R., Singh, H.R. 1988. Diseases of Rape Seed and Mus-
tard Seed and Their Management, pp. 74-77, Today and
Tomorrow’s Printers and Publications, New Delhi, India.
Shafi, N., Hatim, M., Qadir, G. 1994. Crop Production,
pp. 374-376, National Book Foundation, Islamabad,
Pakistan.
Smith, J.P., Knol, W., Tol, I. 1993. Glucosinolate degradation
by Aspergillus clabatus and Fusarium oxysporum in liq-
uid and solid state fermentation. Appl. Microbiol.
Biotechnol. 38: 696-701.
Steel, R.G.D., Torrie, J.H. 1984. Principals and Procedures of
Statistics, McGraw Hill, London, UK.
365Effect of Storage Fungi on Mustard Quality
Pak. J. Sci. Ind. Res. 2005 48(5) 366-367
Evaluation of Commercial and Candidate Bread Wheat Varieties for
Durable Resistance to Rusts in Pakistan
Syed Jawad Ahmad Shah*, Tila Muhammad and Farooq-e-AzamNuclear Institute for Food and Agriculture (NIFA), Tarnab, Peshawar, Pakistan
(received June 22, 2003; revised May 26, 2005; accepted July 26, 2005)
Abstract. Commercial and candidate bread wheat varieties were evaluated for durable resistance to rusts. The candi-
date varieties included genotype lines from the National Uniform Wheat Yield Trial, including Rainfed, Normal and
Short trial programmes. A total of 89 genotype entries were evaluated. The evaluation was done on the basis of well
recognized visual phenotype marker, the adult plant leaf tip necrosis. It was noted that 64% of the genotype varieties
expressed this phenotype marker.
Keywords: bread wheat, candidate wheat varieties, rust resistance, wheat rusts
*Author for correspondence; E-mail: [email protected]
Bread wheat (Triticum aestivum L.) is the staple food of the
population, and an important commodity in the economy of
Pakistan. Therefore, every Government agenda during the last
50 years has received the top-most priority for increasing wheat
production with the objective of attaining self-sufficiency in
this important food crop. Yellow rust (Yr) and leaf rust (Lr)
are the major biotic constraints, which have been the cause of
putting wheat yields at risks in Pakistan. Genetic resistance is
the most effective and environmentally sound method of con-
trolling these diseases (Hussain et al., 1997). Resistance to
rusts in improved wheat cultivars, in the past, was solely based
on major race-specific genes that often succumbed to virulent
pathogenic mutant strains within five to seven years
(CIMMYT, 1992). This was experienced in Northern parts of
Pakistan in the early 1990s when two popular wheat culti-
vars, namely, Pirsabak-85 and Pak-81, became susceptible to
Yr9 resulting in yield losses of up to 40% (Saari et al., 1995).
These losses can be minimized by carefully screening the com-
mercial and future candidate wheat genotypes for slow rust-
ing, before their release for field cultivation. The durability of
resistance can be assessed by careful observation of pheno-
typic traits, and analysis of the area under disease progress
curve (AUDPC). With this objective in view, field screening
experiments on 54 promising genotypes and 35 commercial
wheat varieties, received from the National Wheat Improve-
ment Programme, were carried out at the National Institute
for Food and Agriculture (NIFA), Peshawar, Pakistan. The
candidate wheat genotypes were taken from the National Uni-
form Wheat Yield Trial-Normal (NUWYT-N), National Uni-
form Wheat Yield Trial-Short (NUWYT-S), and National Uni-
form Wheat Yield Trial-Rainfed (NUWYT-R). The commer-
cial wheat varieties were taken from the Trap Nursery (TN).
NUWYT-N, NUWYT-S and NUWYT-R genotypes consisted
of 20, 20, and 14 entries, respectively, which were sown in
replicated trials, whereas the TN included 35 commercial
varieties which were sown next to the candidate genotype tri-
als. Every entry was planted in a single meter row, separated
by 0.3 m. Two spreader rows, with a mixture of susceptible
genotypes (Local White, Morocco and Sonora), were planted
around the trial nurseries. In addition, two rows of suscep-
tible check (Local White) were planted, 20 entries apart, in in
the TN entries.
Despite massive inoculation of the spreader rows, after sun-
set, using Yr and Lr virulent races prevailing in the North-
West Frontier Province of Pakistan, it was not possible to ob-
tain satisfactory epidemic disease levels. This may have been
due to the unfavourable dry weather that had prevailed dur-
ing the whole growing season. Under our experimental field
conditions, adult plant leaf tip necrosis (APLTN), a recog-
nized visual marker for the presence of durable rust resistance
genes (Lr34 and Yr18), was used as the evaluation tool in the
absence of disease response to the virulent rust races in the
experimental field inoculations (CIMMYT, 1992; Singh,
1992).
Out of the 89 genotypes studied, 57 showed APLTN (Table
1). The number of entries with APLTN varied between 6-
22 per experiment. All the promising entries in the
NUWYT-R genotypes showed that trait. In the other three
experiments, up to 75% of the genotypes showed this use-
ful morphological marker. The commercial varieties and
candidate wheat genotypes used in the present study were
either direct selections from CIMMYT (International Maize
and Wheat Improvement Centre, Mexico), or the CIMMYT
Short Communication
366
germplasm-derived varieties. APLTN is expressed in sev-
eral Mexican bread wheat cultivars and numerous bread-
ing lines from CIMMYT’s bread wheat improvement
programme (Singh, 1992). It was noted that 64% of the
entries harboured APLTN, which were associated with du-
rable rust resistance genes (Lr34 and Yr18) responsible for
slow rusting (Drijeondt and Pretorius, 1989). These entries
need to be evaluated again over sites and seasons under
artificial rust epidemic conditions for confirmation of
APLTN before their release for cultivation in the farms and
fields. This is necessary as Inqilab-91 is currently culti-
vated on about 80% of the wheat growing areas in Paki-
stan, thus putting wheat growing production in a vulner-
able situation and increasing the risk of a major epidemic.
Acknowledgement
The first author acknowledges the help of Dr. Etienne M.
Duveiller, Regional CIMMYT Wheat Pathologist for South
Asia based in Nepal, for his guidance and suggestions during
reviewing this paper.
References
CIMMYT. 1992. Improving the productivity of maize and
wheat in developing countries: an assessment of impact.
In: CIMMYT 1991 Annual Report, pp. 36-38, Interna-
tional Maize and Wheat Improvement Center
(CIMMYT), Mexico.
Drijeondt, S.S., Pretorius, Z.A. 1989. Greenhouse evaluation
of adult plant resistance conferred by the gene Lr34 to
leaf rust of wheat. Plant Dis. 73: 669-671.
Singh, R.P. 1992. Association between gene Lr34 for leaf rust
resistance and leaf tip necrosis in wheat. Crop Sci. 32:
874-878.
Saari, E.E., Hashmi, N.I., Kisana, N.S. 1995. Wheat rust and
Pakistan: an update (Year 95 doc.), pp. 1-3, National Ag-
ricultural Research Council (NARC), Islamabad, Paki-
stan.
Hussain, T., Bowden, R.L., Gill, B.S., Cox, T.S., Marshall,
D.S. 1997. Performance of four new leaf rust resistance
genes transferred to common wheat from Aegilops
tauschii and Triticum monococcum. Plant Dis. 81:
582-586.
Table 1. Screening of commercial and candidate bread wheat
varieties for durable rust resistance by using phenotypic marker
of adult plant leaf tip necrosis at National Institute for Food
and Agriculture (NIFA), Peshawar, Pakistan during 2000-2001
Trial Total Varieties expressing
variety genotype adult plant leaf tip
designation* entries necrosis (APLTN)
number %
Commerical 35 22 62.0
NUWYT-R 14 14 100.0
NUWYT-N 20 15 75.0
NUWYT-S 20 06 30.0
Total 89 57 64.0
*NUWYT-R = National Uniform Wheat Yield Trial-Rainfed;
NUWYT-N = National Uniform Wheat Yield Trial-Normal;
NUWYT-S= National Uniform Wheat Yidld Trial-Short
367Short Communication: Durable Wheat Resistance to Rust
Multiple Shoot Bud Formation and Plantlet Regeneration from in vitroCultured Pistacia vera Seeds
Shaista Jabeen and Nasreen Zaidi*Food and Biotechnology Research Centre, PCSIR Laboratories Complex, Lahore-54600, Pakistan
(received October 2, 2003; revised January 8, 2005; accepted August 10, 2005)
Pak. J. Sci. Ind. Res. 2005 48(5) 368-370
Abstract. Studies were carried out on the primordial initiation and development of shoot buds derived from pistachio(Pistacia vera) seedlings, cultured on MS medium with added 6-benzylaminopurin (BA). The in vitro culture of pistachioseeds in the presence of BA (1.0 - 4.0 mg/l), plus kinetin (Kin, 1.0 mg/l) and naphthalene acetic acid (NAA, 0.25-1.0mg/l) stimulated varying degree of seed germination and the number of shoots produced. When excised single shootsfrom these in vitro cultured seeds were subcultured on fresh medium containing high concentration of BA (4.0 mg/l),alongwith Kin and NAA, multiple shoot production was observed. Normal bud growth and shoot elongation were achievedby transferring cultures to the MS medium containing low concentration of the growth regulators BA (1.0 mg/l), plusNAA (0.25 mg/l) and Kin (1.0 mg/l).
Keywords: Pistacia vera, pistachio plantlet regeneration, multiple shoot formation, in vitro seed culture, primordial initiation
Pistacia vera (the pistachio nut plant) is a tree crop, whichgrows naturally in the low rainfall areas where the irrigationwater supply is also insufficient. The plant produces nuts,which are of significant economic importance having highnutritional value. Inspite of these attributes, arboriculturistsare not sufficiently attracted to pistachio orchard growing. Itis partly due to the imbalanced ratio of male and female plantsand the late expression of plant sex, at the age of 6-7 years.The male plants far outnumber the female plants. Thus, theplant nurseries raised from seeds put a serious constraint ontime and money in terms of investment on non-productivestock. In addition to this, the other serious causes that hinderthe propagation of this plant include its lack of resistance topests and some common diseases, as well as the instability ofcharacters in the seed-grown plants. A rapid multiplicationmethod, based on a disease resistant and stable character stock,is likely to help overcome these problems effectively.
The potential of tissue culture techniques for the propagationof woody plants has been extensively reported (Nowak et al.,2004; Quraishi et al., 2004; Ahmed et al., 1995; Philips, 1984;Zimmerman, 1979; Boxus, 1974; Mehra and Mehra, 1974).Although the pistachio tissue culture has also been reported,the success in plant regeneration has been only limited (Ahmedet al., 1995; Jabeen et al., 1995; Baragchi, 1986a; 1986b;Bustamante-Garcia, 1984). The present study reports the re-quirements for successful in vitro multiple shoot formationand plantlet regeneration from excised single juvenile shootsof pistachio raised from in vitro cultured seeds.
Seeds of Pistacia vera were procured from the Department ofAgriculture, Quetta, Pakistan. After removing the testa, thepistachio seeds were submerged in 95% ethanol for a fewseconds and distinfected by immersion in 0.1% HgCl2 solu-tion containing 2-3 drops of Tween-20 per 100 ml of the dis-infectant. After 20 min, the seeds were rinsed three times withsterile distilled water and placed directly on the MS medium(Murashige and Skoog, 1962). Growth regulators, such as6-benzylaminopurin (BA), kinetin (Kin) and naphthalene ace-tic acid (NAA), were added to the defined MS medium indi-vidually (BA), or in combination (BA+NAA, NAA+ Kin,BA+NAA+Kin). To obtain the growth of regulator-treatedseedlings, pistachio seeds were inoculated on the defined MSmedium, supplemented with various concentrations and com-binations of growth regulators. The growth medium pH wasadjusted to 5.7. To the liquid MS medium were added 0.8%agar and 3% sucrose, which was then autoclaved for 20 minat 121 °C at 15 lb psi pressure. The pistachio seeds were asep-tically placed on the solidified culture medium and incubatedin a growth chamber at 20±2 °C with 16 h photoperiod. Coolwhite fluorescent light of 3000 Lux intensity was provided inthe growth chamber.
The effect of addition of BA, alone, or of BA and Kin incombination with NAA on the pistachio seed germination wasexamined. The results showed that the percentage of seedgermination was high (42-47%), whether BA was used alone,or when BA or Kin was used in combination with NAA (Table1). The seed germination, in comparison, was below 30% whenseeds were in vitro cultured on the MS basal medium with noadded growth regulators. The rate of seed germination was*Author for correspondence; Email: [email protected]
Short Communication
368
around 42%, when these were grown on the medium suppliedwith BA (1.0 mg/l) alone, or in combination with the auxinNAA (0.25 mg/l). Average dia of the stem shoots was 1.0 and1.5 mm, and the leaf size (length x width) was 10x7 mm and15x5 mm, respectively. However, no shoot proliferation wasobserved. The addition of Kin (1.0 mg/l), instead of BA incombination with NAA (0.25 mg/l), to the basal MS medium,slightly improved the rate of germination to 47%; furthermore,the average dia of the stem was also noted to increase to 2.0mm and the leaf size to 25x15 mm. However, as with BA andBA+NAA, no shoot proliferation was noted. Seeds culturedon the medium containing higher concentration of BA (4.0mg/l), in combination with NAA (1.00 mg/l) and Kin (1 mg/l), showed an increase in the rate of seed germination to 56%(Table 1). The seedlings were additionally observed to pro-duce multiple shoots with the stem shoot dia 3.0 mm and leafsize 40x30 mm. These observations indicate that the growthregulator combination of BA+NAA+Kin resulted in strongerand multiple shoots in the in vitro cultured pistachioseedlings.
The in vitro cultured seedling gave rise to one main single shoot(Fig. 1), when the seeds were cultured on the MS medium con-taining low concentration of BA (1.0 mg/l) whether alone, orwhen used in combination with NAA (0.25 mg/l) or NAA incombination with Kin (0.25 mg/l + 1.0 mg/l, respectively). Theroot system in these seedlings usually consisted of a long root,which produced extensive lateral roots. In contrast to these ob-servations, at the higher concentration of BA (4.0 mg/l), in thepresence of Kin and NAA (1.0 mg/l and 1.0 mg/l, respectively),the dormant axillary buds present in the axil of cotyledonarystalks became active and produced multiple shoots at the baseof the cotyledonary stalk attachment (Fig 2). The morphologi-cal development of shoot and root apices was also altered andmultiple shoot formation was achieved (Table 1). The observedresponse of pistachio seeds cultured in vitro was similar to thatdescribed for both in vitro and in vivo systems reported forvarious other plants (Purohit et al., 2002; Polisetty et al., 1997;Bhatia et al., 1985). Hence, the developmental behaviour ofseedlings cultured in vitro in the presence of high concentra-tion of BA indicated a definite advantage for the use of excisedshoot segments for the in vitro regenerative expression of mul-tiple shoot bud formation. Conversely, the low concentrationof BA was not effective in the stimulation of shoot bud prolif-eration, though the growth of the primary bud produced normalshoot as also reported by Nadgauda et al. (1978). It was furtherobserved that the excised shoot segments, when subcultured,regenerated multiple shoots in the presence of high concentra-tion of BA (4.0 mg/l), which continued to produce shoot budswithout elongation.
Fig. 1. Pistacia vera seed cultured in vitro on growth me-dium supplementad with 6-benzylaminopurin (1.0 mg/l),and kinetin and naphthalene acetic acid (1.0 mg/l + 0.25mg/l, respectively) producing single main shoot.
Fig. 2. Pistacia vera seed cultured in vitro on growth mediumsupplementad with 6-benzylaminopurin (4.0 mg/l), andnaphthalene acetic acid and kinetin (1.0 mg/l + 1.0 mg/l,respectively), producing multiple shoots.
369Short Communication: Pistachio Plantlet Regeneration in vitro
The pistachio shoot segments obtained from the seedlingscultured in vitro in the presence of various growth regulators,whether singly, or in combination at different concentrations,were subscultured for regeneration studies to optimise theconditions. The optimum conditions for obtaining multipleshoot formation in pistachio seedlings were determined as:(a) in vitro initiation from seeds inoculated on MS mediumsupplemented with growth regulators and auxin (BA, 4.0 mg/l+ Kin, 1.0 mg/l + NAA, 1.0 mg/l) incubated for 4-6 weeks;(b) further stimulation of shoot buds for 4 weeks in fresh me-dium of the same composition as for (a); and (c) stimulationof rhizogenesis of shoots by the transfer of the cultures tofresh MS medium containg an effective auxin. Under theseoptimized conditions, more than 50 shoots per seed were gen-erated within 9-11 weeks.
ReferencesAhmed, Z., Hussain, A., Zaidi, N., Iqbal, M.Z., Shah, F.H.
1995. A study of relationship between growth regulatorsand browning in Pistacia vera calli. Plant Tissue Culture5: 125-129.
Baraghchi, M. 1986a. In vitro culture of mature commercialvarieties of Pistacia vera L. Proc. Inter. Plant Prop. Soc.35: 331-333.
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Bhatia, C.R., Murthy, G. S.S., Mathews, V.H. 1985. Regen-eration of plants from “deembryonated” peanut cotyle-
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Boxus, P.C. 1974. The production of strawberry plants by invitro micropropagation. J. Hortic. Sci. 49: 209-210.
Bustamante-Garcia, M.A. 1984. Micropropagation and Re-juvenation of Pistacia Species, and the Mechanism bywhich Light Influences Root Initiation. Ph. D. Disserta-tion, University of California, Davis, USA.
Jabeen, S., Zaidi, N., Zafar, F., Iqbal, M.Z., Shah, F.H. 1995.Induced histological changes in Pistacia vera L cotyle-donary tissue. Pak. J. Sci. Ind. Res. 38: 264-266.
Mehra, A., Mehra, P.N. 1974. Organogenesis and plantlet for-mation in vitro in almond. Bot. Gaz. 13: 61-73.
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Purohit, V.K., Tamta, S., Chandra, S., Vyas, P., Palni, L.M.S.,Nandi, S.K. 2002. In vitro multiplication of Quercusleucotrichophora and Q. glauca: important Himalayanoaks. Plant Cell, Tissue and Organ Culture 69: 121-133.
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Table 1. The effect of growth regulators on the germinationrate and seedling vigourCombination Germination Shoot growth Leaf sizeof growth (%)* thickness number (mm)**regulators (dia, mm)*(mg/l)
BA NAA Kin
1.00 - - 42.5±1.51 1.0±0.55 single 10x71.00 0.25 - 42.7± 1.92 1.5±0.72 single 15x5- 0.25 1.00 47.5±0.99 2.0±0.68 single 25x154.00 1.00 1.00 55.9±1.95 3.0±0.36 multiple 40x30
*± standard deviation;**length x width; BA= 6-benzy-laminopurin;NAA= naphthalene acetic acid; Kin = kinetin
S. Jabeen and N. Zaidi370
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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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