Spectrophotometric Determination of Kelthane in Environmental Samples

Sensitive spectrophotometric method for determination of kelthane in sub parts per million level is described, which is based on Fujiwara reaction. Kelthane on alkaline hydrolysis gives chloroform, which can be reacted with pyridine to produce red colour. The colour is discharged by addition of glacial acetic acid. Then Benzidine (4,4’-Bianiline) reagent is added due to which a yellowish-red colour is formed which has an absorption maximum at 490nm. Beer’s law is obeyed in the range of 3.3 26.0 μg (0.13 1.04 ppm) of Kelthane per 25ml of final solution. The molar absorptivity and Sandell’s sensitivity were found to be 4.32 × 10 L·mol·cm and 0.022 μg·cm respectively. The method is found to be free from interferences of other organochlorine pesticides and various co-pollutants and can be successfully applied for the determination of kelthane in environmental samples.


Introduction
Kelthane is a well known acaricide of organochlorine group of pesticides, chemically it is known as 4-4'dichloro-alpha trichloromethyl benzhydrol [1] Kelthane appears to be effective against a wide range of mite species and is a well known miticide.It is also effective against tetrachid, mites, cydamen, broad, mites, European red spider, apple-rust, cherry-rust, tomato-rust, and various other fruits and vegetable rusts [2].
Field studies indicate that dicofol persists in soil for at least four years after application.The residue of kelthane accumulates in rotational crops.Due to very long persistency of residue of this material, the use of kelthane is recommended on slow growing crops, i.e. citrus fruits.It has been proved that kelthane is a sever irritant [3,4].The National Cancer Institute suggests the possibility of kelthane as oncogen.The toxic effect of kelthane shows general weakness, comma, affects sex hormones, inhibition of aromatose activity and death in animal.It is a contact herbicide with initial toxicity.It has a moderate acute oral toxicity.The oral LD 50 in rat is 809 mg/kg and 1870 mg/kg body weights for rabbit [2].The tolerance level of dicofol in vegetable is 1 mg/kg [5][6][7].
Several instrumental techniques i.e.GLC with Electron Capture detector [8], Voltammery [9] Neutron activation analysis [10], Gas chromatography [11], Liquid chromatography [12], matrix solid-phase dispersion [13], solid-phase extraction [14], and Spectrophotometry [15][16][17][18] are available in literature, but most of these techniques are costly and require trained staff.Spectrophotometry is a simple, sensitive rapid and versatile technique for quick determination of analyte.A few spectrophotometric methods based on the hydrolysis of kelthane to chloroform and determination of chloroform by Fujiwara method [15][16][17][18] are available, but all these methods require specially constructed apparatus and have poor sensitivity than the present method.
In the present a simple and more sensitive method is developed for the determination of kelthane.The reagent used in the present method is benzidine which increases sensitivity of the Fujiwara reaction.The method has been successfully applied for the determination of kelthane in environmental samples.

Experimental
Apparatus.A Systronics spectrophotometer 104 with 1 cm matched quartz cell was used for all spectral measurement.A Systronics pH meter model 335 was used for pH measurement.
Reagents.All the reagents were of A.R./G.R.grade and double distilled deionised water was used throughout the study.
Stock solution of kelthane.(Tropical Agro system India Ltd.) 1 mg/ml solution or kelthane was prepared in alcohol.Working standards were prepared by appropriate dilution of the stock solution with alcohol.
Procedure.An aliquot containing 2.0 -30 µg of kelthane was taken in a 25 ml-graduated tube.The solution was evaporated off up to 0.5 ml on a water bath.To this 1ml of pyridine and 2 ml of 5 M NaOH were added and thoroughly shaken.The contents were kept in water bath at 70˚C -75˚C for ~3 min.and shaken time to time.The yellowish-red colour solution obtained was cooled in ice-cold water bath and then decolourised with 2 ml glacial acetic acid.To this yellow colour solution 2 ml of 1% benzidine and 1 ml of 10 M HCl were added and the content was allowed to stand for 10 minute.The volume was made up to the mark and the absorbance of the yellowish-red coloured dye was measured at 490 nm against a reagent blank.

Colour Reaction of Kelthane
The reaction was supposed to take place in four steps.
2) In this step chloroform reacted with pyridine in alkaline medium to form Schiff's base of glutaconic aldehyde (II).
3) In the third step, by addition of glacial acetic acid, the pink colour of Schiff's base of glutaconic aldehyde was converted in to the yellow coloured glutaconic aldehyde (III).
4) Yellow coloured Glutaconic aldehyde formed a purple red coloured polymethine dye (IV) with benzidine reagent in the fourth step (Mechanism 1).

Results and Discussion
Spectral Characteristic.All the spectral measurements were carried out against, reagent blank which showed negligible absorbance at 490 nm (Figure 1).
Effect of reagent concentration. 1 ml of pyridine and 2 ml of 5 M NaOH were required for maximum colour intensity.Excess of NaOH made the solution slightly turbid. 2 mL of acetic is necessary for decolourisation of the red colour.Excess amount, however, does not affect the reaction.A minimum of 2 ml of benzidine was required for maximum colour intensity.Excess amount of benzidine decreases the colour intensity (Figure 3).Effect of time and temperature.It was observed that heating the reaction mixture for a ~3 minutes in a water bath at 70˚C -75˚C gave maximum and constant absorbance value.The purple red colour dye was found to be stable for ~10 min.and thereafter showed gradual decrease in intensity with increasing time.(Figures 4 and 5).
Effect of pH.Maximum absorbance of the dye was observed when pH of the final solution was between 3 and 4.
Precision.The precision of the method was checked by seven replicate analysis containing 25 µg kelthane per 10 ml of final solution.The standard deviation and relative standard deviation were found to be ±0.0033 and ± 0.53% respectively.
Effect of foreign species.The validity of the method was assessed by investigating the effect of various co pollutants and polyhalogenated compounds on the determination of Kelthane by the developed method, by adding a known amount of these compounds to a solution containing 25.0 µg Kelthane per 25 ml of the final solution.The tolerance limit in ppm of interfering species was established, as the concentration required for causing an error of not more than ±2.0% in the absorbance for Kelthane.The Results of these experiments are shown in Table 1, which showed that the method was found to be free from interference of various polyhalogenated compounds and metal ions, commonly found in the described samples.Trichloroacetic acid and chloroform gave positive interference.N-hexane and petroleum ether extracts from the vegetables and other samples have no interference.

Application
In Water Sample.100 ml of kelthane free water sample was taken and fortified with known amounts of kelthane   and kept for 3 -4 h.. Then kelthane was extracted in n-hexane.Hexane was evaporated off and kelthane was determined by the present as well the reported method [18].The recoveries are shown in Table 2.
In Milk Sample.To assess the applicability of the method for the determination or kelthane in milk samples, known amounts of dicofol were added to the milk sample.
Kelthane was extracted in n-hexane as reported [6] and determined by present as well as reported method [18].The recoveries are shown in Table 2.
In Vegetables and Fruits Samples.Various vegetables and fruits samples such as tomato, beans grapes were weighed, crushed and then spiked with known amounts of kelthane and kept for 3 -4 h.Kelthane was extracted in n-hexane.Hexane was evaporated off and kelthane was determined by the present as well as reported method [18].The recoveries are shown in Table 2.
The comparison of the present method with other reported [15][16][17][18] method is shown in Table 3.

Conclusions
The data shown in Tables 2 and 3 clearly indicate that the present method is simple, rapid and more sensitive than other reported method for determination of kelthane.It can be successfully applied for the determination of kelthane in various environmental samples.

Figure 3 .
Figure 3.Effect of the concentration of benzidine solution.

Table 2 . Recoveries of kelthane in various environmental samples.
*Mean of three replicate analysis; a Size of sample 100 ml.b Size of Sample 50 gm.

Table 3 . Comparison of the proposed method with other spectrophotometric method.
Method is simple, more sensitive free from inter-ference of other chlorinated hydrocarbon.