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A SIMPLE METHOD FOR THE SPECTROPHOTOMETRIC DETERMINATION 0 ATRAZINE USING P-AMINOACETOPHENONE AND ITS APPLICATION IN ENVIRONMENTAL AND BIOLOGICAL SAMPLES

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A SIMPLE METHOD FOR THE SPECTROPHOTOMETRIC DETERMINATION 0 ATRAZINE USING P-AMINOACETOPHENONE AND ITS APPLICATION IN ENVIRONMENTAL

AND BIOLOGICAL SAMPLES

INTRODUCTION

Atrazine [2 - chloro- 4 ethylamino - 6- isopropylamino - S - triazine] is a widely used,

selective, pre and post emergence herbicide. Atrazine is effective against broad leaf and grassy weeds in many crops including corn, coriander, sugarcane, sorghum, orchards, vineyards and turf grass sod. It is also used for selective weed control in conifer restoration and christmas tree

plantation. For vegetation control in noncrop land atrazine is used as a non-selective herbicide. Atrazine has the following structure ( 1-6)

N C( )NHCH (CH,),

N N v C,H,NH

ATRAZINE

Atrazine is one of the most important and widely used herbicides at present and its residues has been found in many surface and ground water in the world. The effect of atrazine to human and ecosystem has become a focal point of issue. Atrazine has very low water solubility and high binding energy to colloidal soil particles. It penetrates into plant via their roots and leaves therefore it may be applied before the emergence of weeds and to their shoots, spraying them with a suspension (2,7-9).

Atrazine shows phytotoxic effects which include decomposition of chloroplast, inhibition of water photolysis, diminishing the carbohydrate content, supression of tissue respiration, change in the enzyme activity, inhibition of photosythesis and blocking of Hill reaction ( 10,11 ).

Atrazine is toxic to man and animals. It is carcinogenic to humans and produces genotoxic, mutagenic and embryotoxic effects ( 12-14). It produces strong eye irritation including edema of eyelid and conjunctiva. During its exposure weakness, lethargy, anorexia, diarrhea, cardiac arrest, myasthenia, spasticity of muscles of mouth and limbs are reported ( 2,6). Sublethal

effects of atrazine in amphibians showed kidney damage, degenerative changes at proximal

muscles, inflammatory cellular infiltrates in liver and massive increase of melanomacrophages

in liver and lung (15).

Its inhalation and dermal exposure can occur during manufacture and use. It has been

detected in surface water and ground water at levels that exceed U.S. Environmental Protection

Agency's 3 mg I L limit for drinking water (6, 16,17) and affecting aquatic flora and fauna. A work

place airborne control limit (TLV) of 5 mg 1m3 has been recommended by ACGIHC (6). Its tolerance

level in fruits and vegetables is 0.1 mg I kg. (2). The acute oral LD50 for rats proposed for

atrazine is 3080 mg I kg ( 1 , 9).

116

A SIMPLE METHOD FOR THE

SPECTROPHOTOMETRIC DETERMINATION OF ATRAZINE

USING P- AMINOACETOPHENONE AND ITS APPLICATION

IN ENVIRONMENTAL AND BIOLOGICAL SAMPLES.

SUMMARY

A sensitive spectrophotometric determination of the widely used herbicide

atrazine is described. Atrazine reacts readily with pyridine to from a quaternary

halide, which in the presence of alkali gives a carbinol base by addition of hydroxyl

group. The resulting carbinol further undergoes breaking of hetrocyclc ring forming

glutaconic aldehyde. This glutaconic aldehyde is subsequently coupled with

p-aminoacetophenone (P AAP) to give a yellow orange polymethine dye. Beer's law is

obeyed in the range of 0.16 to 1. 6 ppm of atrazine at 470 nm. The method is simple,

sensitive and free from the interference of other common pesticides and ions. The

analytical parameters have been optimised and the method has been successfully

applied to the determination of atrazine in environmental and biological samples.

Part Communicated in TALANTA (U.S.A.) MSS. No. C 97289

115

Because of wide application and toxicity of atrazine a large no of instrumental methods

such as Gas chramatography I Mass spectrometry ( 18-20 ), HPLC (21-23), TLC (24-25), Liquid chomatography (26), Flow injection analysis (9,27,28) Enzyme Immune assay (29-31) Polarography (32), Partial least squares method (33) etc. are reported for its determination. Few spectrophotometric methods (34-37) are also reported for the determination of atrazine. The reagents used are p-aminobenzoic acid (34), ethylcyano acetate (35), picric acid (36) etc.

In the proposed method a simple and sensitive method, based on Koing's reaction is discussed for the determination of atrazine. Atrazine is first reacted with pyridine and converted into a quaternary pyridinium halide, subsequently it adds a hydroxyl group, in the presence of alkali to from a carbinol base. In the second step a glutaconic aldehyde is formed due to breaking of the heterocyclic linkage. Finally the glutaconic aldehyde couples with p-aminoacetophenone in an acidic medium to form a yellow-orange polymethine dye which showed maximum absorbance at470 nm. Different analytical parameters e.g. time, pH, reagent concentration and temperature have been investigated for optimum sensitivity. The method has been successfully applied for the determination of atrazine in grains, fruits, vegetables, soil, water and biological samples.

EXPERIMENTAL

Apparatus

A UV-Vis Spectrophotometer Model108 with matched silica cells was used for all spectral measurements. pH measurements were made with Systronics pH meter model 331.

Reagents

All chemicals used were of AnalaR grade or similar grade. Demineralised, double distilled water was used throughout the experiment.

Atrazine (Northern Minerals Ltd.)

A stock solution of 1 mg ml·' was prepared in methanol. A working standard of 10 flg ml·1

was prepared by appropriate dilution of the stock solution with water.

Pyridine reagent (BDH}

3 ml of concentrated hydrochloric acid was mixed with 18 ml of freshly distilled pyridine and 12 ml of water was added to it (34).

P-aminoacetophenone (PAAP} (Ferak Berlin}

A 1% ( w/v) solution was prepared in 1:4 hydrochloric acid.

Sodium hydroxide

A 2 M aqueous solution ~as used.

117

PROCEDURE

An aliquot of standard solution containing 4 to 40 ~g of atrazine was taken in a graduated tube.To tt 0.2 ml of pyridine reagent was added. This solution was placed on a boiling water bath for 15 min and then allowed to cool at room temperature. Now 1 ml of 2 M sodium hydroxide and 2 ml of p-aminoacetophenone solutions were added. The solution was kept for 5 min for complete colour development and made up to the mark with water. The absorbance was measured at

470 nm against demineralised water as reference.

RESULTS AND DISCUSSION

Spectral characteristics

The colour system shows maximum absorption at 470 nm, whereas the reagent blank has

negligible absorbance at this wave length. (Fig.1)

Adherance to Beer's Law, Molar absorptivity and Sandell's sensitivity

The colour system obeys Beer's law over the range of 4 to 40 ~g of atrazine per 25 ml final solution (0.16 to 1.6 ppm) at 470 nm. (Fig.2). The molar absorptivity and Sandell's sensitivity were found to be 1.1 x 105 (± 100) mol·1 cm·1 and 0.001 ~gem·' respectively.

Effect of reagent concentration

The effect of various reagent concentrations on the colour reaction was studied. It was found that under optimum conditions 0.2 ml of pyridine reagent and 1 ml of 2M sodium hydroxide solution were required for the reaction. It was observed that if the proposed reagent concentration is increased, absorbance value is decreased. It was found that 2 ml of p-aminoacetophenone was sufficient for complete colour development. (Fig. 3, 4)

Effect of time and temperature

Maximum colour intensity was observed when the solution containing pyridine reagent was heated for 15 min on a boiling water bath and then allowed to cool at room temperature. The coloured dye was found to be stable for about 90 min in the temperature range of 15 to 35° C. (Fig. 5)

Effect of pH

The effect of pH on colour reaction was studied and it was found that the polymethine dye was formed only under acidic condition. The pH of final solution was 2 to 2.5.

Effect of foreign species

The effect of various common species and other pesticides on the determination of atrazine was studied to assess the validity of the method. Known amount of foreign species and pesticides

were added to a standard solution containing 10 fig of atrazine per 25 ml prior to analysis and

solution was analysed by the proposed method. The tolerance limit of other pesticides and ions are listed in Table 1.

118

Precision

Precision of the method was checked by analysing 10 fig of atrazine in 25 ml of final solution for a period of 7 days. The standard deviation and relative standard deviation were

found to be ± 0.006 and 3.0 % respectively.

Colour reaction

The probable colour reaction is shown in scheme A and can be explained mainly in three steps-

1. Atrazine is reacted with pyridine and converted into a quaternary pyridinium halide. 2. Pyridinium halide furher undergoes addition of hydroxyl group in the presence of

alkali to form a carbinol base, which further undergoes braking of heterocyclic ring forrning glutaconic aldehyde.

3. Glutaconic aldehyde couples with p-aminoacetophenone in acidic medium to form a yellow orange polymethine dye.

APPLICATION

The proposed method has been satisfactorily applied for the determination of atrazine in various environmental and biological samples.

Determination of atrazine in plant materials

Different samples of fruits and vegetables, free from atrazine were taken. The samples were weighed, crushed and spiked with known amount of atrazine and kept for one day. The samples were washed with 100 ml of water. From this water atrazine was extracted with two 10 ml portions of chloroform. The chloroform extract was evaporated to dryness and the residue was dissolved in 25 ml of methanol. Aliquots were taken and analysed by the proposed and the reported method (34) . Table 2

Determination of atrazine in grains

Since the grain samples obtained from the market were found to be free of atrazine, known amount of atrazine was added to the samples to check the recovery. The samples were kept for one day and then atrazine was extracted with two 10 ml portions of chloroform. The chloroform extract was evaporated to dryness and the residue was dissolved in 25 ml methanol.

Aliquots were taken and analysed by the proposed and the reported method (34) .Table 2

Determination of atrazine in soil samples

Varous soil samples were collected from an agricultural fields where atrazine was sprayed as herbicide. Samples were weighed and finally ground. These samples were washed with two 10 ml portions of methanol. The washings were collected and made up to 25 ml with methanol.

Aliquots were then analysed as described above. Table 3

119

Determination of atrazine in water

Water samples were collected from an agncultural fields where atrazine was sprayed. The samples were extracted with two 10 ml portions of chloroform. The extract was then evaporated

to dryness and the residue was dissolved in methanol (25 ml). Aliquots were then analysed as

described above. Table 3

Determination of atrazine in biological samples

Since the presence of atrazine in blood and urine is reported ( 38 ), synthetic samples were prepared by adding known amount of atrazine to 2 ml aliquot of blood or urine. Samples

containing known amount of atrazine were taken and deproteinzed by adding 2 ml of 1 % trichloroacetic acid (39). After 25 min the mixture was centrifuged and the supernatant solution

was transferred in to a 25 ml graduated tube and analysed as described before. The results of the anlysis are shown in the Table 4. These are in agreement with the reported method. (38)

CONCLUSION

The present methods provides a simple and rapid spectrophotometric procedure for the quantitative determination of atrazine having high sensitivity. The method is fairly reproducible

and free from the interference of a large number of foreign species. The method has also been compared with recently reported spectrophotometric method (34) and found to be more sensitive.

The proposed method can be applied for the determination of atrazine in various enviranmental and biological samples.

120

(I)

(II)

(Ill)

Tr- Cl + (~ Atrazine Pyridine

Tr- 'N8 + NaOH

Quaternary pyridinium

halide

- Tr-'N8 + Cl-

Quaternary pyridinium halide

OH

n"O Carbinol base

l NaOH

Tr - N = CH - CH = CH - CH = CHOH

Tr- N = CH- CH = CH - CH = CHOH

Glutaconic aldehyde l +

Glutaconic aldehyde

NH2

p-aminoacetophenone

Tr- N = CH - CH = CH - CH = CH - NH -0 COCH3

Polymethine dye

(Yellow- Orange)

(A. max- 470 nm)

SCHEME A. COLOUR REACTION OF ATRAZINE

121

w 0 z <( <Il 0:: 0 rn <Il <(

E c:

0 1'-

" w-0 z <( <Il 0:: 0 rn <Il <(

0.9

08

07

0.6

05

04

A 03

0.2

0.1 B

oL_~~~~~~~~~~~~~~~~c~

0.9

0.8

0.7

0.6

05

0.4

0.3

0.2

0.1

390 400 410 420 430 440 450 460 470 480 490 500 510 520 530

WAVELENGTH, nm A- CONCENTRATION OF ATRAZINE- 32 "g /25 ml B- CONCENTRATION OF ATRAZINE- 16" g /25 ml C - REAGENT BLANK

FIG 1 -ABSORPTION SPECTRA OF THE DYE AND REAGENT BLANK

8 16 24 32 40

CONCENTRATION OF ATRAZINE- J.L9 /25 ml

FIG 2- CALIBRATION DATA FOR THE DETERMINATION OF ATRAZINE

122

AMOUNT OF p- AMINOACETOPHENONE , ml, B

05 1 5 2 25 3 35 4 4.5 06

05 8

E c

0 04 "-... w u

03 z A <( 00 (Y

0 A (/) 02

00 B <(

01

0 0 0.5 1.5 2 2.5 3 3.5 4 4.5

AMOUNT OF 2 M SODIUM HYDROXIDE, mi,A

CONCENTRATION OF ATRAZINE • 24 ~g 125 ml

FIG. 3 EFFECT OF AMOUNT OF ALKALI AND p- AMINOACETOPHENONE ON COLOUR REACTION

0.9

0.8 E c 07

0 "-... 06 w u

05 z <( 00 (Y 04 0 (/)

03 00 <(

0.2

01

0 0 0.2 0.4 0.6 0.8 1.2 1.4

AMOUNT OF PYRIDINE REAGENT- ml

CONCENTRATION OF ATRAZINE- 241'9125 ml

FIG. 4- EFFECT OF AMOUNT OF PYRIDINE REAGENT ON COLOUR REACTION

123

08

07 f-

E 06 f-c

0 -r-- 0.5 " w· (_)

0.4 z <>: m cr 03 -0 (/) m <>: 0.2

0.1 -

0 ' ' I I ' ' 0 5 10 15 20 25 30 35 40 45 50

TEMPERATURE, oc CONCENTRATION OF ATRAZINE- 24 "g /25 ml

FIG. 5- EFFECT OF TEMPERATURE ON COLOUR REACTION

124

• •• A-

TABLE -1

Effect of foreign species (Concentration of atrazine 10 J.19 /25 ml)

Foreign Tolerance Foreign Species limit* Species

(ppm)

Ethanol 2000 Mg", Ca"

Carbaryl, 1500 AI+++ , Cd++, Propoxur Cu++ , Fe++ , Fe-+-++ Paraquat, 750 PO-

4 ,N0

3-

Parathion so--4

Aldrin 500 BHC,Dimethoate 400 Kelthane 250 Phenol 200 Formaldehyde 80

Ammonia 50

Tolerance limit* (ppm)

700

300

200 50

* The amount causing the error of ± 2 % in absorbance value.

TABLE- 2

Application of the method for the determination of atrazine in plant materials and grain.

Sample Amount of Amount of atrazine atrazine Found* (J.!g) Added* (J.!g)

[A 1 Pine- 5 4.89 apple** 10 9.90

15 14.85

Potato** 5 4.91 10 9.80 15 14.82

Corn* 5 4.90 10 9.88 15 14.80

Mean of three replicate analysis . Amount of sample- 25 g Proposed method, B- Reported method (34).

125

[ B 1 4.80 9.70

14.50

4.85 9.50

14.30

4.75 9.88

14.60

Recovery %

[A 1 [ B 1 97.80 96.00 99.00 97.00 99.00 96.00

98.20 97.00 98.00 95.00 98.80 95.33

98.00 98.00 98_80 98.80 99.00 97.33

Table 3

Application of the method for the determination of atrazine

in water and soil.

Sample Atrazine Atrazine

originally added

found* (119) (119) (a) (b)

Water•• 4.14 5

Soil***

• •• •••

4.45 10

4.46 15

3.00 5

3.96 10

3.97 15

Mean of three replicate analysis .

Amount of water sample - 100 ml

Amount of soil sample - 50 g

Total

atrazine

found*(l19) (c)

9.09

14.10

19.16

7.75

13.80

18.77

Table 4

Difference Recovery

%

(c-a) x 100

(c-a) b

4.95 99.00

9.65 96.50

14.70 98.00

4.75 95.00

9.84 98.40

14.80 98.60

Application of the method for the determination of

* ••

atrazine in biological samples.

Sample Atrazine added* (119 )

Blood** 5 10 15

Urine** 5 10 15

Mean of three replicate analysis. Amount of sample - 2 mi.

126

Atrazine found* (119 )

4.90 9.81

14.89

4.85 9.80

14.91

Recovery %

98.00 98.10 99.26

97.00 98.00 99.40

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127

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128

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