Atrazine (98.8% purity), ametryn (98.5% purity), chlorpyrifos (99.2% purity) and Mmalathion (99.1% purity) were purchased from Sigma-Aldrich. Aluminium sulfate (Al₂(SO₄)₃.18H₂O, analytical grade) and calcium hydroxide (Ca(OH)₂, analytical grade) were obtained from VETEC Química Fina Ltda. Kaolin was purchased from Prominérios Comércio de Minérios Ltda. and commercial sodium hypochlorite was obtained from Indústrias Anhembi Ltda. The concentration of the sodium hypochlorite solution was confirmed by a standard sodium thiosulfate titration²⁵.

Stock solutions of 800 mg L^-1 of the individual pesticides were prepared in pure acetone. All the stock solutions were stored in amber glass bottles at 4 ºC.

The experimental artificial sample was prepared by adding a kaolin suspension with 100 ± 5 NTU turbidity (TB100p MS Tecnopon) and 450 uH apparent color (Alfakit equipment)²¹. Organic and inorganic compounds were not added to artificial water because the aim was to isolate the variables and verify the treatment without interferents. The effect of a real matrix will be investigated in the next step of this work.

2.2. Experimental procedures

2.2.1 Analytical method and recuperation tests

The method of analysis used to quantify the pesticides and to identify the by-products was based on the extraction/concentration of these substances by means of solid phase extraction. Samples of 50 mL were submitted to solid phase extraction employing OPT 3 mL × 60 mg. Agilent cartridges were conditioned with 6.0 mL of methanol and 6.0 mL of ultrapure water in a 12-port vacuum manifold system. The analytes were then eluted using 7.5 mL of ethyl acetate with subsequent quantification by GC-MS.

Parent compound quantification

The extracts were analyzed by GC-MS (Shimadzu GC - 17A and MS - QP 5050) using selected ion monitoring mode. In the experiments, the injection volume was 1 μL and a VF-5ms capillary column (Varian, 30 m, I.D. 0.25 mm, 0.25 μm) was used. A flow rate of 1 mL min^-1 of helium gas, with a constant pressure of 203 kPa and a 1:10 split ratio was used. The oven temperature started from 100 °C then increased at 25 °C min^-1 to 250 °C and finally increased at 15 °C min^-1 to 270 °C. Temperatures were set at 240 °C in the injector and 230 °C in the interface to the detector. The ions monitored for atrazine detection were: m/z 215, 200 and 173; for ametryn: m/z 227, 212 and 170; for malathion: 173, 125 and 93; for chlorpyrifos: 314, 197 and 97. Quantification was performed by external standardization, with the area obtained compared with the appropriate analytical curve (Table 1)²⁶.

By-product identification

The by-products of the pesticides were also analyzed by GC-MS using similar conditions as described for the parent compounds. A split ratio of 1:10, an injection volume of 1 μL and scan mode were used for this analysis. Compounds were identified using fragmentation analysis, and isotope clustering patterns were found with the aid of the NIST library.

Preliminary tests were carried out to evaluate the recovery of the pesticides in the aqueous matrixes used in the experiments to determine the efficiency of the extraction method. The pesticide recovery ratio was determined (in triplicate) by comparing peak areas from water samples spiked with a known amount of non-extracted pure standards. The linearity of the method, calibration equation, limit of detection (LOD) and limit of quantification (LOQ) were also determined (Table 1). The detection and quantification limits were calculated using the standard deviation of the blank (synthetic water extract after simulated water treatment without pesticides) from the following equations²⁷:

(Equation 1).

(Equation 2)

Note: Slope: Angular coefficient of the analytical curve.

Table 1.
Detection (LOD) and quantification (LOQ) limits, mean percentage recovery (% R), calibration equation* and coefficients of correlation (r²) for the experimental solutions.

*Calibration equation was obtained by mixing the pesticides in synthetic water (100 NTU of turbidity) at concentrations of 0.48, 0.40, 0.32, 0.24, 0.16 and 0.08 mg L^-1.

2.2.2 Determining optimum operating conditions for jar test

The tests were carried out with the artificial sample mentioned in Section 2.1 using jar test Trade Lab Ambiental equipment to obtain the ideal condition of turbidity removal. Then, the pair of values "coagulant dosage × coagulation pH" was varied.

An aluminum sulphate solution (1% w/v) was used to facilitate the coagulation in the water and a calcium hydroxide solution (0.5% w/v) was used for adjusting the pH values²⁵.

2.2.3 Jar test procedures

Bench tests were performed in triplicate using the jar test method to reproduce the conventional water treatment systems, i.e., coagulation-flocculation and decantation, followed by filtration. The pesticides were added to synthetic water (pH corrected according to Section 2.2.2) at a concentration of 0.48 mg L^-1 for each compound, and their removal was verified according to Section 2.2.1.

The jar test was performed according to Brazilian standard NBR 12.216: 1992 and by the Practical manual of water analysis from the National Health Foundation to better represent the operations of the treatment plants of water, as follows:

Rapid mixture: dispersion of aluminum sulphate in 1 L of water sample to be treated, with a maximum speed of 100 rpm for 3 min;

Mechanized flocculation: total time of 10 min, with agitation speed of 50 rpm;

Decantation: the sample was left to decant for ~15 min, which corresponds to a sedimentation rate of 1.74 cm min^-1 (treatment plant with capacity of up to 1,000 m³ day^-1).

Filtration of the samples was done by gravity using 125 mm diameter filter paper. In the last step, chlorination, sodium hypochlorite was added in a dosage that resulted in 5 mg L^-1 of chlorine, a concentration as indicated by the Ministry of Health Ordinance Nº 2914 of 12/12/2011. After this process, an aliquot of each test was removed and submitted to the analysis, as described in the following sections.

2.2.4 Acetylcholinesterase inhibitory activity

Determination of the AChE inhibitory activity was carried out according to the Ellman method, modified as follows²⁸. This method is based on the amount of thiocholine released when AChE hydrolyses the substrate acetylthiocholine iodide. The product thiocholine reacts with Ellman’s reagent (DTNB) to produce a yellow compound [5-thio-2-(nitrobenzoate)], which can be detected at 405 nm. In each well of a 96-well plate, 65 μL of PBS (0.2 mol·L⁻¹ phosphate buffer, pH 7.2), 10 μL of sample* and 10 μL of AchE (1300 U mg⁻¹) were added. The mixture was kept at 37 °C for 3 h. Subsequently, 65 μL of 1.00 mmol L⁻¹ acetylcholine iodide and 65 μL of 1.00 mmol L⁻¹ Ellman’s reagent (DTNB dissolved in 0.2 mol L⁻¹ phosphate buffer pH 7.2) were added. Then, the absorbance was measured at 405 nm using a microplate reader (ELX800 Biotec) in triplicate experiments. The enzymatic activity was calculated as a percentage of the velocities of each sample compared to the control. The inhibitory activity was calculated from one hundred percentage subtracted by the percentage of enzyme activity.

*Control - 10 μL of ultrapure water. Chlorine - 10 μL chlorine solution (5 mg L^-1). After filtration - 10 μL solution after filtration. Postchlorination - 10 μL solution after 30 min reaction time with chlorination.

The optimal conditions for the treatment of 1 L of water at 100 NTU were using 20 mL of aluminum sulphate solution (1% w/v) and pH 10.5, or 15 mL of calcium hydroxide solution (0.5% w/v), conditions which provided a turbidity of 0.24 NTU and 0.0 uH apparent color. All results considered in this study meet the standards for turbidity and apparent color, set forth in Brazilian legislation from the Ministry of Health for drinking water standards. Therefore, these were the parameters used in the pesticide removal test in the simulation of conventional water treatment.

As shown in Figure 2, it was observed that after filtration, the pesticides were not removed efficiently. The organophosphorus compounds were removed in a higher percentage (malathion: 62.21 ± 0.01%, chlorpyrifos: 43.8 ± 0.9%) than the triazines (atrazine: 10.8 ± 0.6%, ametryn: 14.8 ± 0.3%). As the flake formation phenomenon in the treatment of water is carried out by electrostatic attraction, it is inferred that the organophosphorus compounds have been removed in greater percentage due to the presence of the phosphate group, which would allow a greater attraction to the particles, and consequently, greater efficiency in the decantation/filtration process.

Figure 2.
Average percentage of pesticide removal after treatment.

After the chlorination, the removal of pesticides increased, and lower and higher percentages were found for atrazine (15 ± 1%) and ametryn (87.7 ± 0.5%), respectively. The organophosphorus malathion and chlorpyrifos were eliminated by 73.2 ± 0.2% and 62.9 ± 0.8%, respectively. Despite the increased removal of pesticides, according to Figure 3, two by-products were detected in the post-chlorination step. According to the NIST library, the first by-product is malaoxon and the second is an ametryn-derived compound, as shown in Figure 3B. Li et al.²⁹ reported that the oxidation of malathion by chlorination generates malaoxon.

Figure 3.
GC-MS chromatogram scan mode: A – After filtration; B – Post-chlorination.

Using the ametryn-derived fragmentation analysis (Figure 4), we found the molecular ion with a mass to charge ratio of 243, the molecular ametryn ion is m/z = 227, resulting in an increase equal to 16 in its mass value. This difference is consistent with the oxidation product of ametryn, derived from the reaction with sodium hypochlorite, generating ametryn sulfoxide, as reported by Lopez and collaborators³⁰. This by-product formation justifies the marked difference in the removal of ametryn compared with atrazine, due to the methyl sulfide group (R-S-CH₃), which is susceptible to reaction with sodium hypochlorite.

Figure 4.
GC-MS mass spectra for ametryn by-product.

There are other studies on water treatment plant simulation that note that the conventional model is not efficient for the removal of pesticides, such as Soares et al.²¹, whose objective was to verify the removal of endosulfan, ethylenethiourea and 1,2,4-triazole. At the end of the experiments, they verified that 54% of endosulfan, 11% of ethylenethiourea and 18% of 1,2,4-triazole were removed²¹. Li et al.²⁹ also found that after the treatment process, the organophosphorus diazinon and tolcoflos-methyl (initial concentration of 50 μg L^-1) were removed by ca. 63% and 49%, respectively, after filtration. These results are very similar to those found in the removal of the organophosphorus pesticides malathion and chlorpyrifos presented here.

According to Table 2, it is observed that the sample containing chlorine did not show significant difference to the control, in other words, chlorine was not the capable of inhibiting the acetylcholinesterase enzyme. Alternatively, it is known that organophosphates have the ability to inhibit such enzymes^31–33, which explains the inhibition generated after filtration, i.e., this treatment was not effective in removing such compounds. There are not studies demonstrating that atrazine and ametryn alone are capable of inhibiting AChE. However, during toxicity bioassays, the atrazine in combination with chlorpyrifos decreased significantly the acetylcholinesterase activity as compared to chlorpyrifos only treatments³⁴. Studies show that atrazine enhances the uptake and facilitates the biotransformations of chlorpyrifos to chlorpyrifos-oxon³⁵.

Table 2.
Acetylcholinesterase activity (AChE).

After chlorination, 50% enzyme inhibition was found, which is an indication that the compounds formed were more toxic than their parents. Based on Figure 3B, we observed the formation of two by-products, malaoxon and ametryn sulfoxide. It is known that the oxidation of organophosphorus generates more toxic products, the oxons, which are more efficient in inhibiting AChE^27–29. In this perspective, we expected that the enzyme inhibition would be accentuated, since malaoxon was generated after the chlorination. The effect of ametryn sulfoxide is still unknown, however this will be investigated in the next step of this work.