INTRODUCTION:
Nowadays one of the blooming problems of the world is
fungicide traces which are present in edible product causes health hazard on
human being1. Estimation of fungicide to human exposure can be
determined by level present in the surroundings2. It has been
reported that attack of pathogen in crop, vegetables, so that cultivators used
fungicides approximately in daily for the protecting their crop3. It
is appropriate to develop alertness to user for safety concern that to know
what amount of fungicide residue present in their dietary4. The
dithiocarbamate fungicide having a thiocarbonyl group such as thiram which is
broadly used moulds against a range of domestic and farming5.
It
was determine that dithiocarbamate fungicide i.e. thiram act as neurotoxin and
attacking to nervous system of various pathogens (Hodge et al., 1956).
Thiram
fungicide is moderately toxic by inhalation and by dermal absorbing6.
Thiram is used in rubber manufacturing industrial concern and consequence of
fungicide7. Under natural conditions studies report that it
inactivates the enzyme-containing Glutathione reductase8. Its field
of activity is mutagenic and may cause chromosomal abnormality9. The
several procedures have been used for the determination of thiram fungicide10.
These include high-performance liquid chromatography, extraction voltametry,
titrimetry, iodometry in anhydrous solvents, indirect titration with EDTA,
polarography, TLC, GC-MS, Spectrophotometric etc.11. Several
spectrophotometric methods have also been reported for the determination of
thiram fungicide12. The aim of the proposed work is to develop a
simple, sensitive, faster, reliable method of analysis for the determination of
widely used thiram fungicide in microgram levels13. The developed
method is based on acidic hydrolysis of thiram fungicide to carbon disulphide14.
The carbon disulphide is further react with ethanolic sodium hydroxide to form
xanthate, xanthate further reacts with potassium iodate in acidic medium to
librate iodine and this librated iodine oxidizes leucomalachite green to
malachite green15. The developed method has been successfully
applied to determination of thiram in environmental and biological samples,
fruits and vegetables16.
EXPERIMENTAL
SECTION:
Apparatus: -
A Systronics
UV spectrophotometer 105 with 2cm matched silica cells was used for all
spectral measurements. Systronics pH meter model No.335 was used for pH
measurement.
Reagents:-
All
reagents were of analytical reagent grade and double distilled deionised water
was used throughout the experiment.
Prepared
of thiram solution:-
1µg
of thiram fungicide dissolved in 25ml acetonitrile the required working
standard solution was prepared by appropriate dilution of the stock.
Sodium
hydroxide:-
5%
(5g/100ml) solution of ethanolic sodium hydroxide solution was prepared as an
absorbing solution for CS2.
Potassium
iodate:-
0.1M
solution was prepared by dissolving 0.713g of KIO3 in 100ml
water.
N-chlorosuccinimide
Solution:-
250mg
of N-chlorosuccinimide was added to 250ml volumetric flask containing 2.5g of
succinimide and volume was made up to the mark with water.
Leuco
malachite green (LMG):-
0.05%
solution was prepared by dissolving 25mg of LMG, 100 ml of water.
Buffer
solution acetate buffer:-
13.6g
(1M) sodium acetate trihydrate in 80 ml of water, solution PH was
adjusted to 4.5 with acetic acid, and the mixture was diluted to 100 ml with
water.
Sodium
salt of EDTA:-
(5g/100ml)
aqueous solution of EDTA, metaphosphoric acid (3g/100ml).
Procedure:-
A
known 1ml (1µg/25ml) amount of thiram solution prepared in acetonitrile was
introduced in the digestion flask of digestion-absorption apparatus. The hot 9M
H2SO4 was added drop wise to the above solution for the
hydrolysis during reaction which CS2 was released 5ml of ethanolic
sodium hydroxide solution was taken as an absorbing solution. Hydrolysis was
carried out for 35-40şC temperature range. The pale yellow colour solution of
xanthate was obtained and diluted with water as required. This solution 2ml
(1µg/25ml) of thiram were taken in a 25 ml test tube. To it 2 ml acetate buffer
solution, 2ml of potassium iodate, 1ml of N-chlorosuccinimide were added and
shaken for a few seconds. 2ml of 0.05% of LMG solution added to the solution.
The contents were heated 35-40şC in a water bath for 5min, cooled at
temperature and diluted to the mark with distilled water. The reaction was
completed in 10min the mixture was kept for 10min for completion of the
reaction. The absorbance of the formed dye was obtained at 610 nm against the
reagent blank.
RESULT
AND DISCUSSION:
Absorption
spectra of thiram with MG dye:-
This
graph is plotted between wavelength and absorbance. Maximum absorbance (λmax) was obtained at 610 nm. (a) Shows absorption spectrum
of thiram fungicide. (b) Shows absorption spectrum of reagent blank. Thiram is
decomposed under acidic condition to give carbon disulphide which trapped in
methyl potassium hydroxide to give potassium methyl xanthate, which is then
titrated iodometrically, the liberated iodine selectively oxidizes the LMG to
MG dye. The green colour of dye was developed in an acetate buffer (PH
3-4.8) on heating in a water bath (35-40şC) for 5min. A time period of 5min was
required for complete colour development after dilution to 25ml. The pale
yellow colour MG dye showed maximum absorbance (λmax)
at 610 nm, and reagent blank hand absorbance wavelength (Fig.-1).
Fig.-1
Absorption spectra of thiram with LMG dye (a) Absorption spectra of thiram
fungicide and leucomalachite green dye; (b) Absorption spectra of Reagent blank
Colour
reaction:-
1.
Liberation of CS2 from
thiram by acid hydrolysis at temperature ranges 35-40şC.
2.
Formation of xanthate with the
treatment of carbon disulphide with alcoholic sodium hydroxide.
3.
Iodine is liberated as a result of
reaction between potassium iodate act as oxidizing agent and the xanthate in
the presence of N-chlorosuccinimide. Potassium iodate has +5 oxidation states
in iodate it changes into zero oxidation state in free iodine in first step.
But in second step free iodine has zero oxidation state it changes into -1
oxidation state. Here iodine is oxidized.
Formation
of malachite green (MG) dye through selective reduction of leucomalachite green
dye (LMG) by liberated iodine (Scheme-1).
Effect
of time and temperature:-
This
graph is plotted between temperature on X-axis and absorbance on Y-axis.
Temperature between 35-40şC is sufficient for colour development. At range of
this temperature and optimum condition the colour development was rapid and
fast was most suitable [17]. So the reagent system required heating in water
bath for 5min. At above and below this range of temperature absorbance of
thiram decreases. An affect the sensitivity and reproducibility of colour
system, the 10min time was required for complete colour development after
formed dye was stable for seven days [18] (Fig.-4).
Fig.4-Effect
of temperature on absorbance of concentration level of thiram 1ml (1µg/25ml)
fungicide with leucomalachite green dye (25mg/100ml)
Effect
of concentration of LMG Reagent:-
This
graph is plotted between volume of leucomalachite green on X-axis and
absorbance on Y-axis. Minimum requirement of leucomalachite green was
found to be 0.05% of 1ml (25mg/100ml) of leucomalachite green is sufficient for
colour development at the concentration level of thiram 1ml (1µg/25ml)(Fig.-5).
Fig.5-
Effect of concentration of LMG 1ml (25mg/100 ml) on absorbance of concentration
level of thiram 1ml (7µg/25ml)
Effect
of concentration of NaOH Reagent:-
This
graph is plotted between volume of NaOH on X-axis and absorbance on Y-axis. 1.5
ml of (5g/100ml) sodium hydroxide is sufficient for colour development.
Hydrolysis the concentration level of thiram fungicide (1µg/25ml) at
temperature ranges 35-40şC as it was observed that maximum hydrolysis
obtained, it found to be maximum absorbance and stability of the coloured
system (Fig.-6).
Fig.6-Effect
of Concentration of NaOH 1.5ml (5g/100 ml) on absorbance of concentration level
of thiram 1(1µg/25ml) with leucomalachite green dye (25mg/100ml)
Effect
of concentration of potassium Iodate:-
This
graph is plotted between volume of potassium iodate on X-axis and absorbance on
Y-axis. Minimum requirement of 1ml of (0.713g/100ml) potassium iodate is
sufficient for colour development (Fig.-7).
Fig.7-Effect
of concentration of potassium iodate1ml (0.713g/100ml) on absorbance of
concentration level of thiram 1ml (1µg/25ml)
Effect
of concentration of thiram fungicide:-
This
graph is plotted between volume of thiram on X-axis and absorbance on Y-axis.
1ml (1µg/25ml) of thiram fungicide is enough for development of colour
(Fig.-8).
Fig.8-Effect
of concentration of thiram on absorbance of concentration level of thiram 1ml
(1µg/25ml)
Table-1
Spectral characteristics of the proposed method
|
Parameters
|
Thiram Fungicide
|
|
Maximum absorbance (nm)
|
610
|
|
Beer’s law conc. range (µg)
|
(0.7-7)
|
|
Molar absorptivity(lmol-1cm-1)
|
6×10-2
|
|
Sandell’s sensitivity (µgcm-2)
|
4.0073
|
|
Standard deviation
|
4.82147, 0.48214
|
|
Correlation coefficient
|
0.991
|
|
Slope (m)
|
0.1
|
|
Intercept (C)
|
0
|
|
Limit of detection (µgml-1)
|
144.64
|
|
Limit of quantification (µgml-1)
|
48.214
|
|
Precision
|
2.25 ± 6.3
|
|
Accuracy
|
0.008900
|
|
Absolute error
|
0.0089
|
|
Relative error
|
0.008908
|
|
Relative standard deviation
|
1.1856093%, 1.18560%
|
Effect
of possible interference:-
The
tolerance limit of various ions and fungicide, pesticides, insecticides, to be
present with thiram was evaluated and shown in (Table-1). Most commonly used
ions did not interfere in the procedure19. Only evolve of carbon
disulphide compounds interfere acid hydrolysis in this method and such types of
contaminations are rare; H2S also interferes and is eliminated by
its absorption in lead acetate to the passing of carbon disulphide in the
absorbing solution20.
Determination
of thiram in blood and urine samples:-
The
thiram fungicide is reported in blood and urine, the method is applied for
their determination in biological samples; 5ml each of blood and urine samples
were taken from pathology laboratory and 1ml of 5% EDTA, 2ml of 1% TCA
(trichloroacetic acid) and 2ml of 3% metaphosphoric acid were added and known
amount of thiram is added to the sample, centrifuged, the supernatant solution
was diluted to a suitable volume and 1ml sample was analyzed by this method21.
Table-2
Effect of foreign species (1µg of thiram per 25ml)
|
S. No.
|
Foreign
species
|
Tolerance
limit
µgml-1
|
Foreign
species
|
Tolerance
limit
µgml-1
|
|
1.
|
Mancozeb
|
1300
|
Na+ ,
K+ , Ba+
|
2000
|
|
2.
|
Propoxur, Carbary
|
1150
|
F-,Cl-,Br-,
PO43-
|
1800
|
|
3.
|
Phorate
|
800
|
Zn2+,
Fe3+, Al3+
|
400
|
|
4.
|
DDT, BHC
|
950
|
Mg2+,
Na+
|
1000
|
|
5.
|
Malathion
|
600
|
|
|
Tolerance limit is the amount of foreign species that causes an
error of 2% in absorbance value [22].
Table-3
Results of determination of thiram
in different samples
|
Samples
|
Proposed
method
|
Reported method
|
|
Polluted water
|
3.29
3.30
|
3.18
3.21
|
|
Potato tubers
|
4.6
5.67
|
4.30
4.89
|
|
Lettuce
|
2.89
3.86
|
2.32
3.51
|
|
Cucumber
|
3.50
4.99
|
3.39
4.48
|
|
Wheat
|
6.00
6.54
|
5.76
6.56
|
Amount of samples; water samples-100ml, vegetable
samples-100g.
Mean of five replicate analyses [23].
Table-4
Recovery test of thiram in blood
and urine samples
|
Samples
|
Thiram added
in A
|
Thiram found
in µgb B
|
Recovery
B/A  100
|
|
Blooda
|
5.00
5.00
|
4.78
4.82
|
95
96
|
|
Urinea
|
5.00
5.00
|
4.73
4.79
|
94.6
95.8
|
A-Aliquot
of sample-5ml, B-Mean of four replicate analysis.
CONCLUSION:
The
proposed method based on reaction of thiram fungicide and leucomalachite green
in presence of ethanolic sodium hydroxide, potassium iodate,
N-chlorosuccinamide, leucomalachite green dye in acidic medium for the
spectrophotometric determination of thiram. Impact on community to spread
awareness, amongst community, that have this fungicide of harmful to human
health and environment. To give suggestion to the local farmers for tempted used
for these fungicide. In present scenario development of method for analysis of
any harmful chemical is important technique, through many sophisticated
instrumental methods are available but this classical method have their own
importance forever. Significance of method was many analytical parameters have
been studied, results as approved by statiscal analysis accuracy and precision
are determined. Results of application reflect the reliability to the method.
In the development method no any harmful, chemicals were used; the proposed
method is reliable, precise, affordable and easily available in small
laboratory then other sophisticated instrumental reported method.
ACKNOWLEDGEMENT:
I
express my gratitude to Principal Dr. D. N. Verma, Govt. Nagarjuna P. G.
College of Science. Dr. Kishore K. Tiwari my guide Head Department of Chemistry
Shahid Rajiv Pandey Govt. College Bhatagoan Raipur. Dr. Smt. Shipra Verma,
Head, Department of Chemistry Raipur. I am also thankful to University Grant Commission,
New Delhi for providing laboratory facilities and financial assistance to carry
out this experiment.
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10-2l mol-1 cm-1,
and 4.0073µgcm-2, for thiram. Statistical treatment of the
experimental results indicates that the method is precise and accurate. The
method is free from interference of common ions and fungicides. The reliability
of the method was established by parallel determination against reported
method. The method has been successfully applied to the determination of thiram
fungicides in various environmental samples.