5.1 a sensitive derivative spectrophotometric method for the determination of zineb...
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5.1 A sensitive Derivative Spectrophotometric method for the
Determination of Zineb (Zinc ethylenebisdithiocarbamate)
fungicide using Sodium Molybdate
5.1.1 Introduction
Zineb is a light colored ethylenebisdithiocarbamate (EBDC) fungicide used to prevent
crop damage in the field and to protect the harvested crops from deterioration during
storage or transport. It is a non-systemic (surface acting) fungicide, commercially
available as wettable powder, dust formulations and also in mixture with other fungicides
under the trade names like Dithane Z-78, Amitan, etc, Like other EBDCs, it also shows
strong metal binding properties [1, 2] and hence is capable of blocking enzymes and
affecting the biological systems. Even the breakdown products of zineb viz.
ehtylenethiourea (ETU) and ethylene thiuram monosulfide are quite harmful. The US
EPA has classified ETU as a probable human carcinogen. Hence, the residues of Zineb in
food and fodder are harmful and these can also pass through soil and leach into the
ground water.
Figure 5.1.1: Structure of Zineb
Many methods like spectrophotometric [1,3-8], flame absorption spectrometry [9,10],
FTIR [11], gas chromatography [12,13], HPLC [14-16], polarographic [17,18], biosensor
[19] and enzyme linked immuno sorbent assay [20] are available for the determination of
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Zineb and other dithiocarbamates.
Most of the applied methods are using different techniques like spectrophotometry and
gas chromatography [3] based on analysis of CS2, H2S or amines which are evolved by
the decomposition of EBDCs. Dithiocarbamates have been determined in vegetables and
food stuffs using HPLC
[21], extraction voltammetry [22] and titrimetry [23].
Dithiocarbamates (DTCs) can also be determined by methods, like iodometry [24, 25]
and indirect complexometry [26], etc. But the methods discussed above have various
disadvantages like:
1. Methods other then gas chromatography are indirect, time consuming and less
sensitive.
2. Gas chromatographic methods are sensitive but suffer from lack of the selectivity
since all dithiocarbamate pesticides evolve carbon disulphide on acid hydrolysis.
Moreover, the dithiocarbamate decompositions are strongly dependent on
temperature and on individual dithiocarbamates. The evolution of CS2 requires
more than two hours for the decomposition of dithiocarbamate
3. HPLC methods are expensive as these involve the use of the toxic and expensive
solvents.
DTCs are also determined using FTIR spectrometry but the method is less sensitive and
time consuming [27]. Here, a relatively fast, simple, sensitive and selective derivative
spectrophotometric method is presented for the analysis of Zineb by converting it into
molybdenum complex. Zineb reacts with sodium molybdate on heating to form a blue
colored complex. Zineb and sodium molybdate combine in the ratio 1:2 to form complex.
Zineb releases Zn2+
and dithiocarbamate unit, the latter forms complex with sodium
molybdate which is then extracted into methyl isobutyl ketone (MIBK) and determined
144
by derivative spectrophotometry. The significant advantage of this method over the gas
chromatographic methods is that it can be applied for the direct determination of Zineb in
the presence of other dithiocarbamates like Ziram, Thiram and Ferbam.
5.1.2 Experimental
5.1.2.1 Equipment and Reagents
Elico SL-164, Double Beam UV-Vis spectrophotometer was used. Zineb standard was
obtained from Riedel de Haën, Germany. A stock solution was prepared by dissolving
100 mg of Zineb in 100 mL of 0.1M NaOH. Further dilutions were carried out with 0.1M
NaOH as required. A 2% (W/V) solution of sodium molybdate was prepared in doubly
distilled water.
5.1.3 Procedure
5.1.3.1 Absorption spectra
To an aliquot containing 100 g of Zineb were added 2 mL of 2% sodium molybdate
solution and 1 mL of 2 M H2SO4. The mixture was boiled for 5 minutes, cooled and
water was added to make the volume 5 mL. The blue complex formed was extracted into
5 mL of MIBK (methyl isobutyl ketone) with shaking. The organic layer was collected
into a test tube containing fused CaCl2 to remove any moisture. The solution was then
decanted into a 1 cm cell and the spectra were taken against a reagent blank. The
molybdenum complex shows peaks at 670 nm and 956 nm (Figure 5.1.2), but the peak at
956 nm has much higher absorbance; hence all the measurements were made at this
wavelength. The first derivative, second derivative, third derivative and fourth derivative
curves were given in figures 5.1.3-5.1.6 respectively.
145
In DS (Derivative Spectrophotometry), the λ reproducibility and S to N ratio are quite
important. The features like peak height and noise level depend on parameters chosen
like order of derivative; scan speed and integration time during recording of spectra. The
use of optimum parameters will give better resolution and more sensitivity. For the 4th
derivative spectra, Δλ = 9 nm was found to be ideal.
5.1.3.2 Preparation of calibration curve
The known volumes of sample solutions having 10-250 µg of Zineb were analyzed by
general procedure and the derivative spectra were obtained against a reagent blank
prepared under the similar conditions. The characteristics of zero order and derivative
spectra are summarized in Tables 5.1.1 and 5.1.2, respectively.
5.1.4 Results and Discussion
5.1.4.1 Beer’s law and Sensitivity
The optimum wavelength interval was found to be 9 nm for high resolution and
sensitivity. The wavelength range to obtain spectra was selected from 600 nm to 1100
nm. The calibration curve was obtained by measuring the peak height between
wavelength of 850 and 938 nm. Absorbance of sodium molybdate complex with Zineb
recorded against a reagent blank was linear over the concentration range from 2 µg to 40
µg per 5mL of the final solution. The detection limit is 0.006 µg mL-1
for Zineb when
S/N ratio was 3.
5.1.4.2 Effect of heating time
It was observed that the absorbance of the complex increased up to a certain extent on
146
increasing heating time. Therefore, the reaction mixture was heated for different intervals
of time. It was observed that an optimum heating time of 5 minutes was sufficient to
obtain maximum absorbance. Increasing the heating time beyond this did not increase the
absorbance as the complete complexation was achieved by heating for 5 minutes.
5.1.4.3 Effect of acid concentration
Maximum absorbance was observed when volume of acid added was between 1 mL to
1.5 mL. A decrease in absorbance was observed on further increasing the amount of acid
added as higher acid concentration is not conducive for complex formation.
5.1.4.4 Effect of other ions
Sample solutions containing 5 µg mL-1
of Zineb and various amounts of different alkali
metal salts or metal ions were prepared and the general procedure was applied. It was
observed that 20 mg of following foreign anions didn't interfere in the determination of
Zineb: bromide (11 mg), acetate (15 mg), chloride (3.5 mg), fluoride (2 mg), citrate (20
mg) and EDTA (0.06 mg). Of the metal ions examined; Zn(II)(0.066 mg), Mo(IV) (0.14
mg), Ni(II) (0.30 mg), Co(II) (0.009 mg) did not interfere. A 1 mL of 5% NaF solution
was used to mask Fe (II) and Fe (III) ions whereas 2 mL of 5% sodium citrate was used
to mask Hg (II) and Cu (II). Mn (II) was masked using 1.5 mL of 1% potassium bromide
solution.
5.1.4.5 Effect of standing time
It was observed that the absorbance of the solution became constant after 2-3 min so after
extracting into MIBK, a 5 min. standing time was selected. The absorbance of the
complex remained practically constant for about 30 minutes with the R.S.D. values of the
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absorbance varying between 0.69 to 1.6 % for different concentrations.
5.1.5 Applications
5.1.5.1 Determination of Zineb from fortified samples of wheat grains, cabbage and
rice
The method was applied to the determination of Zineb from fortified samples of wheat
grains, cabbage and rice. 20 grams of the foodstuff was finely crushed and solution
containing a known amount of zineb was added, it was mechanically shaken with 100 mL
of Acetonitrile (ACN) for 1 hour. This mixture was filtered and the residue in funnel was
washed with 3 x 10 mL portions of ACN. The extracts were evaporated on a water bath
(70-90oC). The last traces of solvent were removed using a current of dry air. The Zineb
content in the residue was determined by the general procedure and the results indicated
good recoveries in all cases. The results of the determinations were given in Table 5.1.3.
5.1.5.2 Determination of Zineb in commercial samples
The method was applied for determination of Zineb in a commercial sample Dithane Z 78
and Hexathane 75% W.P. The formulated product sample solutions were prepared as
discussed earlier and determined by the general procedure. The results of the
determinations were given in Table 5.1.4.
5.1.5.3 Simultaneous determination of Zineb in presence of Ziram in synthetic
mixtures
The method was applied for the simultaneous determination of Ziram and Zineb in
synthetic mixtures. Ziram forms a yellow complex with sodium molybdate in cold, which
absorbs at 420 nm [28] whereas Zineb forms complex on heating, all other conditions
148
remaining the same. Synthetic mixtures of zineb and ziram were made in different
proportions. To the binary mixture, 0.15 mL of 2 M H2SO4 and 2 mL of 2% sodium
molybdate were added. Molybdenum-ziram complex was extracted into 5 mL MIBK and
zineb remained in aqueous phase. The absorbance of ziram-molybdenum complex was
measured at 420 nm. To the aqueous phase containing zineb was added 1 mL of 4M
H2SO4 and 2 mL of 2% Na2MoO4 solution. The solution was boiled for 5 minutes, cooled
and extracted into 5 mL MIBK, the spectra of blue complex of zineb was taken between
600 nm and 1100nm. Thus, this method was applied for the simultaneous determination
of ziram and zineb. Ziram was determined from the standard calibration curve [28]. The
results of the determinations are given in Table 5.1.5.
5.1.6 Conclusions
The present method is more sensitive than the carbon disulphide evolution methods. By
this method 0.006 µg mL-1
of Zineb can be determined which is equivalent to 0.0033
µgmL-1
of evolved carbon disulphide. Thus, it is superior to the reported methods and the
direct simultaneous determination of ziram and zineb is possible. The sensitivity of the
present method is comparable to other spectrophotometric methods [Table 5.1.6]. The
selectivity of the present method is superior to other methods. The wide applicability of
this method makes it suitable for dithiocarbamate analysis in foodstuffs and in
commercial samples.
149
Figure 5.1.2: Absorption spectra of molybdenum ehtylenebisdithiocarbamate
complex: Zineb (2 mL) with sodium molybdate (2 %) in presence of H2SO4 (2 M),
extracted into MIBK against a reagent blank.
0.2
0.3
0.4
0.5
0.6
0.7
600 700 800 900 1000 1100
Ab
sorb
an
ce
Wavelength, nm
150
Figure 5.1.3: 1
st derivative curve of molybdenum-
ethylenebisdithiocarbamate complex a, b, c, d representing
the amount of Zineb in final solutions (a: 10 µg mL-1,
b:
100 µg mL-1
, c: 200 µg mL-1
, d: 250 µg mL-1
)
Figure 5.1.4: 2nd
derivative curve of molybdenum-
ethylenebisdithiocarbamate complex a, b, c, d
representing the amount of Zineb in final solutions (a:
50 µg mL-1
, b: 100 µg mL-1
, c: 150 µg mL-1
, d: 200 µg
mL-1
)
-0.015
-0.01
-0.005
4E-17
0.005
0.01
0.015
0.02
600 650 700 750 800 850 900 950 1000
dA
/dλ
Wavelength, nm
d
c
b
a
-0.0021
-0.0016
-0.0011
-0.0006
-0.0001
0.0004
0.0009
625 675 725 775 825 875 925 975
d2A
/dλ
2
Wavelength, nm
a
b
c
d
151
Figure 5.1.5 3rd derivative curve molybdenum-
ethylenebisdithiocarbamate complex a, b, c, d
representing the amount of Zineb in final solutions. (a:
10 µg mL-1
, b: 50 µg mL-1
, c: 100 µg mL-1
, d: 200 µg
mL-1
)
Figure 5.1.6 4th derivative curve molybdenum-
ethylenebisdithiocarbamate complex a, b, c, d
representing the amount of Zineb in final solutions.
(a: 10 µg mL-1
, b: 50 µg mL-1
, c: 100 µg mL-1
, d: 200
µg mL-1
)
-0.0002
-0.0001
-1E-18
1E-04
0.0002
0.0003
600 650 700 750 800 850 900 950
d3A
/dλ
3
wavelength(nm)
d c b
a
-5E-05
-3E-05
-1E-05
1E-05
3E-05
5E-05
7E-05
600 650 700 750 800 850 900 950
d4A
/dλ4
wavelength, nm
d
c
b
a
152
Table 5.1.1: Different parameters of zero order spectra
S.No. Parameter
Zero derivative spectra of Zineb-
Sodium molybdate complex
1. Molar Absorptivity (L mol-1
cm-1
) 1.4 x 104
2. Specific Absorptivity (mL g-1
cm-1
) 35.2
3. Sandell’s Sensitivity (μg mL-1
) 2.84x10-2
4. Analytical Sensitivity (μg mL-1
) 2.80x10-2
5. Linear Range 2 μg mL-1
to 40 µg mL-1
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Table 5.1.2: Comparison of calibration curves of Zineb using different derivative spectra
Zineb
complex
Order of
derivative
λ
nm
Regression
Equation R
2
R.S.D.
(%)
S.D. of
slope S.D. of intercept
Analytical
Sensitivity (µg mL-1
)
1. 1st 852 y = 6E-05x+0.007 0.9997 +1.9 1.01 x10
-6 1.4 x 10
-4 1.20
2. 2nd
907 y = 6E-06x +0.009 0.9937 +1.5 8.16 x 10-8
1.6 x 10-4
0.16
3. 3rd
937 y = 8E-07x+9E-05 0.9992 +1.9 8.16 x10-9
8.16 x 10-7
0.20
4 4th
902 y = 3E-07x-9E-06 0.9994 +1.3 5.03 x 10-9
8.16 x 10-8
0.006
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Table 5.1.3: Analysis of Zineb from fortified samples
Sample Crop
Zineb addeda
(μg)
Zineb
found b(μg)
Recovery
%age and
RSD%
Rangaswamy et al30
method
Zineb found Recovery
%age
Wheat 10
9.7
97.0±2.4
9.6
96.0
15
14.8
98.6±2.3
14.7
98.0
Zineb 20
19.8
99.0±1.9
19.8
99.0
Rice 8
7.8
97.5±2.5
7.6
95.0
10
9.9
99.0±2.3
9.6
96.0
12
11.8
98.3±2.2
11.6
96.6
12
11.8
98.3±2.3
11.7
97.5
14
13.8
98.5±2.4
13.7
97.8
Cabbage
18
18.1
100.5±2.2
17.6 97.8
aAmount of crop: 10 g,
bEach result is mean of five experiments.
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Table 5.1.4: Determination of Zineb in commercial samples
Commercial
Samples
Zineb taken
(μg)
Zineb founda
(μg)
RSD
(%)
Rangaswamy et al30
method
Zineb found Recovery (%)
Dithane Z-78 8
20
7.7
19.9
2.1
2.3
7.6
19.8
95.0
99.0
Hexathane
75% W.P
5
15
4.9
15.1
1.9
2.2
4.7
14.6
94.0
97.3
a Each result is mean of five experiments
Table 5.1.5: Determination of Zineb and Ziram in synthetic mixtures
a Each result is mean of five experiments
Zineb
added
( μg)
Ziram
added
( μg)
Zineb found
(μg)
Ziram found
(μg)
Recovery (%) a
and
RSD (%)
Zineb Ziram
60 40 59.0 39.5 98.3±2.3 98.7±2.7
50 50 49.7 49.0 99.4±2.8 98.0±2.5
30 70 29.5 69.5 98.3±3.1 99.3±2.4
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Table 5.1.6: Comparison of Molar Absorptivity of Zineb determination with earlier
methods
Method Molar absorptivity
(L mol-1
cm-1
)
Remarks Reference
Cupric acetate
N.R.
Low sensitivity, long
tedious procedure
[30]
Molybdenum 0.62x104 Less sensitive [28]
Phenylfluorone + CPBa 6.9x10
4
Selective & sensitive [31]
1-(2-pyridylazo)-2-naphthol 5.06x104 Column pre-
concentration factor is
required
[32]
Molybdenum (at 956 nm) and
derivative spectrophotometry
1.4x104 Improved molar
absorptivity
Present Work
a: Cetylpyridinium bromide
N.R. Not reported
157
5.2 A sensitive Derivative Spectrophotometric method for the
Determination of Maneb (Manganese ethylenebis
dithiocarbamate) fungicide using Sodium Molybdate
5.2.1. Introduction
Maneb (manganese ethylenebisdithiocarbamate is a common dithiocarbamate (DTC)
fungicide available as dust and dispersible powder [33]. Maneb is active against a wide
range of bacterial and fungal species, so, it is widely used on vegetable crops, in fields,
orchards and vines, greenhouses, forestry and also for seed treatment [34]. Maneb is not
systemic but rather a protective fungicide. Hence, its residues can be found on the fodder,
fruit and other crops where it is sprayed. Ethylenebisdithiocarbamate (EBDC) fungicides
like maneb have low human toxicity but their degradation to Ethylene thiourea (ETU) is
of great toxicological concern [35, 36, 37] as it is associated with carcinogenic and
teratogenic properties [38]. DTC complexes are toxic and have mutational effect [39].
Rathore et al [40] have studied the mobility of DTC pesticides through soil and this
aspect of their behavior presents severe hazards for the mankind as they can be washed in
to drinking water sources thus entering our food chain. In the wake of above knowledge,
it is imperative that highly sensitive and efficient methods be developed for the residue
analysis of dithiocarbamate pesticides like maneb in various food and environmental
samples. Most of the developed methods like spectrophotometry and gas chromatography
[3] are based on the analysis of CS2, H2S or amines, which are evolved during
decomposition of EBDCs.One of the earliest colorimetric methods for Maneb
determination was developed by Hylin et al [41]. Türker et al devised a flame atomic
absorption spectrometry method for indirect determination of dithiocarbamate fungicides
158
[42]. Crnogorac and Schwack have provided insight into the various methods for the
residue analysis of dithiocarbamate fungicides [43].
Figure 5.2.1 Structure of Maneb
Waseem et al have used a flow-injection method for the analysis of DTC fungicides with
chemiluminescence detection [44]. A microwave assisted extraction method involving
hydrolysis of dithiocarbamates and their analysis in tobacco leaves was developed by
Vryzas et al [45]. A Differential pulse polarographic (DPP) technique has been employed
for the determination of DTC residues by Schwack et al [46]. Lo et al have determined
propineb, zineb, maneb and mancozeb by an HPLC method [47]. Česnik et al have
developed validation for a GC-MS method for the determination of DTCs in foodstuffs
[48]. Cronogorac et al have presented a method using hydrophilic interaction- LC-
Tandem Mass Spectrometry for the determination for DTC fungicide residues in fruit and
vegetables [49]. In the present work, a relatively fast, simple, sensitive and selective
derivative spectrophotometric method is presented for the analysis of Maneb by
converting it into molybdenum complex. Maneb reacts with sodium molybdate on
heating to form a blue coloured complex. Maneb and sodium molybdate combine in the
ratio 1:2 to form complex. Maneb releases Mn2+
and dithiocarbamate unit, the latter
forms complex with sodium molybdate, which is then extracted into methyl isobutyl
159
ketone (MIBK) and determined by derivative spectrophotometry. The significant
advantage of this method over the gas chromatographic methods is that it can be applied
for the direct determination of Maneb in the presence of other dithiocarbamates like
Ziram, Nabam and Zineb.
5.2.2 Experimental
5.2.2.1 Equipment and Reagents
Elico SL-164, Double Beam UV-Vis spectrophotometer was used. Maneb standard was
obtained from Riedel de Haën, Germany. A stock solution was prepared by heating100
mg of Maneb in 100 mL of 0.1 mol L-1
of NaOH. Further dilutions were carried out with
0.1 mol L-1
of NaOH as required. A 2% (m/v) solution of sodium molybdate was
prepared in doubly distilled water.
5.2.3 Procedures
5.2.3.1 Absorption spectra
To an aliquot containing 100 g of Maneb were added 2 mL of 2% sodium molybdate
solution and 1 mL of 2 mol L-1
of H2SO4. The mixture was boiled for 5 minutes, cooled
and water was added to make the volume 5 mL. The blue complex formed was extracted
into 5 mL of MIBK (methyl isobutyl ketone) with shaking. The organic layer was
collected into a test tube containing fused CaCl2 to remove any moisture. The solution
was then decanted into a 1 cm cell and the spectra were taken against a reagent blank.
The molybdenum complex shows peaks at 670 nm and 956 nm [Figure 5.2.2], but the
peak at 956 nm has much higher absorbance; hence all the measurements were made at
this wavelength. The first derivative, second derivative, third derivative and fourth
160
derivative curves were given in figures 5.2.3-5.2.6 respectively. In DS (Derivative
Spectrophotometry), the λ reproducibility and S to N ratio are quite important. The
features like peak height and noise level depend on parameters chosen like order of
derivative; scan speed and integration time during recording of spectra. The use of
optimum parameters will give better resolution and more sensitivity. For the 4th
derivative
spectra, Δλ = 9 nm was found to be ideal.
5.2.3.2 Preparation of calibration curve
The known volumes of sample solutions having 10-250 µg of Maneb were analyzed by
general procedure and the derivative spectra were obtained against a reagent blank
prepared under the similar conditions. The characteristics of zero order spectrum are
summarized in Table 5.2.1. The figures 5.2.3-5.2.6 show the derivative spectra of
different orders. The comparison of calibration curves of different derivative spectra are
provided in the Table 5.2.2.
5.2.4 Results and Discussion
5.2.4.1 Beer’s law and Sensitivity
The optimum wavelength interval was found to be 9 nm for high resolution and
sensitivity. The wavelength range to obtain spectra was selected from 600 nm to 1100
nm. The calibration curve was obtained by measuring the peak height between
wavelength of 850 and 938 nm. Absorbance of sodium molybdate complex with Maneb
recorded against a reagent blank was linear over the concentration range from 2 µg 5
mL-1
to 40 µg 5mL-1
of the final solution. The detection limit is 0.0011 µg mL-1
for
Maneb when S/N ratio was 3.
161
5.2.4.2 Effect of heating time
It was observed that the absorbance of the complex increased up to a certain extent on
increasing the heating time. Therefore, the reaction mixture was heated for different
intervals of time. It was observed that an optimum heating time of 5 minutes was
sufficient to obtain maximum absorbance. Increasing the heating time beyond this did not
increase the absorbance as the complete complexation was achieved by heating for 5 min.
5.2.4.3 Effect of acid concentration
Maximum absorbance was observed when volume of acid added was between 1 mL to
1.5 mL. A decrease in absorbance was observed on further increasing the amount of acid
added as higher acid concentration is not conducive for complex formation.
5.2.4.4 Effect of other ions
Sample solutions containing 5 µg mL-1
of Maneb and various amounts of different alkali
metal salts or metal ions were prepared and the general procedure was applied. It was
observed that 20 mg of following foreign anions didn't interfere in the determination of
Maneb: bromide (11 mg), acetate (15 mg), chloride (3.5 mg), fluoride (2 mg), citrate (20
mg) and EDTA (0.06 mg). Of the metal ions examined, Mn (II) (0.066 mg), Mo(IV)
(0.14 mg), Ni(II) (0.30 mg), Co(II) (0.009 mg) did not interfere. Fe (II) and Zn(II) were
masked with 1 mL of 5% NaF solution and 1 mL of 5% KCN solution respectively. 1 mL
of 5% potassium iodide and thiourea were used to mask Hg (II) and Cu (II) respectively.
It is thus clear that several ions like Fe, Zn, Hg etc. interfered in maneb determination.
5.2.4.5 Effect of standing time
It was observed that the absorbance of the solution became constant after 2-3 min. so
162
after extracting into MIBK, a 5 min. standing time was selected. The absorbance of the
complex remained practically constant for about 30 minutes with the R.S.D. of
absorbance values varying between 0.69 to 1.6 % for different concentrations.
5.2.5 Applications (Determination of Maneb from fortified samples of wheat grains
and soft drinks)
The method was applied to the determination of Maneb from fortified samples of wheat
grains, cabbage and soft drink samples.
5.2.5.1 Sample preparation for food stuffs
The foodstuff (20 g) taken was finely crushed and a solution containing known amount of
maneb was added, it was mechanically shaken with 100 mL of ACN (Acetonitrile) for 1
hour. This mixture was filtered and the residue in funnel was washed with 3 x 10mL
portions of ACN. The extracts were evaporated on a water bath (70-90oC). The last traces
of solvent were removed using a current of dry air. The Maneb content in the residue was
determined by the general procedure and the results indicated good recoveries in all
cases. The results of the determinations were given in Table 5.2.3.
5.2.5.2 Sample preparation for soft drinks
The soft drinks (coke and limca) were locally obtained. These were diluted ten times,
filtered and spiked with known amount of maneb solution. The maneb content was
determined by the general procedure using a reagent blank prepared under similar
conditions.
163
5.2.5.3 Determination of Maneb in commercial samples
The method was applied for determination of Maneb in commercial samples. The
formulated product sample solutions were prepared as discussed earlier and determined
by the general procedure. The results of the determinations were given in Table 5.2.4.
5.2.5.4 Simultaneous determination of Maneb in presence of Ziram in synthetic
mixtures
The method was applied for the simultaneous determination of Ziram and Maneb in
synthetic mixtures. Ziram forms a yellow complex with sodium molybdate in cold, which
absorbs at 420 nm [26] whereas Maneb forms complex on heating, all other conditions
remaining the same. Synthetic mixtures of maneb and ziram were made in different
proportions. To the binary mixture, 0.15 mL of 2 mol L-1
of H2SO4 and 2 mL of 2%
sodium molybdate were added. Molybdenum-ziram complex was extracted into 5 mL
MIBK and maneb remained in aqueous phase. The absorbance of ziram-molybdenum
complex was measured at 420 nm. To the aqueous phase containing maneb was added
1mL 4 mol L-1
H2SO4 and 2mL of 2% Na2MoO4 solution. The solution was boiled for 5
minutes, cooled and extracted into 5 mL MIBK, the spectra of blue complex of maneb
was taken between 600 nm and 1100nm. Thus, this method was applied for the
simultaneous determination of ziram and maneb. Ziram was determined from the
standard calibration curve. The results of the determinations are given in Table 5.2.5.
5.2.6 Conclusions
The present method is more sensitive than the carbon disulphide evolution methods. It is
superior to the reported methods and the direct simultaneous determination of ziram and
164
maneb is possible. The sensitivity of the present method is comparable to other
spectrophotometric methods [Table 5.2.6]. The selectivity of the present method is
superior to other methods. The wide applicability of this method makes it suitable for
dithiocarbamate analysis in foodstuffs and in commercial samples.
165
Figure 5.2.2: Absorption spectrum of Maneb-molybdate complex: Maneb (2 mL)
with sodium molybdate (2%) in presence of H2SO4 (2M), extracted into MIBK
against a reagent blank.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
600 650 700 750 800 850 900 950 1000
Ab
sorb
ance
Wavelength (nm)
166
Figure 5.2.3: 1st derivative curves of molybdenum- ethylenebisdithiocarbamate
complex: a, b, c, d representing the amounts of Maneb in final solutions (a: 10, b:
20, c: 30 and d: 40 μg mL-1
)
Figure 5.2.4: 2nd
derivative curves of molybdenum- ethylenebisdithiocarbamate
complex: a, b, c, d representing the amounts of Maneb in final solutions (a: 10, b:
20, c: 30 and d: 40 μg mL-1
)
d c b a
a b c d
167
Figure 5.2.5: 3rd
derivative curves of molybdenum- ethylenebisdithiocarbamate
complex: a, b, c, d representing the amounts of Maneb in final solutions (a: 10, b:
20, c: 30 and d: 40 μg mL-1
)
Figure 5.2.6: 4th
derivative curves of molybdenum- ethylenebisdithiocarbamate
complex: a, b, c, d representing the amounts of Maneb in final solutions (a: 10, b:
20, c: 30 and d: 40 μg mL-1
)
168
Table 5.2.1: Different parameters of zero order spectrum
Serial
No.
Parameter Zero order spectrum of
maneb- sodium molybdate
complex
1.
Molar Absorptivity
(L.mol-1
cm—1)
8.16 x 104
2.
Sandell’s Sensitivity (μg mL
-1)
0.0049
3.
Analytical Sensitivity (μg mL
-1)
0.0011
4.
Linear Range (μg mL
-1)
2 to 40
Table 5.2.2: Comparison of calibration curves of Maneb using different derivative
spectra
Zineb
Complex
Order of
Derivative
λ
(nm)
Regression Equation R2
S.D. of
Slope
Analytical
Sensitivity
µg/mL
1.
1st
814 y= 4 X 10-5
x -0.0001
0.9997
1.2 X 10-6
1.4
2. 2nd
950 y= 7 X 10-6
x -0.0002 0.9992 2.4 X 10-7
0.9
3.
3rd
932
y= 3 X 10-6
x -5 X10-5
0.9995
1.4 X 10-8
0.032
4. 4th
904 y= 8 X 10-7
x +7 X10-6
0.9994 2.3 X 10-9
0.0011
169
Table 5.2.3: Determination of Maneb in fortified samples (mean values and
standard deviations, n=5)
Fungicide Sample Maneb added
(μg)
Maneb found
(μg)
Recovery (%)
± RSD (%)
Wheat 10 9.8 98 ± 2.1
15 14.9 99.3 ±1.9
20 19.8 99 ± 1.9
Cabbage 8 7.8 97.5 ± 2.2
Maneb 10 9.7 97 ± 1.9
Coke 10 9.1 91 ± 2.4
20 18.6 93 ± 2.1
25 23.9 95.6 ± 2.5
Limca 10 9.0 90 ± 2.2
20 18.2 91 ± 1.8
25 23.6 94.4 ± 1.8
170
Table 5.2.4: Determination of Maneb in commercial samples (mean values and
standard deviations, n=5)
Commercial
Sample
Maneb
taken (μg)
Maneb
found (μg)
Recovery (%)
± RSD(%)
Rangaswamy et al
method22
Maneb found Recovery %
(μg)
Dithane M-45
(80% W.P.) 20 19.9 99.5 ±1.5 19.7 98.5
30 30 100 ± 1.8 29.8 99.3
40 39.9 99.8 ± 1.6 39.8 99.5
Dithane M-22
(80% W.P.) 20 19.8 99 ± 1.6 19.7 98.5
30 29.8 99.3 ± 1.4 29.6 98.7
40 39.9 99.8 ± 1.5 39.8 99.5
171
Table 5.2.5: Determination of Maneb and Ziram in synthetic mixtures (mean values
and standard deviations, n=5)
Maneb
added (μg)
Ziram added
(μg)
Maneb
found (μg)
Ziram found
(μg)
Recovery (%) ± RSD (%)
Maneb Ziram
30 40 29.5 38.8 98.3 ± 2.3 97 ± 2.3
20 30 19.6 29.0 98.0 ± 2.6 96.7 ± 2.5
15 20 14.5 19.0 96.7 ± 2.1 95 ± 2.6
Table 5.2.6 Comparison of molar absorptivities of Maneb complexes
Procedure
Molar
Absorptivity
(L.mol-1
cm-1
)
Remarks Reference
Molybdenum
0.4 x 10 4
Low sensitivity and
selectivity
28
Rangaswamy et al method
_
Low sensitivity, long
tedious procedure
30
Complex formation with
diphenylcarbazone and
pyridine
6.5 x 104
Extraction not rapid
and uses toxic pyridine
51
Complex formation with
PAN
4.1 x 104
Less sensitive than
present method
52
Molybdenum (at 956 nm)
and derivative
spectrophotometry 8.1 x 10
4 Better sensitivity Present work
172
5.3 A sensitive Derivative Spectrophotometric method for the
Determination of Propineb (Zinc propylene bis
dithiocarbamate) fungicide using Sodium Molybdate
5.3.1 Introduction
Propineb [polymeric zinc propylenebis dithiocarbamate)] [(C5H8N2S4Zn)x] is a polymeric
dithiocarbamate substance that acts as a potent, nonsystemic, protective fungicide.
Figure 5.3.1: Structure of Propineb
The dithiocarbamate pesticides like propineb are favored worldwide as their properties
like low toxicity, high effectiveness and low production costs make them a popular
choice for fungal crop protection on various crops like potato, tomatoes, apples, grapes
etc [53, 54]. A new, commercially available fungicide formulation, Propineb 70% WP,
belonging to the group of propylenebisdithiocarbamates is also suitable for above
purposes [55, 56]. Though dithiocarbamates are known to display low acute and chronic
toxicities in human and experimental animals [57], but propineb and its residues like PTU
(phenyl thiourea) are extremely reactive due to their metal-chelating ability [58], and
high affinity for proteins containing the -SH group. DTC complexes are toxic and have
mutational effect [59], several researchers have studied the adverse effects of DTC
173
compounds including propineb [60-65]. The mobility of dithiocarbamate fungicides such
as mancozeb, sodium diethyldithiocarbamate, ziram, propineb and zineb through the soil
was studied by Rathore et al [40], whereas, Aktar et al studied the persistence and
dissipation of propineb in potato tubers [66]. A number of methods are available in the
literature for propineb analysis. Banerjee et al [67] and Schwack et al [68] determined the
Propineb residues in terms of CS2. A new cellular biosensor was developed for
determining the propineb residues by Flampouri et al [69] whereas Lo et al have
described HPLC and Atomic absorption methods for distinguishing between
dithiocarbamate fungicides like propineb, zineb, maneb and mancozeb [46]. Kazos et al
[70] developed a GC–MS and an improved HPLC method. Gas chromatographic
methods were also developed by Royer et al 1 and Česnik et al [48]. Nakazawa et al
[16] developed a reversed-phase ion-pair liquid chromatography with chemiluminescence
method for the determination of dithiocarbamates like propineb and mancozeb. Sharma et
al [71] have discussed various analytical methods for thiram analysis. The above
discussed methods have several disadvantages like the methods apart from gas
chromatography are less sensitive, indirect and time inefficient. Gas chromatographic
methods on the other hand have better sensitivity but are not selective as all
dithiocarbamates evolve CS2. The liquid chromatographic methods are comparatively
expensive and further use toxic and costly solvents. The present method is based on the
complexation of dihtiocarbamate fungicide zineb with sodium molybdate as developed
by Rao et al [5]. This method was also applied by Sharma et al [73] for the determination
of fungicide thiram in grains and commercial samples. In this method the
dithiocarbamate unit of the fungicide forms coloured complex with the molybdate ion
which is then extracted into the methyl isobutyl ketone (MIBK) for spectrophotometric
174
analysis. The present work describes a sensitive, efficient and more selective derivative
spectrophotometric method for the determination of dithiocarbamate fungicide, propineb
where it is complexed with sodium molybdate. Propineb can be determined in presence
of dithiocarbamate fungicides ziram or thiram by this method.
5.3.2 Experimental
5.3.2.1 Equipment and Reagents
Elico SL-164, Double Beam UV-Vis spectrophotometer was used. Propineb standard was
obtained from Riedel de Haën, Germany. A stock solution was prepared by heating100
mg of Propineb in 100 mL of 0.1 mol L-1
of NaOH. Further dilutions were carried out
with 0.1 mol L-1
of NaOH as required. A 2% (m/v) solution of sodium molybdate was
prepared in doubly distilled water.
5.3.3 Procedures
5.3.3.1 Absorption spectra
2 mL of 2% sodium molybdate solution and 1 mL of 2 mol L-1
of H2SO4 were added to an
aliquot containing 100 µg of Propineb. The mixture was boiled for 5 minutes, cooled and
water was added to make the volume 5 mL. The blue complex formed was extracted into
5 mL of MIBK (methyl isobutyl ketone) with shaking. The organic layer was collected
into a test tube containing fused CaCl2 to remove any moisture. The solution was then
decanted into a 1 cm cell and the spectra were taken against a reagent blank. The
molybdenum complex shows peaks at 670 nm and 956 nm (Figure 5.3.2), but the peak at
956 nm has much higher absorbance; hence all the measurements were done at this
wavelength. The fourth derivative curve is given in figure 3 and the calibration curve for
175
fourth order spectra is presented in figure 5.3.3. In DS (Derivative Spectrophotometry),
the λ reproducibility and S to N ratio are quite important. The features like peak height
and noise level depend on parameters chosen like order of derivative; scan speed and
integration time during recording of spectra. The use of optimum parameters will give
better resolution and more sensitivity. For the 4th
derivative spectra, Δλ = 9 nm was found
to be ideal.
5.3.3.2 Preparation of calibration curve
The known volumes of sample solutions having 20-300 µg 5mL-1
of Propineb were
analyzed by general procedure and the derivative spectra were obtained against a reagent
blank prepared under the similar conditions. The characteristics of 1st to 4
th order spectra
are summarized in Table 5.3.1.
5.3.4 Applications
5.3.4.1 Determination of Propineb from fortified food stuff samples
The method was applied to the determination of Propineb from fortified samples of
apples, grapes, potato, tomato and aubergines (egg plant).
5.3.4.1.1 Sample preparation for food stuffs
20 grams of the foodstuff was finely crushed and solution containing a known amount of
Propineb was added, it was mechanically shaken with 100 mL of Acetonitrile for 1 hour.
This mixture was filtered and the residue in funnel was washed with 3 x 10mL portions
of ACN. The extracts were evaporated on a water bath (70-90oC). The last traces of
solvent were removed using a current of dry air. The Propineb content in the residue was
176
determined by the general procedure and the results indicated good recoveries in all
cases. The recoveries from the fruit and vegetable samples show minor decrease due to
slight decomposition of pesticide during extraction [30]. The results of the determinations
were given in Table 5.3.2.
5.3.4.2 Determination of Propineb in commercial samples
The method was applied for determination of propineb in commercial samples. The
formulated product sample solutions were prepared as discussed earlier and determined
by the general procedure. The results of the determinations were given in Table 5.3.3.
5.3.4.3 Simultaneous determination of Propineb in synthetic mixtures
The method was applied for the simultaneous determination of Ziram and Propineb and
thiram and propineb in synthetic mixtures. Ziram forms a yellow complex with sodium
molybdate in cold, which absorbs at 420 nm [28] whereas Propineb forms blue complex
on heating, all other conditions remaining the same. Synthetic mixtures of Propineb and
Ziram were made in different proportions. To the binary mixture, 0.15 mL of 2 mol L-1
of
H2SO4 and 2 mL of 2% sodium molybdate were added. Molybdenum-ziram complex was
extracted into 5 mL MIBK and propineb remained in aqueous phase. The absorbance of
ziram-molybdenum complex was measured at 420 nm. 1 mL of 4 mol L-1
H2SO4 and 2
mL of 2% Na2MoO4 solution were added to the aqueous phase containing propineb was
added. The solution was boiled for 5 minutes, cooled and extracted into 5 mL MIBK, the
spectra of blue complex of propineb was taken between 600 nm and 1100 nm. Thiram
forms complex with sodium molybdate without heating which has absorption maxima at
360 nm. Thiram and propineb were determined simultaneously by the above method.
177
Thus, this method was applied for the simultaneous determination of propineb with ziram
or thiram. The results of the determinations are given in Table 5.3.4.
5.3.5 Results and Discussion
5.3.5.1 Analytical Performance
The optimum wavelength interval was found to be 9 nm for high resolution and
sensitivity. The wavelength range to obtain spectra was selected from 600 nm to 1100
nm. The calibration curve for 4th
derivative spectra was obtained by measuring the peak
height at 956 nm. Absorbance of sodium molybdate complex with Propineb recorded
against a reagent blank was linear over the concentration range from 5µg mL-1
to 60 µg
mL-1
of the final solution. The detection limit has been found to be 0.0088 µg mL-1
from
4th
order spectra for Propineb when S/N ratio was 3.
5.3.5.2 Effect of heating time
It was observed that the absorbance of the complex increased up to a certain extent on
increasing the heating time. Therefore, the reaction mixture was heated for different
intervals of time. It was observed that an optimum heating time of 5 minutes was
sufficient to obtain maximum absorbance. Increasing the heating time beyond this did not
increase the absorbance as the complete complexation was achieved by heating for 5
minutes
5.3.5.3 Effect of acid concentration
Maximum absorbance was observed when volume of acid added was between 1 mL to1.5
mL. A decrease in absorbance was observed on further increasing the amount of the acid
178
added as higher acid concentration is not conducive for complex formation.
5.3.5.4 Interferences
Sample solutions containing 5 µg mL-1
of Propineb and various amounts of different
alkali metal salts or metal ions were prepared and the general procedure was applied. It
was observed that given amount of the following foreign anions didn't interfere in the
determination of Propineb : bromide (11 mg), acetate (15 mg), chloride (3.5 mg), fluoride
(2 mg), citrate (20 mg) and EDTA (0.06 mg). Of the metal ions examined, Mn (II) (0.066
mg), Mo (IV) (0.14 mg), Ni (II) (0.30 mg), Co (II) (0.009 mg) did not interfere. The
tolerable amounts of foreign species during determination of propineb are summarized in
table 5.3.5. Fe (II) and Zn (II) were masked with 1 mL of 5% NaF solution and 1 mL of
5% KCN solution respectively. 1 mL of 5% potassium iodide and thiourea were used to
mask Hg (II) and Cu (II) respectively. It is thus clear that several ions like Fe, Zn, Hg etc.
interfered in propineb determination. Thiram, ziram and ferbam if present could be
separated by extraction with chloroform followed by the propineb determination.
Propineb can be determined in presence of Thiram or ziram, as both form complexes with
sodium molybdate without heating, hence their complex can be extracted and propineb
left can then be determined by the method. Zineb and maneb interfere if present.
5.3.5.5 Effect of standing time
It was observed that the absorbance of the solution became constant after 2-3 min. so
after extracting into MIBK a 5 min standing time was selected. The absorbance of the
complex remained practically constant for about 30 minutes with R.S.D. of absorbance
values varying between 0.69 to 1.6 % for different concentrations.
179
5.3.6 Conclusion
The present method is more sensitive than the carbon disulphide evolution methods. It is
superior to the reported methods and the direct simultaneous determination of ziram and
propineb is possible. The sensitivity of the present method is good when compared to
other methods (Table 5.3.6) and has wide applicability as it doesn’t require expensive
reagents and equipments. The present method permits the determination of propineb in
presence of other dithiocarbamate fungicides Ziram or Thiram which is not possible with
gas chromatographic and liquid chromatographic methods. The recoveries were found to
be good during the simultaneous determinations. The large scope and application of this
method makes it suitable for dithiocarbamate analysis in foodstuffs and in commercial
samples.
180
Figure 5.3.2: Absorption spectrum of Propineb-molybdate complex: Propineb (2
mL) with sodium molybdate (2%) in presence of H2SO4 (2M), extracted into MIBK
against a reagent blank.
Figure 5.3.3: 4th derivative curves of molybdenum-propylenebisdithiocarbamate
complex: a, b, c, d, representing the amounts of propineb in final solutions ( a:30, b:
40, c: 50 and d: 60 µg mL-1
)
0.15
0.2
0.25
0.3
0.35
0.4
0.45
600 700 800 900 1000
Ab
so
rban
ce
Wavelength (nm)
-0.001
-0.0005
0
0.0005
0.001
0.0015
605 705 805 905
d4/d
λ4
Wavelength (nm)
d
c
b
a
181
Table 5.3.1: Comparison of Calibration curves of propineb-molybdate complex using different derivative spectra
Propineb
Complex
Order of
derivative
λ
(nm)
Linear Range
(µg mL-1
) Regression Equation R
2
Standard
Deviation of
Slope
LOD
(µg mL-1
)
1. 1st 872 5-60 y=5×10
-5x-0.0003 0.999 2.1×10
-4 13.86
2. 2nd 901 5-60 y=2×10
-5x+0.00025 0.994 2.1×10
-5 3.3
3. 3rd
928 5-60 y=6×10-6
x-0.0001 0.998 1.2×10-6
6.6
4. 4th
956 5-60 y=2×10-5
x-5×10-6 0.999 2.3×10
-6 0.38
182
Table 5.3.2: Determination of Propineb in fortified samples (n=5)
Fungicide Sample Propineb added
(µg )
Propineb
found (µg ) Recovery % ± R.S.D.
Propineb Tomato 10 8.9 89.0 ± 2.2
15 13.1 87.3 ± 2.2
20 17.5 87.5 ± 1.9
Potato 10 8.9 89.0 ± 2.1
15 13.0 86.7 ± 2.4
20 18.0 90.0 ± 1.9
Grapes 15 13.5 90.0 ± 2.5
20 17.8 89.0 ± 2.2
Apple 20 18.0 90.0 ± 2.1
25 22.6 90.4 ± 2.3
Aubergines 10 9.3 93.0 ± 1.8
20 18.3 91.5 ± 1.8
183
Table 5.3.3: Determination of Propineb in commercial samples (mean values and standard deviation, n=5)
Commercial Sample Propineb taken(µg) Propineb found
(µg) Recovery % ± R.S.D. (%)
Antracol (70% W.P.) 20 18.6 93.0±2.5
30 28.7 95.7±2.6
40 38.4 96.0±2.0
Propineb 70% WP 20 19.3 96.5±1.6
30 29.2 97.3±1.6
40 38.7 96.7±1.8
184
Table 5.3.4: Determination of Propineb in synthetic mixtures, (mean values and standard deviations n=5)
Propineb
added (µg)
Ziram
added (µg)
Propineb
found (µg)
Ziram
found (µg)
Recovery % ± R.S.D. %
Propineb Ziram
30 40 27.8 38.7 92.7±3.7 96.75±3.8
20 30 18.7 28.8 93.5±4.2 96.0±3.4
25 25 23.2 24 92.8±4.3 96.0±3.3
Propineb
added (µg)
Thiram
added (µg)
Propineb
found (µg)
Thiram
found (µg)
Recovery% ± R.S.D.%
Propineb Thiram
30 50 28.2 47.6 94.0±2.3 95.2±2.5
40 40 38.1 38.2 95.3±2.1 95.5±2.1
185
Table 5.3.5: Effect of Foreign ions on Propineb determination
S. No. Foreign species Tolerable Amount (mg)
1. Bromide 11.0
2. Acetate 15.0
3. Chloride 3.5
4. Fluoride 2.0
5. Citrate 20.0
6. EDTA 0.06
7. Mn(II) 0.07
8. Mo(IV) 0.14
9. Ni(II) 0.30
10. Co(II) 0.01
186
Table 5.3.6: Comparison of limit of detection of propineb from different methods
S. No. Method Matrix LOD (µg mL-1
) Remarks Reference
1. Spectrophometric: CS2 evolution Tea leaves 0.10 Low sensitivity 67
2.
Spectrophometric: CS2 evolution Onion 0.20 (LOQ) Low sensitivity
77
3.
Photometric
determination
Miscellaneous 0.05 Low sensitivity
76
4.
HILIC column (LC-MS/MS) Apples,
tomatoes, etc.
0.001
Good sensitivity
but expensive
equipment
71
5.
Ion pair LC with
chemiluminescence detection using
Luminol
Vegetables 0.00042 High sensitivity but
expensive method
16
6. Derivative spectrophotometric
using sodium molybdate
Apples,grapes,
tomatoes, etc.
0.38,
0.0088
(analytical
sensitivity)
High sensitivity Present
method
187
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