A new strategy for the determination of dinobuton fungicide by square wave stripping voltammetry on the multi-walled carbon nanotube electrode

Dinobuton is a fungicide with a dinitrophenol group pesticide, and its electrochemical behavior was investigated by cyclic voltammetry (CV) and square wave stripping voltammetry (SWSV) on a multi-walled carbon nanotube paste electrode (MWCNTPE). First of all, optimum parameters such as pH, step potential, frequency, puls amplitude, deposition time and, deposition potential were specified by using SWSV on the MWCNTPE. In the negative potential scans, two cathodic peaks appeared at nearly −480 mV and –760 mV due to the nitro groups on the molecule and the second sharp one appeared at −760 mV (versus Ag/AgCl) was used for analytical purposes. The linear working range was found to be within 3.74–25.8 μM on the MWCNTPE by SWSV in pH 7.0 Britton Robinson (BR) buffer solution. The limit of detection (LOD) and limit of quantification (LOQ) values were found to be 0.73 μM and 2.43 μM, respectively. The interference study was conducted in the presence of some pesticides such as triasulfuron, azinphos-methyl, bromoxynil-octanoate, dialifos, fipronil, vinclozolin, iprodione, procymidone, and some selected metal ions withal. Furthermore, the proposed method was also applied to apple juice, tap water, and grape juice, and percent recoveries (%) were detected as 105.9 ± 4.3; 98.3 ± 0.9; 103.7 ± 2.5% with relative standard deviations of 4.0, 1.0, and 2.4%, respectively. On the other hand, percent relative errors were calculated as 5.90, 1.65, and 3.74%, respectively. High recoveries and low relative standard deviations indicate that the applicability of the proposed method in both matrix and real samples is satisfying.


Introduction
Having a diet including vegetables and fruits reduce the risk of high blood pressure, heart disease, diabetes, stroke, many cancers, and other chronic disease.Higher productivity of agricultural products leads to a healthier population and a good harvest of vegetables and fruits plays a vital role in our lives.At that point, using pesticides effectively is one of the important factors resulting in maximum efficiency for agricultural products [1].There is some kind of organisms that harms farm products like insects, fungi, etc and pesticides have a role that prevents these harmful organisms from damaging them.However, pesticides have harmful effects on the environment as well as benefits [2].Therefore, the highest efficiency with the lowest concentration of pesticides in crops may decrease the damage to the environment.At that point, the quantification of pesticides at very low concentrations creates the need for sensitive and efficient new analytical methods.
Dinobuton ((2-butan-2-yl-4,6-dinitrophenyl) propan-2-yl carbonate) is a fungicide as a subtype of pesticide and has a dinitrophenol functional group therefore included in the dinitrophenol pesticide class.Dinobuton is a bio-activated fungicide/acaricides group of dinitrophenyl group of pesticide [1] and the molecular structure of dinobuton is shown in Scheme 1.
The degradation of dinobuton in soils, plants, and animals involves the reduction of the nitro group to produce the amino group, N-acetylation, deamination, and carboxylation of the sec-butyl group.The metabolic pathways of dinobuton in plants and animals are; hydrolytic cleavage of the carbonate-dinitrophenyl linkage, dinoseb is produced with hydrolytic cleavage of the carbonate-dinitrophenyl linkage, reduction of nitro groups of dinoseb, production of monoamine and diamino analogs, acetylation and deamination (via hydroxylation/ elimination), oxidation of the sec-butyl moiety and N-conjugation and O-conjugation as glucosides and glucuronides [3].
So far, chromatographic and spectrophotometric methods have been discussed and the gel permeation chromatography (GPC) treat with gas chromatography-mass spectrometry (GC-MS) [4], liquid chromatography-tandem mass spectrometry (LC-MS-MS), high-performance liquid chromatography with photodiode-array detection (HPLC-DAD) [5], stopped-flow pneumatic system [6] and micellar modified UVvis spectroscopy [7] was used in dinobuton determination.However, these methods have some shortcomings such as the need for long preprocessing and for expertise, expensive equipment, and using too much solvent for the analysis of dinobuton substance.Therefore, in recent years, alternative methods have been sought for such determinations.Electrochemical methods are the most remarkable alternative for the identify of numerous substances due to their superior properties [8,9].Electrochemical analyses in particular have attracted great interest in the last two decades because of their fast, stable, inexpensive, portable and sensitive characters.Due to the extraordinary properties exhibited by electrochemical techniques, they are preferred in the qualitative and quantitative determination of numerous substances [10][11][12][13][14].However, to our knowledge, an electrochemical study for the determination of dinobuton has been carried out using the drop mercury electrode (DME) [15], which is often claimed to be toxic, and extremely important electrochemical models such as electrochemical behavior, kinetic model and electrode mechanism have not yet been clarified.In addition, there is still a need to develop alternative indicator electrodes in terms of green chemistry to avoid a highly toxic substance such as mercury.
The aim of this study is to investigate the electrochemical behavior, electrode mechanism, kinetic model and analytical application of dinobuton fungicide in natural samples using the square wave stripping voltammetry (SWSV) technique on a multi-walled carbon paste electrode (MWCNTPE).For this purpose, the pH effect and the operating parameters in the SWSV technique have been optimized on the MWCNTPE.Then, the working range, limit of detection, selectivity, and other validation data were investigated in detail.Finally, recommended method has been successfully applied to tap water and commercial fruit juices.Consequently, a new detection strategy for dinobuton, which has not yet had a comprehensive electrochemical investigation, was developed for the first time on a MWCNTPE nanomaterial by square wave stripping voltammetry (SWSV).

Apparatus and reagents
The BAS Epsilon model electrochemical analyzer (Bioanalytical Systems, Epsilon potentiostat/galvanostat, IN 47906, US) was used for recording voltammetric measurements.The electrochemical analyzer consists of a potentiostat power supply, a cell stand, and a computer.The cell stand consists of three electrodes which are MWCNTP electrode (BASi, MF-2012) as the working electrode, Ag/AgCl (BASi, MF-2052) as the reference electrode, and platinum wire (BASi, MW-1032) as a counter electrode.To prepare the working electrode, MWCNTs and mineral oil were mixed at a ratio of 60:40 (m/m), and then the MWCNT paste was squeezed into the bare carbon paste electrode (BASi, MF-2012) using a spatula.Then, the surface of the pasta was polished with a spatula until it achieved a shiny appearance and was ready for use.The pH values of the prepared solutions were measured by using a portable HANNA 211 model, microprocessor pH meter with a combined glass electrode.All samples were accurately weighted with portable, Sartorius analytical balance (precision to ± 0.0001).
Dinobuton was provided as analytical grade.The stock solution of dinobuton fungicide was prepared by precisely weighing 0.0049 g in 10 ml of acetone.For pH 1.0 studies, 0.1 M H 2 SO 4 was preferred and voltammograms between pH 2.0 and pH 12.0 were recorded in Britton Robinson (BR) buffer solution.The 0.04 M BR buffer solution was prepared from a mixture of 2.5 g boric acid (99.8%), 2.3 ml acetic acid (Glacial, ReagentPlus ® , 99%), and 2.7 ml phosphoric acid (85%) made up to 1 l volume with distilled water and the pH of this solution is around 2.0.To bring these prepared support electrolyte solutions to the desired pH values (between approximately pH 2.0 and 12.0), 2.0 M NaOH or 2.0 M HCl was added with a pH-meter control.Distilled water was used for the preparation, and dilution of all solutions, and also cleaning process throughout the experiments.The solutions were stored in a dark refrigerator when not in use.In addition, experimental measurements were made at an ambient temperature (25 °C ± 2).

Analytical procedures
The electrochemical behavior of dinobuton fungicide was investigated in various pH and Britton Robinson buffer solution with a pH 7.0 was suitable as the optimum supporting electrolyte.Then, cyclic voltammetric measurements were carried out with at different scanning rates to find the kind of transport of the molecules to the electrode surface and the number of transferred electrons.In the square wave stripping voltammetry (SWSV), some parameters such as accumulation time (t acc ), accumulation potential (E acc ), frequency ( f ), pulse amplitude (ΔE), and step potential (ΔE s ) which are significantly affect on the peak current and peak potential have been optimized in detail.The optimum parameters were found as t acc = 50 s, E acc = -500 mV, f = 30 Hz, ΔE = 20 mV and ΔE s = 5 mV, respectively.Then, using the optimum modules of the SWS voltammetric technique, the calibration curve was constructed by standard addition in pH 7.0 BR buffer solutions.Finally, validation studies such as accuracy, analytical application, detection limits (LOQ and LOD), precision, selectivity, and linear dynamic range were carried out.
Precision and accuracy tests are the most prominent validation parameters for a new determination method.Therefore, analytical applications were performed in apple juice, tap water, and grape juices to demonstrate the accuracy and precision of the recommended SWS voltammetric method.The spiked real samples of apple juice, tap water and grape juice were prepared with specific amounts, by adding 1 mL from dinobuton stock solution to 9 ml real samples.Then, theses spiked natural samples mixed homogeneously were stored in the dark at 4 °C until analyzed by SWS voltammetry on the MWCNTPE.

Electrode characterization and sensor performance
Cyclic voltammogram of ferricyanide and scanning electron microscope measurements were performed for the analytical performance and surface characterization of the constructed MWCNTP electrode to be used for the analysis of dinobuton fungicide.At the outset, the electroactive surface area of MWCNTP electrode was calculated by recording cyclic voltammetric measurements (100 mV s −1 ) of 1.0 mM ferricyanide in 0.1 KCl based on the Randles-Sevcik equation (I p = 2.69 × 10 5 n 3/2 A C D 1/2 v 1/2 ) (figure 1(A)).The terms A, C, D, I p , n, and v in this equation just given indicate the concentration of ferrocyanide solutions, the diffusion coefficient of ferricyanide, the peak current, the electron transfer number, and the scanning rate, respectively.The electroactive surface area of MWCNTP electrode was found to be 0.149 cm 2 .Moreover, the surface morphology of the constructed MWCNTP electrode was clarified by scanning electron microscopy (SEM) (figure 1(B)) and it was concluded that it had a large surface area in a mesh structure.As a result, an electrochemical sensor based on carbon-based material with unique properties was developed for the analysis of dinobuton molecule.

Voltammetric behavior of dinobuton
The voltammetric behavior of dinobuton was examined by cyclic voltammetry (CV) on the MWCNTP electrode in pH 7.0 BR buffer solutions and to achieve it voltammograms were recorded by scanning potential between 1.0 V and -1.2 V in both cathodic and anodic directions.Cyclic voltammograms (figure 2) of the 136 μM dinobuton recorded between scan rates of 10 mV s −1 and 80 mV/s showed a single peak at approximately -0.8 V in the cathodic direction, while no peaks appeared in the anodic direction.This is evidence that dinobuton exhibits irreversible electrochemical properties on the MWCNTP electrode.
In addition, the potential scanning rate with cyclic voltammetry provides valuable data whether the electroactive species is transported to the electrode interface by diffusion-controlled or adsorption-controlled process [16].The graph the logarithm of peak currents (I p ) versus the logarithm of the scanning rate (log v) was plotted from voltammograms recorded at various scanning rates (equation ( 1)).
The slope of the graph between log v and log I p obtained for the cathodic peak was found to be 0.65.On the other hand, if the transport of an electroactive species to an electrode interface is diffusion-controlled, it is known that this slope should theoretically be around 0.5, whereas 1.0, adsorption-controlled [16].Since the δI p /δlogv slope is seen between 0.5 and 1.0 in the evaluations made with cyclic voltammetry, it can be concluded that the transfer of dinobuton to the electrode surface is both diffusion and adsorption controlled or mixed controlled.However, since it is closer to the theoretical value of 0.5, it can be interpreted that the diffusion effect is more dominant than the adsorption effect.
In addition, CV measurements provide extremely important findings about the electron(s) transferred in the electrode reaction within the Nernst diffusion layer [13].Accordingly, cyclic voltametric data collected in pH 7.0 BR buffer solution on the MWCNTP electrode at different scan rates within 10 mV s −1 to 80 mV s −1 and the recorded voltammograms revealed that the cathodic peak potentials of the dinobuton shifted to more negative positions due to the increased scanning rate.A linear equation of E p versus log v with a slope of -0.068 prove that the electro-reduction of dinobuton includes some electrons in its Faradaic process to be used for the electrode mechanism [9,13].
Since the dinobuton was studied only with a mercury drop electrode, we could not reach any data about the electrode mechanism yet.In our studies, we used an extremely common tool, cyclic voltammetry (CV), to elucidate the mechanism of electrode reactions of electroactive organic species.In order to explain the reduction mechanism of dinobuton on the MWCNTP electrode, we applied the CV technique in pH 7.0 BR buffer solutions at different scanning rates from 10 mV s −1 to 80 mV s −1 .When we replace the slope value (68 mV) obtained from the E p versus log v graph in the Laviron equation accepted for the irreversible reaction, the 'αn' value is calculated as 0.869 [17][18][19].
303 log The symbol of R, T, n, F, k°, α, and v in Laviron equation accentuates universal gas constant (8.314J K −1 mol −1 ), temperature (K), number of electrons in cathodic electrode reaction (n), Faraday constant (96485 C mol −1 ), standard heterogeneous reaction rate constant (k°), electron transfer coefficient (α) and potential scan rate (v), respectively.Since 'α' is accepted as 0.5 in a non-reversible electrode reaction, this value was substituted in the slope equation and the number 'n' was found to be 1.74 and rounded to 2. Since a shift of about 27.8 mV is observed in dinobuton reduction potentials depending on the pH increasing by one unit, the electrode reaction mechanism includes an equal number of electrons and protons (H + ).The shift of the dinobuton cathodic peak signals depending on pH is also evidence of the involvement of the proton in the electrode reaction [9,20].This is valid for the reduction of only one nitro group in the chemical structure of dinobuton fungicide thus, the possible reduction mechanism of dinobuton in Scheme 2 is postulated for the first time on the carbon based MWCNTP electrode in the pH 7.0 BR buffer solutions.

Effect of pH on the electro-reduction of dinobuton
The pH and type of supporting electrolyte has a significant effect on both the peak potential and the peak signal of the electroactive substance so that electrochemical measurements should be taken in the buffer solutions prepared in different pH solutions [20].Towards this goal, square wave stripping voltammograms were recorded at various supporting electrolytes between pH 1.0 and pH 12.0 to find the optimum supporting electrolyte (figure 3).The highest and sharpest peak for 14.8 μM dinobuton was observed in BR buffer pH 7.0.Thus, even the most uniform and well-defined peak was obtained, so pH 7.0 was considered as the ideal supporting electrolyte.The shift of dinobuton peaks to lower negative potentials due to increasing pH, that is, the potentials being dependent on the pH of the supporting electrolyte, is the most important evidence of the

Optimization of instrumental parameters
Electroanalytical variables such as step potential, pulse amplitude, deposition time, deposition potential and frequency in SWS voltammetric technique affect both on the peak current and the potential of the analyte (figure 4).Therefore, SWS voltammograms for the 14.8 μM dinobuton in the pH 7.0 BR buffer solution on MWCNTP electrode were recorded at different voltammetric parameters, and optimizations of instrumental parameters were performed, accordingly.Firstly, SWS voltammograms were obtained at different step potentials between 2 mV and 10 mV to optimize the step potential.According to the obtained SWSV, irregular increases on the peak current of the dinobuton were observed due to the increasing step potentials.The ideal step potential of 5 mV was chosen to achieve the highest and well-defined peak current for the dinobuton reduction.Then, different frequencies ranging from 10 Hz to 100 Hz were applied to optimize the frequency module and SWS voltammograms were recorded in pH 7.0 BR buffer solutions.While there was a regular increase on the cathodic signal of the dinobuton up to 30 Hz, irregular decreases were observed on the peak current after 30 Hz.The 30 Hz, where the highest cathodic signal is obtained, was preferred as the optimum frequency.In order to find the pulse amplitude, SWSV measurements were carried out with amplitudes ranging from 10 mV to 40 mV.Up to 20 mV, there was an increase on the peak signal of the dinobuton, however very small decreases in pulse amplitudes were observed, aiming to we can say that the peak current remained constant.Therefore, the optimum pulse amplitude of the validated SWSV technique for dinobuton determination was accepted as 20 mV.In stripping techniques, it is the accumulation time and accumulation potential modules that have a great influence on the peak signal and peak potential of analyte.Therefore, these two parameters have been meticulously optimized as with all SWS operation parameters.In the accumulation potential optimization study, a potential between -500 mV and + 500 mV was applied and SWSV measurements were recorded on the constructed MWCNTP electrode.A linear decrease in the cathodic peak signal was monitored from -500 mV to +500 mV and therefore -500 mV, where the highest peak signal was obtained, was chosen as the optimum accumulation potential.During the accumulation time applied to the electrode surface an increase in the cathodic peak was observed from 20 to 50 s and thereafter remained nearly constant, which is an indication of electrode surface saturation.In the light of the above findings, 50 s, when the peak current reached its maximum, was accepted as the optimum accumulation time.Consequently, the table 1 summarizes the optimized SWS voltammetric parameters, including the supporting electrolyte and the peak potential.

Calibration and validation study
Validation studies of a newly developed method are of high priority, and therefore, studies such as linear range, the limit of quantification (LOQ) and limit of detection (LOD), intra-day and inter-day repeatability, precision, and accuracy were performed under the optimized experimental and instrumental parameters.Firstly, SWS voltammograms were recorded by standard addition method under optimum operating conditions on the MWCNTPE (figure 5) and a linear operating range was obtained between 3.74 μM and 25.8 μM depending on the measured peak currents of dinobuton fungicide.The linear equation for the dinobuton calibration plot was constructed as follows.
The s m 3 and s m 10 equations were applied respectively to calculate the limit of detection (LOD) and limit of quantity (LOQ), which are two important validation parameters.[21,22].The term 's' in these equations represents standard deviation of seven measurements of the blank solution obtained at the lowest quantitative concentration of dinobuton, and 'm' is the slope of the constructed calibration plot.These obtained data were inserted into the relevant equations and LOD and LOQ were calculated as 0.73 μM and 2.43 μM, respectively.To calculate the precision of the method, intraday reproducibility was performed by recording seven voltammetric measurements for 15.9 μM dinobuton within day, and the relative standard deviations (%RSD) of I p and E p were calculated to be less than an acceptable limit of 5%.In addition, a decrease in the dinobuton signal within the 10% tolerance limits was observed after approximately four weeks of use with the created MWCNTPE electrode.This proves the high stability and excellent reproducibility of the cheap, sensitive, environmentally friendly MWCNTP electrode recommended for dinobuton determination.These low %RSD values obtained show that the sensitivity of the proposed voltammetric method is acceptable, and all validation parameters are also summarized in table 2.

Interference effects
Percent recovery studies were performed in the presence of some well-known pesticides such as triasulfuron, azinphos-methyl, bromoxynil-octanoate, dialifos, fipronil, vinclozolin, iprodione, procymidone, and also various ions to investigate the selectivity of the proposed voltammetric method.In order to examine the influences of interference SWS voltammetric measurements were recorded under conditions where the mass ratio of other substances to dinobuton was 1:1, 1:5 and 1:10 (m/m).The recovery results were calculated as the % recovery of dinobuton in the presence of coexisting species.In the 1:1 (mol/mol) interference ratio of dinobuton/interfering pesticide and dinobuton/metal ions, the interference effect was within the 5% tolerance limit.In the 1:5 interference ratio (mol/mol), only bromoxyniloctanoate and dialifos showed a significant interference effect (higher than the 5% tolerance limit).These interference effects can be attributed to dispersion forces or Van der Waals forces depending on the molecular structures.No interference effect was observed significantly when other pesticides and metal ions were used as matrices.In the 1:10 interference ratio (mol/mol), no significant interference effect was observed when iprodione and procymidone were used as interfering species, but the metal ions showed an interference effect in that determination.As a result, when it is a 1:1 interference type in mass, dinobuton could be detected at a tolerance limit of less than 5% with high recovery and low relative error, but when it is a 1:1 interference type, these determinations could be made at a tolerance limit of 10%.To minimize the interference effects of pesticides at other concentrations, it may be recommended to change the pH value or eliminate metal interactions by pretreatments such as adding ethylene diamine tetra acetic acid (EDTA), which can form metal complexes.All the recordings were repeated in triplicate and percent relative standard deviations together with the percent recoveries were summarized in table 3.

Application of the method to water and juice samples
Analytical application contributes to the accuracy and precision of a novel method and therefore analytical determinations for dinobuton have been performed in natural samples such as spiked apple juice, tap water and grape juice using square wave stripping voltammetry (SWSV) on MWCNTPE in pH 7.0 BR buffer solutions.The recovery studies in apple juice were performed from the 10 ml of juice solution containing 12.25 μM dinobuton with the standard additions of 20, 40, 60, and 80 μl from 6.25 mM dinobuton stock solution.Square wave stripping voltammograms of dinobuton peak increments with their standard additions are presented in figure 6.
The validity of the proposed method was also evaluated by the recoveries of the spiked tap water.In this procedure, 0.0209 grams of dinobuton was dissolved in 10.0 ml of acetone and a 6.40 mM dinobuton stock solution was obtained.1.0 ml of dinobuton stock solution was transferred to the 10.0 ml of volumetric flask and the volume was completed with tap water thus, 0.64 mM spiked dinobuton sample was obtained.200 μl of dinobuton spiked tap water was delivered into the cell containing 10.0 ml of pH 7.0 BR buffer solution, and standard additions of 60 μl of 6.40 mM dinobuton stock solution were made four times.
Finally, validation studies in grape juice were performed and validity of the recommended method was investigated with recoveries of spiked grape juice.As a process, 6.2 mM dinobuton stock solution was prepared by dissolving 0.0202 grams of dinobuton in 10 ml of acetone, and 1.0 ml of dinobuton stock solution was transferred to a 10.0 ml volumetric flask and made up to volume with grape juice obtained from the market, thus, 0.62 mM dinobuton in grape juice stock solution was obtained.400 μl of 62 mM dinobuton-grape juice stock solution was transferred to 10.0 ml of pH 7.0 BR buffer in a voltammetric cell, whereupon sequential standard additions from 40 μl of dinobuton stock solution were performed four times.The recovery percentages presented as percent relative standard deviation (RSD%) and percent relative error (%RE) (table 4) exhibited within the tolerance limits of 10% and the relative standard deviation values vary between 0.97%-2.43%,and the relative standard deviations vary between -1.65 percent and 5.9 percent.

Conclusion
The pesticide dinobuton is a potentially non-systemic acaricide and fungicide and has been used to control mites resistant to organophosphorus compounds, especially those found in cotton, vegetables, tomatoes and fruits.In this study, the electrochemical behavior of dinobuton was investigated by using square wave stripping voltammetry and cyclic voltammetry on multi-walled carbon nanotube electrodes.The first peak obtained was not well defined and the second peak could be used for analytical purposes to a large extent.The second sharp peak appeared at −760 mV (versus Ag/AgCl) was used for analytical purposes.It has been shown that the proposed method can be successfully applied to natural samples and the % recovery values for spiked apple juice, tap water, and grape juice samples were found to be 105.9± 4.3, 98.3 ± 0.9, and 103.7 ± 2.5 respectively.Low relative standard deviation (4%, 0.97%, and 2.43%, respectively) and low relative errors (5.8%, −1.65%, and 3.74%, respectively) indicate that the method is precise and accurate.To demonstrate the selectivity of the method, interference effects of some pesticides such as triasulfuron, azinphos-methyl, bromoxynil-octanoate, dialifos, fipronil, vinclozolin, iprodione, and procymidone were investigated.Dinobuton was successfully detected within the 10% tolerance limit in the presence of interacting pesticides at a ratio of 1:1 (m/m).

Figure 1 .
Figure 1.For the constructed MWNPT electrode (a) cyclic voltammogram of ferricyanide demonstrating sensor performance (b) SEM image for the surface characterization.

Scheme 2 .
Scheme 2. The possible reduction mechanism of dinobuton.

Figure 4 .
Figure 4. Optimization of instrumental parameters for the determination of dinobuton by SWSV, The influence of (A) step potential, (B) frequency, (C) pulse amplitude, (D) accumulation time and, (E) accumulation potential.

Table 1 .
Optimum parameters for determination of dinobuton by SWSV on the MWCNTPE.

Table 2 .
Parameters obtained by dinobuton calibration graph.

Table 3 .
Interference of some pesticides and percent recoveries a.
a n = 3