Page 1
..:llAPTbR - VIII
PART -A
A SI!YiPLE SPECTROPHOTOME.TIUC DETERMINATION OF
ENOOSULF AN IN Rl VER WATER AND SOIL
SUMMARY
A simple spectrophotometric determination of
endosulfan (thiodan), a sulphur containing chlorinated
pesticide is described. The method is based on the
liberation of sulphur dioxide into an absorbing reagent,
malonyldihydrazide and estimated by using p-aminoazo
benzene and formaldehyde in an acidic medium to give a
pink coloured dye which has an absorbance maxima at
505 nm. Beer's law is obeyed in the range of 1-6 ppm
for a standard solution of endosulfan. The method can
~e easily applied in river water and soil samples to
determine endosulfan level as low as 0.05 ppm and
0.25 ppm in river water and soil respectively. The
method is free from the interference of most of the
commonly used pesticides and other common ions.
Page 2
,; il l'.i'l.!; ~WJ;;(,'TrtOt'III)TVMl:.T!U C Q£TERJ:IlNAT 101'1 Of
V.!«iMJ:'M IN IU M WATE,K MP 1!11.
1-)ldosultan ( thiodao), a broad specti'Ulll chlorinated
inltcticide as w~ll as ~ticide contains both sulphur and
oxyun as heterocyclic ato• and 1a 6,7,8,9,10,10-hexachloro-
1,~,5a,6,9,9a-hexahydro-6,9,-•ethano-2,4,,-benzo
dioxathiepin-,-oxide, also known as 5462. Endosul!an is
very effective against many different types o! insects
such as potato infesting insects, insects in!esting alfalfa,
:lover, cotton and shade tobacco, aphids on ornamentals,
and weevils on seed peas etc. It also shows a promising
control on spit bugs, white flies, tse-tse flies and
lea! hoppers and is approved to use on certain fruit and
vegetable crops (1-6). It acts as a synergistic insecti
cide and acaricide (7,8). Endosulfan has controlled a
wide range of wood boring insects, hence it is used as a
demanding •.vood preservative (9). Consequently, the
residues of endosulfan are found in se.veral environmental
samples leading to the constant ingestion of residual
':iuant.i ties of the chemical, posing a hazardo>.t.s problem
for the heal til of man and animals ( 10-12).
It is highly toxic to animals, and is reported to
·:oe a suggestive carcinogenic compound ( 13, 14). Endo
sulfan is metabolized, in plants and animals, to the
sulphate which is toxicologically similar to sul,.phi te( 15).
Page 3
Syl!ptoms of poisoning by endosulfan are rather similar
to those caused by 'YBHC. Hyper excitability, tremor,
dypsnea and salivation are the common signs of endosulfan
toxicity (16,17). The acute oral LD50 in rats is
40-50 mg/kg and the median letllal concentrations Lc50 in
fishes is 5 ~g/1 for 24 hours and 0.6 ~g/1 for 48 hours
( 14, 15). Hesidues of endosulfan in soil was estimated to
be around 0.3!:l-4.60 ppm and in water about 1S7.0 - 3,400
ng/1 in various locations in u.s.A. (14).
The toxicity and persistent nature of endosulfan
in the environmental samples necessitated simple and
reliable ruethods for its determination in residues. Very
few analytical techniques such as gas chromatography (18),
gas liquid chromatography (19), thin layer chromato-
graphy (20), high performance liquid chromatography(21,22),
mass spectrophotometry (23) and visual spectrophoto-
metry (24,25) are available in literature for its determi
nation. The commonly used spectrophotometric method
reported in literature (24) is based on the liberation of
sulphur dioxide from endosulfan using p-toluene sulphonic
acid. The liberated sulphur dioxide is subsequently
absorbed in glycerol-alkali solu+ion ;md estimated by
using p-rosaniline (PRA) and formaldehyde in an acidic
medium following West & Gaeke's method (26). But this
method has been criticized due to certain disadvantages
such as instability of sulphur dioxide in glycerol-alkali
solution (27), variable purity of the reagent PRA (28),
Page 4
the !ormation o! unstable coloured complex (29), and lack
o! reproducibility and reliability. Hence, in the present
work, a simple spectrophotometric determination of endo
sul!an is described to overcome the above detects. In
this method the liberated sulphur dioxide froa1 endosulfan
is absorbed in a simple absorbing reagent, malonyl
dihydrazide and subsequently estimated by using p-amino
azobenzene and formaldehyde in hydrochloric acid medium
to give a pink coloured dye, having absorbance maxima at
J05 nm. The proposed method has been successfully applied
for the determination of endosulfan in river water and
soil samples.
Apparatus:
A Carl Zeiss spekol with 1 em matched glass cells
was used for spectral measurements. Fritted midget
impingers of 35 ml capacity, a flow rate adjustable
calibrated rotameter and a vacuum pump were used for the
liberation and absorption of sulphur qioxide from
endosulfan.
Reagents:
Standard endosulfan solution (Tata Fision Ltd., India):
A 1% stock solution of endosulfan was prepared in ethanol.
A working standard of 20 pg/ml was prepared daily by
appropriate dilution of the stock.
Page 5
3tar.dard sulphite solution: ~ulphite solution contain-
ing about 320 - 400 ~g of so2/ml was prepared by dissol
ving 0,2 g of pre-dried sodium sulphite in 250 ml of
0.01 M malonyl dihydrazide in water. Sulphite solution
was standardized iodometrically. Working standards were
prepared by appropriate dilution of the stock in 0.01 M
malony ldihydrazide,
Acid reagent (24): An acid reagent was prepared by
dissolving 304 g of p-toluene sulphonic acid in 1 litre
of isopropanol plus 200 ml of demineralized water.
J>,alonyl dihydrazide (MI.lH) : The reagent was prepared as
earlier rE~ported method ( 30), described in Chapter II,
using diethyl malonate and hydrazine hydrate.
0.01 M solution of 1"1!lf, prepared in demineralized
water was used as absorbing reagent.
p-Aminoazobenzene (31): A 0,02% (w/v) solution of recrystallized p-aminoazobenzene was prepared in 25%
ethanol.
.2ormaldehyde: A 0.2% (v/v) solution of formaldehyde was . prepared by diluting 0. 5 ml of 40% f orrnal dehyde to 100 ml
with demineralized water.
2% Alcoholic potassium hydroxide and concentrated
hydrochloric acid were also made use of.
All reagents unless mentioned otherwise used were
of An alaR grade.
Page 6
Procedurt>:
Preparation of calibration curve with endosulfan:
Liberation/adsorption of sulphur dioxide:
An aliquot of the standard solution containing
25 - 150 ~g of endosulfan was taken in an impinger. To
this 5 ml of acid reagent and 1 m1 of alcoholic pot.:.ssium
hydroxide were added. The impinger was then kept in a
water bath and connected serially to two impingers kept
outside the water bath containing 5 ml each of MDH solu
tion. The third impinger was then connected to a source
of suction through a rotameter. Temperature of the water
beth was raised to r-J 90°C end the air was drawn through
the impingers at a rate of 0.75 lit/min for 15 minutes.
Analysis:
The absorbed solution was transferred into a
25 ml volumetric flask. To it 1 ml of p-aminoazobenzene
was added and acidity was maintained between 0.02 - 0.16 M
hydrochloric acid. Then 1 ml of formaldehyde and 1 ml of
concentrated hydrochloric acid were added, The volume was
made upto the mark with demineralized water. After
15 minutes, the absorbance of the pink coloured dye was
measured at 505 nm against demineralized water. The
actual absorbance of sample solution was calculated by
substracting the absorbance value of the blank from the
value of the sample.
Page 7
Preparation of standard extinction curve1
An aliquot of standard sodium sulphite solution
in 0.01 M MUi containing 4-24 }Jg of sulphur dioxide was
taken in 25 m1 volumetric flask and colour was developed
as described above. The amount of sulphur dioxide was
multiplied by 6.35 to convert to endosulfan equivalent(24).
The resulting values were then plotted against the proper
absorbance values to obtain a standard curve (Fig. 5).
Determination of recovery in water and soil samples:
Water samples (500 ml) or 100 g of soil sample of
finely ground soil samples were spiked with known amount
of endosulfan and extracted with 2 x 100 ml portions of
petroleum ether (60-80°C) in a glass bottle. The extract
was decanted and combined in a separatory funnel and
washed with 2 x 200 ml portions of demineralized water,
discarding the water layer. Extract was dried over
anhydrous sodium sulphate in a filter funnel and collected
into a 250 ml calibrated flask. The filter funnel was
washed with 20 ml of petroleum ether and the volume was
made upto the mark. Sui table aliquot of washed extract
containing 25 - 150 }Jg of endosulfan was evaporated off
under reduced pressure using moderate suction. The residue
was mixed with 5 ml of iso-propanol and the sulphur dioxide
was liberated/absorbed for the subsequent colour develop
ment as described in the procedure for the preparation of
calibration curve with endosulfan. Recoveries from samples
were found to be~ 99% (Table I).
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TAIJLE - I
RE<X>VERY OF ENJXJSULFAN FROM SPIKE!J
RI Vffi WATffi MlD SOIL SAMPLES
-----------------------------------------------------------Sample Amount of
endosulfan added *
(p.g)
Amount found by proposed method *
(p.g)
% liecovery
Amount found by reported method *
(,ug)
% Recovery
-----------------------------------------------------------
** Soil
*** Water
25
50
75
100
25
50
75
100
24,62
48.65
?3.65
98.60
24.57
49.00
73.65
99.10
98.5
97.3
98.2
98.6
98.3
98.0
98.2
99.1
24.55
48.55
73.50
98.50
24.57
49.00
73.57
98.80
98.2
97.1
98.0
98.5
98.3
98.0
98.1
98,8
-----------------------------------------------------------* **
Mean of three replicate analyses
Amount of sample = 100 g
Amount of sample "' 500 ml
Page 9
~ESULTS AND DISCUSSION
Spectral characteristics:
The pink coloured dye has a maximum absorbance
at 50~ nm. The reagent blank has also same wave length
of absorption (Fig. 1). The actual absorbance of sample
was therefore calculated by substracting the value of
reagent bl.ank from the value of the sample (31).
Absorption efficiency:
The important analytical parameters such as
absorption efficiency of the proposed absorbing reagent
(MDH), effect of various concentration of absorbing solu
tion, flow rate and temperature on absorption efficiency
and stability of liberated sulphur dioxide from endosulfan
in absorbing medium were studied by liberating sulphur
dioxide from standard sodium sulphite solution following
the reported methods (31).
Two midget impingers containing 10 ml of 0.01 M
MDH in each were connected in series and rJ 38.2 litres of
air containing various amounts of sulphur dioxide was
passed at different flow rates ranging from 0.05-2,0
lit/min. through the impingers. (If the sample size is
38.2 litres, then each microgram of sulphur dioxide
obtained is equivalent to 0.01 ppm of sulphur dioxide in
air (26,31)). After sampling sulphur dioxide was analysed
by tne proposed procedure. Almost 100~ absorption effi
ciency was obtained in the first impinger, while the
Page 10
0·8......--------------------.
0·7
8 0·6
0·5
0·4
0·3 A
0·2
0 ·1
QL---~ ____ _L ____ J_ ____ L_ __ ~----~----~--~
450 470 490 510 5 30
WAVElENGTH, nm
FIG.1.ABSOR BANCE SPECTRA OF THE DYE
A. REAGENT BLANK.
550 570
8. CONCENTRATION OF ENOOSULFAN= 125).lg/25ml.
5 971
Page 11
second impinger gave negative test !or sulphur dioxide.
E.!!ect of various concentrations of MI:il solution on absor
ption efficiency was studied • 0.005 - 0,1 M MDH solution
were found to have ,..... 100% absorption efficiency (Table II) •
Flow rate variation from 0.25 - 2.0 lit/min and temper
ature variation from 15° - 4o°C had no effect on the
absorption efficiency (Table III).
Stability of collected sulphur dioxide samples:
Stability of collected sulphur dioxide in 0.01 M
MDH solution as well as 0.05 M sodium hydroxide solution
containing 2% glycerol were studied by preparing standard
sodium sulphite solution in 0.01 N MDH solution and 0.05 1'11
sodium hydroxide solution containing 2% glycerol respect
ively. The ali~ots of above solution were analysed for
30 days by proposed procedure. It was observed that
sulphite solution was stable forr-> 30 days in 0.01 M MDH
without any deterioration when kept in refrigerator,
whereas sulphite solution in 0.05 M sodium hydroxide
containing 2% glycerol was found to be very unstable at
room temperature (Fig, 2),
Effect of variables on colour development:
Acidity:
Acidity of the colour reaction was maintained in
two steps. It was found that the maximum colour intensity
was obtained when the acidity was adjusted to o.o2-o.16 M
with hydrochloric acid after the addition of p-aminoazo
benzene in the first step (Fig. 3) ar:d then 0.2-1.5 N
Page 12
flow rate
TJJ.JLE - II
El''r'ECT OF COt.CE.N'l'RATION OF Kill ON
AdSORPTION EFFICIENCY
•
Volume of air sampled in each case •
0. 75 lit/min
38.2 11 tres.
-----------------------------------------------------------S.No. Concen
tration MDH
so2 passed of
(}lg)
so2 found in * first impinger
(}.lg)
Absorption %
------------------------------------------------------------1. 0.005
2. 0.01
3. 0.02
4. 0.08
5. 0.1
4.0 8.0 12 .o 24.0
4.0 8.0
12.0 24.0
4.0 8.0
12.0 24.0
4.0 s.o
12.0 24.0
4.0 8.0
12.0 24.0
3.97 + 0.012 7.96 + 0.180
11.98..! 0.05 23.99 + o.oe
3. 94 .:!: 0 • 0 3 7.99 + 0.07
11. 99 ..! 0. 04 23.94 + 0.06
3. 99 .:!: 0.04 7.9<) + 0.04
11.94 + 0.03 23.98 + 0.06
3.96.:!: 0.01 7.98.:!: 0.015
11.97.:!: 0.054 23.80 + 0.020
3.95.:!: 0.015 7.87 + 0.031
11.89 + 0.06 23.96 + 0.07
99.26 99.60 99.84 99.97
98.70 99.90 99.98 99.76
99.80 99.94 99.50 99.92
99.20 99.84 99.76 99.20
98.80 98.40 99.10 99.84
------------------------------------------------------------* Mean of three repetitive analyses. In each case sulphur dioxide found in 2nd impinger was negligible.
Page 13
TABLE - III
FJ.o'n;CT OF TEI>iPEHATUHE ON AoSOHPTlON
EFr'ICIENCY
Ancentration of MOO - 0.01 M
Flow rate -1olume of air sampled in each case •
0. 75 11 t/min.
38.2 11 tres
--------------------------------------------------------S.No. Temper
ature oc
so2 passed so2 found in
first * impinger
().I g)
Absorption %
--------------------------------------------------------1. 15 10 9.96 + 0.021 99.60
20 19.99..! 0.028 99.96
30 29.97.! 0.049 99.90
2. 25 10 9.99 + 0.031 99.90
20 19.99 .± 0.054 99.95
30 29.99 + 0.019 99.99
3. 40 10 9.95 + Q.050 99.50
20 19.98 + 0.015 99.90
30 29.88 + 0.030 99.60
--------------------------------------------------------*Mean of four repi ti ti ve analyses.
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0.7......----------------------,
0.6r--
O.Sf-
E (j, 0.41-0 ..,., ~
w u ~ 0.3r-CD 0:: 0 C/l CD < 0.2f-
0. 11-
,...._ -..... --· -..... --......... _
-...... --,.._ ----- -
OL----~·----~L----~'----L-'--~'-----L-'--~'~--~ 5 10 15 20 25 30 35
NUMBER OF DAYS,S02 ANALYSED
FIG.2. STABILITY CURVE FOR S02 DIS SOLVED
0 0 IN MALONYL DIHYDRAZIDE (MDH)
------e IN GLYCEROL ALKALI
CONCENTRATION OF S02 LIBERA TED FROM
100J..tg ENDOSULFAN =l5.75J..tg/25ml.
40
Page 15
wlth hydrochlorlc acid after the addition of formaldehyde
solution in the second step (Fig. 4).
Ti•e:
It was noted that 20 minutes were needed for full
colour development and the pink coloured dye was stable
for ,.J 12 hours at 20 - 30°C,
Beer's law, Molar absorptivity and Sandell's sensitivity:
Beer's law is obeyed in the range of 25-150 pg
( 1 - 6 ppm) per 25 ml of standard endosulfan solution
(Fig. 5).
vity were
The molar absorptivity
found to be 4.8x104 lit
6 -2 0.008 pg em respectively.
Reoroduci bili ty of the method:
and Sandell's sensiti-
-1 -1 ( ) mol em .:!: 100 and
The reproducibility of the method was checked by
replicate analysis of a standard solution containing
100 pg per 25 ml (4 ppm) of endosulfan over a period of
7 days, The standard deviation and relative standard
deviation were found to be .:!: 0.0071 and .:!: 1. 5% respect
ively (Table IV).
Effect of foreign species:
Effect of foreign species commonly present with
endosulfan was studied to check the applicability of the
method. Other organochlorine pesticide, organophosphorus
pesticides, carbamates, organomercurials, ammonia, phenol,
nitrate and phosphate do not interfere with the reaction.
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o.er---------------------1 0.7
~
w u o., z <t IJ) 0. 3 a:: 0
:£ 0.2 <t
0.1
0.02 o.o, 0.06 0.08 0.10 0.12 0.1, 0.16 0.18 0.20
CONCENTRATION OF HYDROCHLORIC ACID, M
FIG.3. EFFECT OF ACIDITY ON COLOUR REACTION (AFTER THE
ADDITION OF P-AMINO AZO BENZENE ) -
CONCENTRATION OF ENDOSULFAN: 100}.lg/25mL
0.7'r-------------------------------------------.
E 0.6 c:: ~ 0.5 Lll .., w 0.4 u z ~ 0.3 a:: ~ 0.2 m <t 0.1
OL-~L---~--~--~---L---L--~--~--~L_----~ 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7
CONCENTRATION OF HYDROCHLORIC ACID,M
FIG.t.. EFFECT OF ACrDITY ON COLOUR DEVELOPMENT (AFTER
THE ADDITION OF FORMALDEHYDE)
CONCENTRATION OF ENDOSULFAN: 100)..lg/25mL
Page 17
: 0.&.--------------------------r
f 0.7
0.6
0.5
: . ' ) 0.4 , ~ )
' i· D 3 ~ . : ) l l ( 0.2
h
25
/, /,
I.
/ /
30
/
~ ;·
/ /
75
I I
/ t
I I
I
100
/
~ / .
125
I
/.A I
150
CONCENTRATION OF ENDOSULFAN IN ~g /25m!.
17 5
•
FIG.S.CALIBRATION DATA FOR THE DETERMINATION OF ENDOSULFAN.
A.~ CALIBRATION CURVE FOR ENDOSULFAN THROUGH
LIBERATION APPARATUS.
8. ·•----•·CALIBRATION CURVE FOR ENDOSULFAN AS
SOz x6.35 NOT LIBERATED THROUGH APPARATUS.
Page 18
TABLE _ IV
REPRODUCIBILITY Or' THE METHOD
Concentration of endosulfan - 100 pg/25 ml (4 ppm)
--------------------------------------------------------No. of days Absorbance,
505 nm --------------------------------------------------------
1 0.475
2 0.478
3 0.480
4 0.475
5 0.460
6 0.478
7 0.481
Mean "' 0.475
Standard deviation = + 0.0071
Relative standard deviation = .!: 1.5 %
--------------------------------------------------------
Page 19
Aramite and other sulphur containing compounds, which
easily liberate sulphur dioxide interfere with ttJe react
ion. Nitrogen dioxide does not interfere upto 8 ppm.
Reaction mechanism:
The sulphur dioxide (I) liberated from endosulfan
is absorbed in MDH to form hydrazino sulphinic acid (II)
similar to one obtained by the reaction between phenyl
bydrazine and sulphur dioxide (32,33).
Cl ( 1) H
H
Cl
Endosulfan
Phenyl hydrazine
H
---CH2 - 0
~ H CH2 - 0
"' I s~o
Hydrazino sulphinic acid
(II)
The sulphur dioxide absorbed in MDH is released
quantitatively by the addition of 0.1 J'll hydrochloric
acid, The released sulphur dioxide combines in situ with
p-aminoazobenzene (containing only one amino group) in
presence of formaldehyde to give pink coloured dye having
aminomethane sulphonic acid structure (III).
Page 20
(111) @- N-N-@- Nli2 + SOz + HCHO
p-aminoazo benzene l @- N • N - @ -NHCH2so 3H
(III)
CONCLUSION
Malonyl dihydrazide (MDH) as an absorbing medium
in combination with p-aminoazobenzene and formaldehyde
provides a simple, selective and reproducible spectre-
photometric method for the determination of endosulfan.
The pink coloured dye is stable for,....., 12 hours and has
the reproducibility. Method is free from the inter-
ference of most commonly associated foreign species,
hence can be applied for the determination of endosulfan
in environmental samples.
Page 21
CliAP'fER - VI li
PAR'f - B
A NO VEL TEST FOR THE DETECTION AND SEMiqUANTITATl VE
DETERMINATION OF ENOOSULFAN IN ENVIRONMENTAL SAMPLES
SUfo'.MARY
A fast and simple method for the detection and
semi quanti tat! ve determination of endosulfan ( thiodan)
is described. The method is based on the liberation of
sulphur dioxide from endosulfan and its subsequent esti
mation using zinc acetate, sodium nitroprusside and
malonyldihydrazide (MDH) to form a brick red coloured
sulphi to nitroprusside ion [ ~'e (CN) 5
Noso3
] 4-. The
intensity of the red colour is enhanced by the use of
zinc ion and malonyldihydrazide. This reaction has been
successfully applied to detect and semiquantitatively
determine the endosulfan as low as 0. 3 ppm in soil and
0.1 ppm in water.
Page 22
A NOVl::L TEST r'UR THE DE'n.:CTlOt. AND SOOQUAATITATI VE
LlETEi\Mlt.ATION Or' Ei'IOOSULF AJ'; lN El' Vl RONMl:l'lTAL SAl'!PLES
Endosul!an, a sul~hur containing chlorinated
pesticide is well known !or its toxicity. Its common
and frequent usage has rendered it ubi qui to us in the
environment. In view of its toxic effects on health,
vegetation and property, a fast, simple and reliable
method for the detection of traces of endosulfan would
be of imn,ense value. Various methods have been suggested
for its detection and determination in trace amounts.
Toxicity and methods of determination of endosulfan has
already beE'Il discussed in Part 'A 1 • Few methods for
the detection of E'Ildosulfan are cited in the litera-
ture (34-38),
In the present investigation a fast and simple
method for the detection and semiquantitative determina
tion of endosulfan based on the liberation of sulphur
dioxide from endosulfan is described. The subsequent
estimation of liberated sulphur dioxide is based on
Bodeker' s reaction for sulphite ion with sodium nitro
prusside (39,4o) in which a red colouration develops due
to the formation of sulphito nitroprusside ion
[Fe (CN) 5 NOS0 3 ]4
- (41). The intensity of the coloured
product in Bodeker reaction was found to increase by the
addition of metals such as zinc, cadmium, iron, nickel,
Page 23
copper and organic bases like hexamine, pyridine, 2-amino
pyridine, quinoline and thiourea (41-43). Later, this
reaction was used in the preparation of test papers for
detection of sulphur dioxide (44). Bourbon et al (45)
have used pyridine with theae reagents to tnhance the • colour reaction. o<:, oe bipyridyl, 1: 10 phenanthroline
and rv.re have been reported for the detection of sulphur
dioxide by Gupta et al (46-48) using similar method,
This reaction has now been successfully applied to
detect and semiquantitatively determine the endosulfan
as low as 0.3 ppm in soil and 0,1 ppm in water.
EXPEiUiv.ENTAL
Standard endosulfan solution (Tata !:"ison Ltd., India):
A standard solution containing 10 ~g/ml of endosulfan
was prepared in ethanol.
Acid reagent: Acid reagent was prepared as described
in Part A.
Malonyldihydrazide (l-1DH): Mil-l was prepared by the
reported method (30).
Test reagents:
1, Zinc acetate: 1 M zinc acetate solution was
prepared in 5% v/v glycerol.
2. Sodiuu; nitroprusside: 2;lt w/v solution of sodium
nitroprusside was prepared in demineralized water,
3. fvlDH solution: 2% w/v solution of MDi-! was
prepared in demineralized water.
Page 24
All reagents unless mentioned otherwise used
were of AnalaR grade.
Preparation of test solution:
Test solution was pre~ared fresh daily by mixing
equal volumes of each of the above mpntioned test reagents,
zinc acetate, sodium nitroprusside and MDH. A cream
coloured colloidal solution was formed.
Preparation of test papers:
Whatman No. 1 filter paper strips (1x5 ems) were
dipped in each of the reagents zinc acetate, sodium nitro
prusside and l'•lli serially. These papers were dried after 0
each dip in a temperature controlled oven at 50 - 60 C.
These papers were cream coloured and stable for few weeks
when kept in well stoppered dark brown bottles.
Procedure:.
50 g of finely ground soil or 100 ml water sample
was spiked with known amounts of endosulfan and extracted
twice with 2x50 ml petroleum ether (60-80°C) in a glass
bottle. The extract was decanted and combined in a sepa-
ratory funnel and washed with two portions of 200 ml of
water in a separatory funnel, discarding the water layer.
Extract was then dried over anhydrous sodium sulphate.
Suitable aliquot of the washed extract containing 10 pg
or above endosulfan was evaporated off in an impinger or
test tube and was tested for endosulfan in two ways: by
using test solution or using test papers.
Page 25
Using test solution:
Tbe endosulfan residue taken in an impinger, was
dissolved in 10 ml of ethanol. To it 1 ml of 2~ alcoholic
potassium hydroxide and 2 m1 of acid reagent were added.
The impinger was then kept on a water bath and connected
to a second impinger containing test solution kept out
side the water bath. The second impinger was then
connected to a source of suction. Temperature of the
water bath was raised to,......, 90° C and the air was drawn
through the second impinger. The colour of colloidal
test solution turned from cream to brick red due to
evolved sulphur dioxide indicatin&the presence of endo
sulfan in the residue.
Using test paper:
The endosulfan residue taken in a test tube was
dissolved in 10 ml of ethanol. To it 1 ml of alcoholic
potassium hydroxide and 2 ml of acid reagent were added.
The test tube was then kept on a water bath (,...., 90°C).
The test paper when kept on the mouth of the test tube .
turned from cream to brick red immediately, which indi-
cated the presence of endosulfan in the residue.
For semiquantitative determination J the colour
produced in the test solution or test paper was compared
with that of obtained from the standard solution contain
ing 10 - 60 pg of endosulfan following the same procedure.
.....
Page 26
ttESULTS AND DlSCUSS!Ot.
The brick red colloidal :;elution was stable
for more than 24 hours. The pH of the colloidal solu
tion after colour formation was found to be between 5.5
and 5.8. Increase in intensity of the colour by addition
of MDH was supposed to be due to the formation of
[ Zn (MDH) x) 2 Fe (CN) 5 No.so3 J (48). Brick red preci
pitate was not soluble in organic solvents like chloro
form, carbontetrachloride, benzene, butanol, methyl
acetate, etc. The coloured colloidal solution was either
decomposed or remained undissolved in these solvents.
Semiquantitative detern1ination of endosulfan in
soil or water is possible by comparing the colour of
test solution produced by different known amounts of
endosulfan with that produced by the unknown following
the above procedure.
Other organochlorine pesticides, organophosphorus
pesticides, carbamates, chloride, ammonia, nitrate and
phosphate do not interfere with this reaction. Aramite . and other sulphur containing aromatic compounds which
easily liberate sulphur dioxide, interfere with this
reaction.
Page 27
OON eLUSION
The proposed method is fast, simple and reason
ably sensitive for the determination and semiquantitative
determination of endosulfan. The method is also found
to be free of co-pollutants and can be successfully
applied for the detection or semiquantitative determi
nation of endosulfan in environmental samples.
Page 28
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