Spectrophotometric Detection of Glyphosate in Water by Complex Formation between Bis 5-Phenyldipyrrinate of Nickel (II) and Glyphosate

: A spectrophotometric method for the determination of glyphosate based on the monitoring of a complex formation between bis 5-phenyldipyrrinate of nickel (II) and the herbicide was developed. The method showed a short response time (10 s), high selectivity (very low interference from other pesticides and salts), and high sensitivity (LOD 2.07 × 10 − 7 mol / L, LOQ 9.87 × 10 − 7 mol / L, and a K d from 1.75 × 10 − 6 to 6.95 × 10 − 6 mol / L). The Job plot showed that complex formation occurs with a 1:1 stoichiometry. The method was successfully applied in potable, urban, groundwater, and residual-treated water samples, showing high precision (0.34–2.9%) and accuracy (87.20–119.04%). The structure of the complex was elucidated through theoretical studies demonstrating that the nickel in the bis 5-phenyldipyrrinate forms a distorted octahedral molecular geometry by expanding its coordination number through one bond with the nitrogen and another with the oxygen of the glyphosate’ carboxyl group, at distances between 1.89–2.08 Å.


Introduction
Glyphosate (N-(phosphonomethyl)glycine) (glyp) (Figure 1c) is the most intensively used herbicide worldwide because of its high effectiveness against annual grasses and aquatic weeds [1,2], with a global annual production estimated to be over 825,804 tons in 2014 [3]. Its use is allowed in agricultural, urban, and domestic activities [4], with the agricultural application being the most intensive one. The physical and chemical properties of glyp allow its distribution in the environmental compartments [5][6][7]. Furthermore, its chelating ability and the absorption constant in soil allows for its accumulation in several types of soils, mainly clays [8]; it is considered a stable compound in a pH range between 4 and 9 for hydrolysis and photolysis [9,10]. Glyp pollution in water, soil, and food samples are becoming water, soil, and food samples are becoming a serious health concern [11][12][13]. In addition, its metabolite, aminomethylphosphonic acid (AMPA), also represents a potential danger to human and animal health [14,15]. Both compounds have been detected in groundwater and surface water in several countries [6,16,17]. As glyp has been labeled as a global pollutant [18,19], assessing its presence in several environmental matrices is a vigorous area of research [20][21][22].
There are some analytical methods to determine the presence of glyp and the metabolite AMPA in different media, such as water, urine, and serums. The official method to determine glyp in water, the EPA-547 [23], requires herbicide derivatization post-column with o-phathdialdehyde (OPA) [24]. Other methods employ high-performance liquid chromatography (HPLC) [25] coupled with mass spectrometry [26][27][28][29], fluorescence [30], or capillary electrophoresis [31]. However, these methods are complex, as they require pre-treatment steps for the samples and lengthy analysis times, by which it is not always possible to analyze massive samples in situ [32,33]. Recently, other methods have been reported as alternative tools for monitoring environmental samples in situ with the added benefit of short analysis times. Furthermore, some other analytical methods can have remarkably low detection limits, such as spectrophotometric [34][35][36][37], electrochemical [38][39][40], and Enzyme-Linked ImmunoSorbent Assay (ELISA) techniques [17,41]. The development of new methodologies that are quick, sensitive, reproducible, and inexpensive represent a viable alternative to the current methods and instruments by allowing the analysis of a larger number of samples either on the field or at the lab [42][43][44].
Dipyrromethenes are chelators of bipyrrolic monoacids that can form stable complexes with metals due to their coordination chemistry and optic and fluorescent properties [45]. These ligands are structurally rigid, completely conjugated, and capable of functionalizing several positions of their structure (1, 5, and 9) (Figure 1a). Hence, the coordination with metallic ions of meso-substituted dipyrromethenes (position 5) has been applied to the design of sensors. For instance, there is a study based on fluorescent probes on a boron dipyrromethene functionalized with a group of phenylboronic acids (BODIPY-PBAs) that can detect several monosaccharides in a concentration range of 0.1-100 mM, with good reproducibility and photostability [46]. In another study, an electrochemical biosensor was developed using a dipyrromethene-Cu(II) to determine the oligomeric form of amyloid beta (Aβ16-23) with concentrations in the range of 0.001-1.00 µM, which induces the neuronal dysfunction associated with Alzheimer´s disease (AD) [47]. Another paper reported on the electroactive dipyrromethene-Cu biosensor to detect antibodies against avian influenza virus type H5N1 in hen sera [48].
In this study, the chelating capacity of glyp was exploited to bind the metallic moiety of the compound bis 5-phenyldipyrrinate of nickel (II) (Ni(PhDP)2) (Figure 1b). Based on this molecular association, a method for glyp determination in several water samples is developed for the quantification of the complex formed between glyp and (Ni(PhDP)2). The resulting method is fast, sensitive, accurate, and useful for the quantification of glyp in drinking, urban, and ground waters, and residual-treated wastewater.

Apparatus
Electronic absorbance spectra measurements were obtained using a Varian Cary 50 spectrophotometer equipped with a xenon lamp (Australia). To perform electronic spectra measurements, quartz cuvettes (1 cm path length) were used.

Synthesis of the (Ni(PhDP) 2 )
The procedure used by Brückner et al. [49] was followed to synthesize (Ni(PhDP) 2 ). First, the 5-phenyldipyrrometane with benzaldehyde (1 mmol), pyrrole (1 mmol), and trifluoroacetic acid were synthesized. Then 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (1 mmol) was added and stirred for 30 min at room temperature. After that Et 3 N (0.5 mL) was added to the solution and stirred for another 30 min at room temperature. After the elimination of the solvent, the residue was dissolved in CH 2 Cl 2 , and the solution was filtered to remove precipitates. The solvent was removed, and the product was purified by short silica gel column chromatography using ethyl acetate as an eluent. The first eluted yellow fraction was evaporated to afford the crude product. Later, 2 mmol of 5-Phenyldipyrromethene and 1 mmol of nickel sulfate hexahydrate were dissolved in a mixture of CHCl 3 and CH 3 OH. The solution was agitated and heated under reflux for 6 h, and then 0.5 mL of Et 3 N were added and heated again under reflux for 4 h. The solution was evaporated in a rotary evaporator until a dark brown solid was obtained. The dried remainder was dissolved in CH 2 Cl 2 and CH 3 OH (1:1), leaving it to slowly evaporate until crystals were obtained.

Complex Stoichiometry
The Job method of continuous variation [50] was used to determine the stoichiometry of (NiGlyp(PhDP) 2 ). Two equimolar stock solutions were prepared and mixed in a way that the total concentration was kept constant (5 × 10 −5 and 1 × 10 −4 mol/L for two different assays). The absorbance at 362 nm was measured after mixing for 10 s in a 1 mL reaction mixture (99% CH 3 OH-1% water).
The absorbance changes at 362 nm of the (Ni(PhDP) 2 ) complex at different concentrations of glyp were transformed to a percentage of change and adjusted to the one-site binding model [51] (Equation (1)) to determine K d : where ∆AG is the percentage of absorbance change at 362 nm upon adding each glyp concentration, ∆A max is the maximum percentage of change (100%) when the (Ni(PhDP) 2 ) is saturated with glyp, and G 0 is the total concentration of glyp. The reported values are the mean of the three replicates. The data were fit to the Hill equation using an iteration procedure following the Marquardt-Levenberg nonlinear least-squares algorithm, using Origin 8.0 software (Originlab Corporation, Northampton, MA, USA).

The Analysis in Water Samples
Four different water samples were analyzed to assess the potential of the methodology: potable, treated wastewater, urban, and groundwater, with each of them containing concentrations of glyp (4.1 × 10 −6 and 5.9 × 10 −6 mol/L) added intentionally. The analyses were carried out, in 1 mL of a 99% methanol, 1% water solution. Treated wastewater was filtered to remove suspended solids. The four water samples were stored at 4 • C until used and physicochemically characterized by following conventional methods: pH, specific conductance, Chemical Oxygen Demand (COD), and Biochemical Oxygen Demand (BOD). Several anion and cation analyses were performed as well: Ca 2+ , Fe 2+ , SO 4 2− , Mg 2+ , NO 3− , NO 3 -N, PO 4 3− , P 2 O 5 , and free chlorine.
All the experimental assays were made in triplicate to assess the repeatability of the results. The statistical analysis of the data was performed using Origin Software V 8.0.

Theoretical Structure of the (NiGlyp(PhDP) 2 ) Complex
All calculations were performed using Density Functional Theory (DFT) [52,53] implemented in Gaussian 16 [54]. Geometry optimizations and frequency analysis were done in a vacuum with B3LYP functional and LANLD2Z basis sets. The local minima were identified with zero number of imaginary frequencies (NIMAG = 0). All calculations were made with no symmetry constraints.

The Interaction between Glyp with (Ni(PhDP) 2 )
As mentioned, the electronic absorption spectrum of (Ni(PhDP) 2 ) showed a characteristic band at 480 nm, indicative of double-bond electron transition metals ( Figure 2, line), which significantly increased in the presence of glyp (1.1 × 10 −5 mol/L); in addition, a new band at 362 nm was observed. The change in absorbance at 480 nm, as well as the formation of a new absorption band, is attributed to the formation of a complex between both species, with measurable characteristics in the ultraviolet-visible boundary region. The complex formation was almost instantaneous, and no additional changes in the absorbance were detected after mixing and measuring immediately (average time 10 seconds).
Water 2019, 11, x FOR PEER REVIEW 5 of 14

The Interaction between Glyp with (Ni(PhDP)2)
As mentioned, the electronic absorption spectrum of (Ni(PhDP)2) showed a characteristic band at 480 nm, indicative of double-bond electron transition metals (Figure 2, line), which significantly increased in the presence of glyp (1.1 × 10 −5 mol/L); in addition, a new band at 362 nm was observed. The change in absorbance at 480 nm, as well as the formation of a new absorption band, is attributed to the formation of a complex between both species, with measurable characteristics in the ultraviolet-visible boundary region. The complex formation was almost instantaneous, and no additional changes in the absorbance were detected after mixing and measuring immediately (average time 10 seconds). Changes in the pH of the glyphosate solution, or the (Ni(PhDPD)2) concentration, did not lead to improved results. Therefore, the pH of the water was kept at pH 7.0, (Ni(PhDPD)2) of 1.08 × 10 −4 mol/L, and incubation time of 10 s.
As the purpose of the present study is the quantification of glyp in water samples, the dependence of the absorbance change on glyp concentration was determined. As can be seen in Figure 3, the absorbance at 362 nm was dependent on the glyp concentration, with a linear range from 5.9 × 10 −7 to 1.1 × 10 −5 mol/L of glyp.  Changes in the pH of the glyphosate solution, or the (Ni(PhDPD) 2 ) concentration, did not lead to improved results. Therefore, the pH of the water was kept at pH 7.0, (Ni(PhDPD) 2 ) of 1.08 × 10 −4 mol/L, and incubation time of 10 s.
As the purpose of the present study is the quantification of glyp in water samples, the dependence of the absorbance change on glyp concentration was determined. As can be seen in Figure 3, the absorbance at 362 nm was dependent on the glyp concentration, with a linear range from 5.9 × 10 −7 to 1.1 × 10 −5 mol/L of glyp.

The Interaction between Glyp with (Ni(PhDP)2)
As mentioned, the electronic absorption spectrum of (Ni(PhDP)2) showed a characteristic band at 480 nm, indicative of double-bond electron transition metals (Figure 2, line), which significantly increased in the presence of glyp (1.1 × 10 −5 mol/L); in addition, a new band at 362 nm was observed. The change in absorbance at 480 nm, as well as the formation of a new absorption band, is attributed to the formation of a complex between both species, with measurable characteristics in the ultraviolet-visible boundary region. The complex formation was almost instantaneous, and no additional changes in the absorbance were detected after mixing and measuring immediately (average time 10 seconds). Changes in the pH of the glyphosate solution, or the (Ni(PhDPD)2) concentration, did not lead to improved results. Therefore, the pH of the water was kept at pH 7.0, (Ni(PhDPD)2) of 1.08 × 10 −4 mol/L, and incubation time of 10 s.
As the purpose of the present study is the quantification of glyp in water samples, the dependence of the absorbance change on glyp concentration was determined. As can be seen in Figure 3, the absorbance at 362 nm was dependent on the glyp concentration, with a linear range from 5.9 × 10 −7 to 1.1 × 10 −5 mol/L of glyp.  Equation (2) describes the relationship between the two variables, where A is the absorbance at 362 nm, and G is the concentration of glyp (mol/L). The linear regression model fits the data very well, with a correlation coefficient higher than 0.99. Also, other statistical results (analysis of variance and dynamic graphs, i.e., normal probability plot of the standardized residuals, scatter plots of the standardized residual against the predictor variable, and the index plot of the standardized residuals) support the quality of the linear model.
Using Equation (2), the detection limit (LOD) and the quantification limit (LOQ) were calculated. The LOD and the LOQ are numerically equal to 3 and 10 times the standard deviation of the mean absorbance of (Ni(PhDP) 2 ) without glyp (blank absorbance). Substituting these values in Equation (1), a LOD of 2.01 × 10 −7 mol/L (34.98 µg/L) and an LOQ of 9.87 × 10 −7 mol/L (166 µg/L) were determined. These values are good enough for glyp determination in drinking water in the USA, as the EPA Maximum Contaminant Level (the highest level of a contaminant that is allowed in drinking water) of 700 µg/L has been set [56], and, in principle, should be higher for environmental water samples. A study by Botta et al. [57] reported concentrations of glyp of 4.4 × 10 −7 -5.32 × 10 −7 mol/L (75-90 µg/L) in surface water. Therefore, it is possible to find high concentrations of glyp in environmental water that are within the LOD and LOQ obtained in this work. However, an improvement in detection is needed for application of the method in European countries, where the maximum level of glyp must not be higher than 0.1 µg/L [58]. In addition, our LOD and LOQ values are within the range of other alternative methods to detect the herbicide in a water sample (Table 1), with the advantages that the response time is shorter and there is no need for derivatization or the addition of reaction precursors.

Stoichiometry
For the complex stoichiometry, determination of the absorbance changes was plotted as a function of the mole fraction of the Glyp or (Ni(PhDP) 2 ) ( Figure 4) at two total concentrations. The Job plots show a triangular shape, which according to literature suggests a strong molecular interaction between the compounds; also, the maximum point in the curves takes place at 0.5 mole fraction, indicating that the molecular association occurs with stoichiometry 1:1 [50,61].

Dissociation Constant
The Kd was determined by fitting the data to the one-site binding model using nonlinear regression analysis ( Figure 5). The obtained Kd values were 1.75 × 10 −6 and 6.9 × 10 −6 mol/L at two (Ni(PhDP)2) concentrations (3.6 × 10 −5 and 1.08 × 10 −4 mol/L), which account for the affinity of the dipyrrinate ligand to glyphosate. According to Chenprakhon et al. and Pan et al. [51,62], a small Kd value refers to a high binding affinity of the ligand to its target. Although the affinity values are three orders of magnitude lower compared to the antibody-antigen system, the dipirrinate ligand has the advantage that the analytic response does not need additional steps for separation and quantification, as in the immunoassay format.

Method Selectivity
The interference caused by other components that are usually present in water was determined to know the potential applicability of the method. It is well known that glyp can form strong coordination bonds with metal ions Fe 2+ , Ca 2+ , and Mg +2 [63,64]. The results showed that the presence of individual cations and its mixture did not interfere significantly in the complexation of glyp with (Ni(PhDP)2). The same set of experiments were carried out with other organophosphorus pesticides, which are continuously used in agriculture, such as parathion [65], dimethoate [66], and diclofenthion [67], as well as their mixtures. The absorbance values of the (NiGlyp(PhDP)2) complex in the presence of the organophosphorus pesticides and its mixtures did not show an interference

Dissociation Constant
The K d was determined by fitting the data to the one-site binding model using nonlinear regression analysis ( Figure 5). The obtained K d values were 1.75 × 10 −6 and 6.9 × 10 −6 mol/L at two (Ni(PhDP) 2 ) concentrations (3.6 × 10 −5 and 1.08 × 10 −4 mol/L), which account for the affinity of the dipyrrinate ligand to glyphosate. According to Chenprakhon et al. and Pan et al. [51,62], a small K d value refers to a high binding affinity of the ligand to its target. Although the affinity values are three orders of magnitude lower compared to the antibody-antigen system, the dipirrinate ligand has the advantage that the analytic response does not need additional steps for separation and quantification, as in the immunoassay format.

Dissociation Constant
The Kd was determined by fitting the data to the one-site binding model using nonlinear regression analysis ( Figure 5). The obtained Kd values were 1.75 × 10 −6 and 6.9 × 10 −6 mol/L at two (Ni(PhDP)2) concentrations (3.6 × 10 −5 and 1.08 × 10 −4 mol/L), which account for the affinity of the dipyrrinate ligand to glyphosate. According to Chenprakhon et al. and Pan et al. [51,62], a small Kd value refers to a high binding affinity of the ligand to its target. Although the affinity values are three orders of magnitude lower compared to the antibody-antigen system, the dipirrinate ligand has the advantage that the analytic response does not need additional steps for separation and quantification, as in the immunoassay format.

Method Selectivity
The interference caused by other components that are usually present in water was determined to know the potential applicability of the method. It is well known that glyp can form strong coordination bonds with metal ions Fe 2+ , Ca 2+ , and Mg +2 [63,64]. The results showed that the presence of individual cations and its mixture did not interfere significantly in the complexation of glyp with (Ni(PhDP)2). The same set of experiments were carried out with other organophosphorus pesticides, which are continuously used in agriculture, such as parathion [65], dimethoate [66], and diclofenthion [67], as well as their mixtures. The absorbance values of the (NiGlyp(PhDP)2) complex in the presence of the organophosphorus pesticides and its mixtures did not show an interference

Method Selectivity
The interference caused by other components that are usually present in water was determined to know the potential applicability of the method. It is well known that glyp can form strong coordination bonds with metal ions Fe 2+ , Ca 2+ , and Mg +2 [63,64]. The results showed that the presence of individual cations and its mixture did not interfere significantly in the complexation of glyp with (Ni(PhDP) 2 ). The same set of experiments were carried out with other organophosphorus pesticides, which are continuously used in agriculture, such as parathion [65], dimethoate [66], and diclofenthion [67], as well as their mixtures. The absorbance values of the (NiGlyp(PhDP) 2 ) complex in the presence of the organophosphorus pesticides and its mixtures did not show an interference greater than 10% and thus are discarded as interferents. A mixture was tested between (NiGlyp(PhDP) 2 ) and AMPA because AMPA is frequently detected in water together with glyp [68,69]. Interestingly, the metabolite showed no additional change in the detection of glyp. Finally, different phosphate salts were tested, as it is already known that the phosphate ion may form complexes with nickel and other metals [70][71][72], which also showed no interference. All the assays suggest a good selectivity of the method (Table 2). 1.08 × 10 −4 mol/L and glyp 4.1 × 10 −6 mol/L as 0% of interference, minus the absorbance of the complex in the presence of interference between the absorbance of the complex, multiplied by one hundred. Table 3 shows the results of the physicochemical parameters of the four water matrices used to check the applicability of the proposed method. The parameter values are indicative of the different sources; as expected, treated wastewater shows the highest COD and BOD. The amount of dication metals was determined because of their possible interference behavior. No glyp was detected using the commercially available ELISA method (PN 500086) (Abraxis LLC, Warminster, PA, USA). Then, the samples were spiked with glyp (4.1 × 10 −6 and 5.9 × 10 −6 mol/L) and added to the (Ni(PhDP) 2 ) solution. The glyp concentrations in the samples were calculated using Equation (2).

Analysis of Spiked Water Samples
The percentages of recovery vary between 87.20-119.04% (Table 4), which indicates good accuracy of the methodology. Furthermore, a high precision was obtained, as reflected by the coefficients of variation (0.34-2.89%). Overall, it seems that the presence of several salts and metals at different concentrations did not affect the detection, and the method may be applied for different water sources. It is important to note that the LOD and LOQ reported here suggests that the method may be used for screening purposes in heavily polluted water samples, such as those in agricultural lands or treatment facilities, where the pollutants are concentrated. Regarding urban or groundwater, where pollutants are in lower concentrations, a preconcentration step should be necessary, as it is usually carried out with other methods.

Theoretical Results
To predict the possible complex between (Ni(PhDP) 2 ) and glyp quantum (QM), calculations were undertaken, starting the geometry optimization of probable compounds at the DFT level.
The structure complex shown in Figure 6 is the most probable compound found for the reaction of (Ni(PhDP) 2 ) with glyp, where the nickel in the (NiGlyp(PhDP) 2 ) complex expands its coordination number from 4 to 6 with a nitrogen and an oxygen atom of the glyp' carboxyl group, at distances between 1.89-2.08 Å in a distorted octahedral molecular geometry. The calculated bond distances are reported in Table 5. As can be seen, the distance values are similar to those reported in experimental conditions in the literature [55,70]. The oxygen atoms of phosphonic did not interact with the nickel atom because during the optimization this complex is not stable. Although it is known that the glyp could coordinate to nickel in a tridentated fashion, it appears that a heptacoordinate compound, in this case, is not stable. The shorter distance of the oxygen of the carboxyl group indicates a stronger interaction of the glyp with the title compound. Table 5. Bond distances (Å) for the coordination atoms of the optimized structure (NiGlyp(PhDP)2). Experimental values of (Ni(PhDP)2) fragment (a) and Ni(Glyp)2 (b) from the literature are provided for comparison [55,70].

Conclusions
It has been possible to detect glyp by complex formation with stoichiometry 1:1, achieving a LOD of 2.07 × 10 −7 mol/L and an LOQ of 9.8 × 10 −7 mol/L. The method developed assures data repeatability, high sensitivity, and quick detection (10 s).
The method was applied to determine known concentrations of glyp (4.1 × 10 −6 and 5.9 × 10 −6 mol/L) in potable and urban water, as well as groundwater and treated wastewater. The recovery percentages and the coefficients of variation obtained show good precision and accuracy of the method to be applied in environmental samples. The presence of the salts, other organophosphorus pesticides, and phosphates, as well as their mixtures in the water, do not interfere with glyp detection. The theoretical results show that the nickel of (Ni(PhDP)2) forms coordination bonds with the nitrogen and oxygen atoms of glyp in a distorted octahedral molecular geometry.  The oxygen atoms of phosphonic did not interact with the nickel atom because during the optimization this complex is not stable. Although it is known that the glyp could coordinate to nickel in a tridentated fashion, it appears that a heptacoordinate compound, in this case, is not stable. The shorter distance of the oxygen of the carboxyl group indicates a stronger interaction of the glyp with the title compound. Table 5. Bond distances (Å) for the coordination atoms of the optimized structure (NiGlyp(PhDP) 2 ). Experimental values of (Ni(PhDP) 2 ) fragment (a) and Ni(Glyp) 2 (b) from the literature are provided for comparison [55,70].

Conclusions
It has been possible to detect glyp by complex formation with stoichiometry 1:1, achieving a LOD of 2.07 × 10 −7 mol/L and an LOQ of 9.8 × 10 −7 mol/L. The method developed assures data repeatability, high sensitivity, and quick detection (10 s).
The method was applied to determine known concentrations of glyp (4.1 × 10 −6 and 5.9 × 10 −6 mol/L) in potable and urban water, as well as groundwater and treated wastewater. The recovery percentages and the coefficients of variation obtained show good precision and accuracy of the method to be applied in environmental samples. The presence of the salts, other organophosphorus pesticides, and phosphates, as well as their mixtures in the water, do not interfere with glyp detection. The theoretical results show that the nickel of (Ni(PhDP) 2 ) forms coordination bonds with the nitrogen and oxygen atoms of glyp in a distorted octahedral molecular geometry.

Acknowledgments:
Thanks to Marcela Ayala for allowing the use of UNAM supercomputing resources to perform the theoretical calculations, geometry optimizations, and frequency analysis (Project LANCAD-UNAM-DGTIC-293).

Conflicts of Interest:
The authors declare no conflict of interest.