Nano Scale Potentiometric and Spectrophotometric Assays for 2,4-Dichlorophenoxyacetic Acid

2,4-D is one of the most widely used herbicides in the world and is characterized as low-cost, quite efficient even at low concentrations, and moderately hazardous (class II) according to the World Health Organization [1] Although this herbicide easily moves though the soil, its leaching into the groundwater can be minimized due to the degradation by microorganisms and also the absorption by plants. Its primary route of degradation in the environment is by microorganisms, which increases with temperature, humidity, pH, and organic matter content [2,3]. Microbial degradation is a possible route for the breakdown of 2,4-D, but it is very dependent on the characteristics of the water. Laboratory studies have shown that in warm, nutrient rich water that has been previously treated with 2,4-D microbial degradation can be a major factor for dissipation [4]. Residues of 2,4-D and its salts or esters in water are commonly measure by extraction, chemical derivatization, separation by gas-liquid chromatography and electron capture detection [4]. Other methods used in the determination of 2,4-D residues include high-performance liquid and thin-layer chromatography [5-7].


Introduction
2,4-D is one of the most widely used herbicides in the world and is characterized as low-cost, quite efficient even at low concentrations, and moderately hazardous (class II) according to the World Health Organization [1] Although this herbicide easily moves though the soil, its leaching into the groundwater can be minimized due to the degradation by microorganisms and also the absorption by plants. Its primary route of degradation in the environment is by microorganisms, which increases with temperature, humidity, pH, and organic matter content [2,3]. Microbial degradation is a possible route for the breakdown of 2,4-D, but it is very dependent on the characteristics of the water. Laboratory studies have shown that in warm, nutrient rich water that has been previously treated with 2,4-D microbial degradation can be a major factor for dissipation [4]. Residues of 2,4-D and its salts or esters in water are commonly measure by extraction, chemical derivatization, separation by gas-liquid chromatography and electron capture detection [4]. Other methods used in the determination of 2,4-D residues include high-performance liquid and thin-layer chromatography [5][6][7].
Several methods have been reported for the analysis of 2,4-D in food and environmental samples. These methods include gas chromatography (GC) [8][9][10]. High-Performance Liquid Chromatography (HPLC) and UV-spectrophotometry [11,12]. However the chromatographic techniques are expensive and not available in many quality laboratories worldwide. Spectrophotometry is probably the most convenient analytical technique for routine analysis because of its inherent simplicity, low cost and wide availability in quality control laboratories [13].

Instrumentation
The electrochemical measurements were carried out with HANNA instrument 211 (EZODO) for measuring pH. Sensitive balance model AG204 (METTLER TOLEDO) was used for measurements. Spectral measurements were carried out by using a single beam UV (OPTIMA) SP-SPECTROPHOTOMETER, with quartz cells of 1 cm optical path length.

Stock standard solution of 2,4-D
An accurately 0.01 M solution was prepared by dissolving 0.212 g of 2,4-D standard in 25% ethanol, transferred into a 100 mL volumetric flask and diluted to the mark with 25% ethanol and mixed well. This stock solution was further diluted with 25% ethanol to obtain working solutions in the ranges of 10 -3 :10 -11 M, reservation solutions in brown bottles inside the refrigerator.

Construction of wire ion selective electrode
Membrane was prepared by mixing well of 96% sodium silicate, 1% active coal, 3% dioctylephethalate with 1 cm of fusible polyethylene tube. The mixture was poured into 3 cm diameter petri dish with 10 ml tetrahydrofuran (THF). A pure silver wire electrode was dipped in the coating membrane solution several time, allow the film to dry for about five min repeat the process until 2.0 mm diameter. Wire electrode was soaked about 3 h in 1 × 10 -3 M of 2,4-D. All potentiometric determination were performed using the analytical cell, Ag /membrane/2,4-D test solution//Reference electrode.

Electrode calibration
Connect the wire electrode to the pH meter in the presence of reference electrode. The calibration of the electrode was preceded using standard solutions of 2,4-D ranging from 10 -7 :10 -2 M.
Separate and spiking addition method was used by the sequence from low to a higher concentration. The measured potential was plotted against logarithm of 2,4-D concentration after one min ( Figure 1). The electrode was washed with distilled water and dried with tissue paper and back to base line by immersing in water between measurements, stored in a dark place immersed in 10 -5 M.

Response times
Calibration curve was measured from the lower (10 -7 M 2,4-D) to the higher concentrated (10 -2 M of 2,4-D) solutions the time traces of the calibration curves of silicate electrode was represented in Figure 2.

Selectivity
Selectivity coefficients K pot 2,4-D of the electrodes towards different species were determined by the standard addition technique method [14] in which the following equation was applied where a`A known activity of primary ion, aA fixed activity of primary ion and aB activity of interfering ions. The results are appear by potting mV in the presence of different interference ( Figure 3).

Effect of pH
The effect of pH of the potential of electrode was investigated. The potential was measured at a 2,4-D concentration solution (10 -5 M) from the pH value of 3 up to 9. The solution was acidified by the addition of very small volumes of 0.2 N HCl then the pH value was increased gradually using 0.2 N NaOH for each pH value, two pH/mV meters were used to measure the potential. The potential was recorded and plotted against different pH values ( Figure 4).

Analytical application
Growing watercress: Watercress seeds were germinated in 250 gm clay dry soil with about 3-10 seeds per pot, after two weak. The temperature of the plants was maintained between 20-25°C.    them in a 50 ml ethanol half an hour. Ethanol is a water-miscible solvent, removes all pigments from soft-leafed. The blank solution was prepared by 1 ml of chlorophyll extract and 11 ml distilled water and sample stock solutions were prepared by mixing aliquots of different concentration (10 -11 , 10 -5 and 10 -3 ) of 2,4-D were precisely pipette into 10 ml distelled water and 1 ml chlorophyll extract. The absorbance of solutions was measured at 286, 440, 662 nm and plotted against different concentration of 2,4-D in sample solutions ( Figure 10).

Time considerations:
Effect of time on the absorbance of watercress leaf chlorophyll extract with 1 ml different concentration of (10 -11 , 10 -5 and 10 -3 M) of 2,4-D solutions was determined and plotted ( Figure 11).

Result and Discussion
2,4-D was classified among the phenoxy acid herbicides MCPA as a class-2B carcinogen possibly carcinogenic to humans. The movement of auxin, (2,4-D) is a common herbicide,. Denmark, Norway, Kuwait and the Canadian provinces of Québec and Ontario, 2,4-D use is severely restricted in the country of Belize [15].
2,4-D concentration is one of the affecting factor to the rate of reaction, so that affects to the performance of silicate ion selective electrode. It has been found that separate technique is the best way to apply Nernst equation. Response characteristics of the silicate ion selective electrode appear in Table 1, the results showed that the highest sensitivity is 42.1 ng/ml. The purpose of this study was to investigate the working electrode conditions to obtain the maximum performance. The research studied of the electrode character was pH and response time. The pH optimum of 2,4-D at a range from 2.5 to 5.5. Response time is a measure of the rate of degradation reaction of 2,4-D. The response time with concentration higher than 10 -4 M were of the order of 15 seconds. The silicate electrode has good selectivity to 2,4-D, in the presence of double-charged metal ions, as Fe +2 and Glutamic acid, and has poor sensitivity in the presence of Na 2 HPO 4 .
The residual pesticide was decreases to 1.005, 0.663, 0.221 μg ml -1 , after two, three, and five days respectively from the original solution (2.21 μg ml -1 ) added on the pots of watercress. The number of plants did not effect on the residual of the pesticide concentration. The residual of 2,4-D degradation of (10.6 μg ml -1 ) by the temperature effect was varied from -5 to 43°C (Figure 5), the large amount of residual of 2,4-D was at 43°C, for a period of time following treatment. It is therefore to be expected that temperature may influence the rate of degradation response and govern to some extent, Plants that put out the pesticide at low temperature longer than the age of the plants that put them at a temperature of exterminator other heat. This research study reports the concentrations of residual 2,4-D (pH 2.5-9) taken up by watercress grown. This study showed that the pH 4 of 2,4-D solution is the lower pesticide absorption by soil and watercress plants [16,17].
It is very important to see the effect of different solvent as methanol, ethanol, 1,2-dichloroethane and 25% ethanol on the photodegradation of 10 -11 M of 2,4-D (Figure 1). It is observed that the degradation of 2,4-D in the ethanol was the maximum and the degradation in methanol was the minimum. The velocity of photodegradation of 10 -3 M of 2,4-D in 25% ethanol is very high (Figure 3).  chain. This is followed by ring cleavage and degradation to produce aliphatic acids such as succinic acid. The rate of microbial degradation is dependent upon the water potential 17, the absorbance increased and obeyed Beer's law from 10 -5 :10 -2 M. The molar absorptivity coefficient equal to 1000 l mol -1 cm -1 . Pigment content of chlorophyll were increased after adding , the reason is photodegradation of 2,4-D in this region.10 -3 M < 10 -5 M < 10 -11 M of 2,4-D ( Figure 10) at 286,440 and 662 nm. The absorption decreasing with the effect of time ( Figure 11).

Conclusion
Silicate wire electrodes was suitable for the determination of 2,4-D with regard to working concentration range, slope, pH range, response time, and selectivity . The electrodes exhibited good reproducibility over a useful lifetime of 2 months. Spectrophotometric study allows understand 2,4-D toxicity and it's excellent deleterious effect. The reproducibility, recovery, and operational simplicity of the methods make them suitable to determined 2,4-D. The detection limit was 2.21 μg ml -1 .