The objective of this study is to develop a simple and economic spectrophotometric method for the quantitative determination of atrazine. This method is based on the complexation of atrazine derivatization (dechlorinated atrazine [DA]) with a mixture of formaldehyde and ketone compound, as described by Mannich reaction. The complex was determined by UV-Vis absorption measurement and the ketone compound used was the uranine due to its high coefficient absorption. The UV spectrum of the complex shows maxima of absorption at 207 nm and at 227 nm. An internal standard was used to quantify the atrazine. There is a good linearity between the absorbance and the concentration in the range of 0.1 – 10 μg.mL-1 of atrazine. The recovery value was 97 % and the limit of detection was 0.01µg.mL-1. Real samples collected from irrigation local area were analyzed using this method and the estimated concentration of atrazine found in the mentioned river is 0.29 ± 0.011 μg.mL-1.
Keywords: Atrazine, dechlorination by zero valent iron, Mannich reaction, Spectrophotometer, quantification, internal standard, real samples measurements.
Atrazine are widely used in agriculture, and their heavy use has resulted in the environmental pollution. Their persistent presence had been a serious problem, especially in surface and ground water systems. Atrazine herbicides were often used especially in Europe and the United States (1, 2), as important atrazine and simazine have been greatly used in maize cultivation and forestry. Their solubility in soil is low, and then can migrate along the food chains, and their intense use and presence in the environment have created a health threat to human beings. Recently, they have been considered as a group to be endocrine disrupting chemicals (3). The European Union Drinking Water Directive sets official regulations on the maximum admissible concentrations in drinking water as 0.1 mg.L-1 for an individual herbicide and 0.5 mg L-1 for total pesticides (4) whereas in surface water the alert and alarm threshold values are typically 1 and 3 mg.L-1 (5). Hence, the development of sensitive and economic analytical methods is very crucial for screening the presence and amounts of atrazine and preventing toxicological risks. In general, gas chromatography (GC) and high performance liquid chromatography (HPLC) are the techniques popularly used for the determination of atrazine and simazine (6-9). Gas chromatography-mass spectrometry (GC-MS), amperometric immunosensor, and adsorptive stripping voltammetric determination were developed for the analysis of atrazine and simazine, (10-13).
In general, these techniques are expensive and involve time-consuming separation steps. These methods are unsuitable for field testing, for continuous monitoring or for screening high numbers of samples as required in mapping pesticide pollution in time and space.
The objective of this work is to analyse the atrazine by a economic and rapid method. The proposed method in this work is based on the dechlorination of atrazine [DA] by zero valent iron powder (ZVIP), according to the reference fourteen (14) and using the [ DA] obtained in the Mannich reaction in order to give rise a by product having an extinction coefficient absorption more intense than atrazine compound.
Materials and methods
Chemicals and reagents
All chemicals and solvents used were of analytical grade or of a higher grade when available. Formaldehyde, hydrochloric acid were purchased from Fisher, (MA, USA). Atrazine was purchased from Rodel-dehein, zero valent iron powder (350 mesh) was purchased from Sigma Aldrich Ultra pure water was prepared using a multi-Q filter system (Millipore, MA, USA).
The UV absorption measurements were performed on a Shimadzu UV- 1650 PC. With 10 mm quartz cells were used for spectrophotometric measurements. The pH values are measured using METTLER TOLEDO pH-meter.
Stock solution of atrazine was prepared into a volumetric flask at a concentration of 10 μg.mL-1, 10 mL of this solution were mixed with 20 mL of acidified di-ionized water (pH = 4) and transferred into a flask of 100 mL. 2.5 g of zero valent iron powder were also added into the flask and shacked for 15 minutes. A complete dechlorinatation of atrazine must be achieved according to the previous work (14). This solution was in the Mannich reaction.
Samples for analysis were prepared by mixing uranine , formaldehyde and dechlorinated atrazine solutions. De-ionized water was transferred into each sample to reach a final volume of 10 mL. Calibration curves were built for quantitative measurements using the samples prepared according the table 1.
Composition of samples used to build regression curve of the absorbance of the complex obtained by Mannich reaction and atrazine dechlorinated [DA]
Internal standard curve addition and Recovery
Atrazine dechlorinated was added as an internal standard for the calibration of the measurement, according to the method described in (Muel and Lacroix, 1960; Rima, Lamotte and Joussot-Dubien, 1982) (16, 17). Determination of the pH was done using a Mettler Toledo (OH, USA) pH-meter.
Samples for analysis were prepared by mixing 0.5 ml of uranine (10 μg.mL-1), 1mL of pure formaldehyde and different volumes of dechlorinated atrazine stock solutions diluted to [0.75 μg.mL-1] (1-1.5 -2- 2.5 and 3 mL). De-ionized water was transferred to each sample to reach a final volume of 5 mL. Table 2 describes the preparation of the standard curve.
Recovery experiments were performed by standard addition method: 0.15 μg.mL-1 of
Atrazine dechlorinated was added to samples and percentage of recovery (R%) was calculated as follows:
R% = [(Cr-Cf)/Cr] – 100
Cr = Real concentration of atrazine in the fortified samples
Cf = Concentration of atrazine obtained by the internal standard addition curve
River water analysis
The water analyzed was collected from River in the north of Lebanon.[agricultural area] 100mL of the polluted water were treated by zero valent iron powder according to the protocol mentioned above. The samples were fortified by solutions of dechlorinated atrazine having an initial concentration of 0.75 μg.ml-1 in order to build the internal standard curve. Table 3 summarizes the volumes of different solutions used in the mixtures.
Results and Discussion
Mechanism of the dechlorination of atrazine
Most halogenated hydrocarbons, RX, can be reduced by iron metal. The overall reaction (Equation 1) results in dehalogenation of RX. Three general pathways by which this process may occur have been proposed (Matheson and tratnyek,) (18). The first involves direct reaction of the metal surface, in which case equation (A) alone adequately represents the pathway of reduction. The other two possible pathways do not involve the metal surface directly. Instead, Fe2+ and H2, which are products of corrosion by water, serve as the reductants that are directly responsible for dehalogenation of RX equation B and C.
Fe0 + RX + H+ ↔ Fe2+ + RH + X- A
2Fe2+ + RX + H+ ↔ 2Fe3+ + RH + X- B
H2 + RX ↔ RH + H+ + X- C
More specifically the atrazine can be written as RX ( X = Cl ).
With zero valent iron powder in the acidic aqueous solution the chlorine can be replaced by the hydrogen.
According the following reaction:
Effect of the dechlorination of atrazine on its extinction coefficient absorption.
UV absorption spectra of atrazine in aqueous solution (1.5μg.mL-1) were recorded and compared to the spectra of the by-product obtained by the treatment of atrazine as shown in figure 1. Atrazine was dechlorination by zero valent iron powder according to the method described by Matheson, L.J et al (18).
The extinction coefficient of the atrazine at the 220 nm (maximum of absorption) was estimated the value of 35200 M-1.L; whereas the extinction coefficient of the dechlorinated atrazine which give a maximum at 210 nm was found equal to 169000 M-1.L. Figure 1.
The experimental observations revealed that by the removal of the chlorine atoms an hyperchromic and hypsochromic effects were observed. An increasing of the extinction coefficient and a blue shift from 220 nm to 210 nm of the maximum of absorption were detected.
According to the table 4 , the proton that accompanies the formation of the free amine in Equilibrium 1 is available to protonate other reactants in the solution (Equilibria 2 and 3). Addition of the free amine to a protonated molecule of formaldehyde leads to the formation of the iminium ion shown at the right of (Equilibria 4). The enol of acetone then adds to the carbon atom of the iminium ion in (Equilibrium 5).
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In the equilibrium 1 as shown in the table 1, the nitrogen is enriched by electrons of the CH3 group and this nitrogen will react with H+ as base/acid reaction. However, when the electrons of the nitrogen, are deprived under the influence of the electron affinity of chlorine, this nitrogen will lose its basic character, then the équilibre1 should be disturbed .In the other hand the nitrogen of the Equilibrium 4 play a nucleophilic role and ,if this nitrogen is disadvantaged by a chlorine attractive effect; it will lose the nucleophilic characteristics and the reaction with the carbon of the aldehyde in the Equilibrium 4. cannot be obtained
In the case of atrazine it well known that the chlorine plays the role of the electrons donor to the nitrogen of the cycle and the electrons attractive of the aliphatic nitrogen. Since the Mannich reaction must take place at the aliphatic nitrogen not at the aromatic nitrogen and when the chlorine was removed by dechlorination process, the electrons at the aliphatic nitrogen become more dense and then it will be more able to play the role of nucleophilic atoms as mentioned in Mannich reation.
In conclusion when the chlorine is removed from atrazine, the Mannich reaction can be observed as we demonstrated experimentally.
Identification of the complex obtained in the mixture atrazine dechlorinated-formaldehyd and uranine
The Mannich reaction is an organic reaction which consists of an amino alkylation of an acidic proton placed next to a carbonyl functional group with formaldehyde and ammonia or any primary or secondary amine. The final product is a β-amino-carbonyl compound also known as a Mannich base. Reactions between aldimines and α-methylene carbonyls are also considered Mannich reactions because these imines form between amines and aldehydes.
Equation D (15).
We hypothesized that the reaction between uranine, formaldehyde and dechlorinated atrazine must be similar to reaction E. The mechanism of the reaction is the following:
Atrazine compound presents a UV spectrum with a maximum absorbance at 220 nm whereas dechlorinated atrazine presents a maximum of absorption at 210 nm Spectra of atrazine and dechlorinated atrazine are presented in Figure 1. The mixture of dechlorinated atrazine, formaldehyde and uranine give rise to the formation of a complex described by the Mannich reaction. It is obviously that formaldehyde does not have any UV spectrum. The UV spectra of the complex give rise to a spectrum with two maxima at at 207 nm and 227 nm respectively. Figure 2 presents the spectra of atrazine. dechlorinted atrazine and the complex obtained by Mannich reaction.
Atrazine 2 μg.ml-1 (A)
(DA : dechlorinated atrazine by zero valent iron powder),
(DA+ H2CO : dechlorinated atrazine by zero valent iron powder with formaldehyde)
(DA+ H2CO + uranine : : dechlorinated atrazine by zero valent iron powder with formaldehyde)
Regression curve between the complex formation
A calibration curve of the complex was built to examine the linearity of the complex absorbance and atrazine concentrations. The least square method was used to calculate the regression equation. A strong linear correlation was obtained between the absorbance of the complex and the concentrations of atrazine. Figure 4 shows the regression curve of complex absorbance in function of atrazine concentrations. Correlation coefficients were higher than 0.99 in a concentration range of 0.15 μg.mL-1 to 0.75μg.mL-1.The precision of the method was evaluated with relative standard deviations (RSD) of atrazine determination in five samples. RSD was 3 %. The limit of detection of the method was 0.01µg.mL-1 as defined by a signal-to-noise ration of 3:1 (19).
Spectrophotometric method for quantification of atrazine e using the internal standard addition model
A spectrophotometric method using the internal standard addition was examined to quantitatively determine melamine concentrations in samples. A calibration curve was described by the following equation: A*= aC + b, which is equivalent to A* = (A0* /C0) x Cadd + A0*, with A* = (A/ A0) normalized absorbance intensity (arbitrary values), is equal to the ratio of the absorbance intensity after adding the internal standard A to the absorbance intensity before adding the internal standard (A0)
C0: solute concentration to be estimated. C0 is determined by the negative intercept of the curve with the abscissa axis (16, 17).
A0*: normalized absorbance intensity of the starting solution
Cadd: known added concentrations.
The plot of A* vs. Cadd is shown in Figure 5. The internal standard used in this method was the atrazine that we would like to determine (C0). To this initial solution, different known concentrations (Cadd) were added.
The average recovery for five samples spiked with melamine as described above in Table 1 was estimated to be 97% ± 3. Table 5 summarizes the validation parameters of this method.
Spectrophotometric method for the quantification of atrazine in a sample taken from an agricultural zone, using the internal standard method.
Samples taken from a water source contaminated with atrazine, are processed by zero valent iron powder for the dechlorination of atrazine molecules. A definite volume of this solution is mixed with the same concentrations of formaldehyde and uranine. Different volumes of standard solution of dechlorinated atrazine were added to the solutions to be analyzed. The composition of these solutions are summarized in Table 3.
The UV-Vis spectra of the solutions were recorded to follow the evolution of the complex obtained after the mixture called Mannich mixture.
Using the evolution of UV-Vis spectra, an internal standard curve could be constructed and the intersection of this curve with the axis of abscises gives the concentration of atrazine in the waters of Agriculture .
The concentration of atrazine in these waters is estimated at 0.29± 0.011 μg.mL-1 (n = 5).
The spectrophotometric method used to analyse the atrazine in agriculture water was based on Mannich reaction .This new method is a specific and simple method for the quantitative determination of atrazine in the contaminated water. Often the determination of atrazine is measured by sophisticated and expensive methods like HPLC, GC/MS. However the proposed method is easy to use, rapid and economic and it showed high accuracy, but it was restricted by the potentiality of the spectrophotometer which reaches a limit of detection of 0.01 μg.mL-1 as described in the manuscript.
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