Different methods for physical and chemical elimination of strobilurin fungicides from wastewater

Molecular imprinting was developed as a method for selective adsorption. This makes molecular imprinting, a good option for elimination of different environmental interferents especially from wastewater. However, bio-polymeric adsorbents e.g. calcium alginate and chitosan-calcium alginate beads were used in wastewater treatment in different occasions, molecular imprints were found to be of superior elimination capacity than natural adsorbents. Conditions for optimum elimination were concluded to be 4 hrs shaking (150 rpm) at room temperature and pH 10 for 1 g alginate beads to eliminate 210 mg pesticide, pH 4 or 10 for 1 g chitosan-alginate beads to eliminate 480 and 1140 mg pesticide, respectively. However, with the same conditions at pH range 2-8, each gram of molecularly imprinted polymers (MIPs) could be used in elimination of 3290-3760 mg pesticide, and 1 g of molecularly imprinted resin (MIR) could eliminate 3280-3320 mg pesticide, which increased with increasing the shaking time to one day. Also, resins, either alone or in combination with graphene oxide (GO) succeeded to act as a photocatalyst in chemical degradation of more than 65% of strobilurins under visible light, with high elimination capacity.


Introduction
Fungicides, like other pesticides, play a very important role in protection of different crops from many diseases caused by a wide variety of microorganisms.
Although, the innovation of new molecules from natural origin, in order to eliminate or even reduce the environmental impact of toxic hazardous residues remaining after using these fungicides, they still have some toxicity on environment, and sometimes on human being e.g.kresoxim-methyl and pyraclostrobin cause a rapid raise in intracellular calcium and strong depolarization of mitochondrial membrane potential [1]{Regueiro, 2015 #1}{Regueiro, 2015 #1} {Noegrohati, 2018 #9}.The introduction of any contaminants affecting quality of water, is considered pollution [2].{Regueiro, 2015 #1}Therefore, many efforts are needed to reduce and/or eliminate these toxic chemicals from environment safely as possible.
Strobilurin fungicides also when applied at environmentally relevant concentrations may cause mortality to larval and meta morph amphibians, however, chronic exposure effects in amphibians are still unclear [3].
Studies on strobilurins toxicity proved that low concentrations of azoxystrobin residues in the environment can be toxic to many aquatic animals, and invertebrates [4,5].Also showed that pyraclostrobin is potentially genotoxic and cytotoxic to human peripheral blood lymphocytes, neurotoxic to primary cultured cutaneous neurons.This may interfere with some disorders in humans that may lead to autism and other neural diseases [6].
Although a relatively low toxicity has been claimed for this kind of compounds, information about their neurotoxicity is still scarce [1,7,8].
Regarding domestic animals and human being toxicity, kresoxim-methyl and pyraclostrobin were the most neurotoxic compounds causing a rapid raise in intracellular calcium [Ca 2+ ] and strong depolarization of mitochondrial membrane potential [1].Also, it has been approved that strobilurins can decrease mitochondrial function and thus diminished growth and movement in zebra sh [9].
Wastewater treatment techniques are diverse and of different nature, chemical, biological, physical or combination of two or more techniques [2].It is important to choose a technique with good elimination and regeneration properties.Polymeric adsorbents were used as a good candidate for physical elimination of pesticides.
Alginate is a natural polyelectrolyte extracted from seaweed, used in the form of sodium salt, to form hydrogel with water.Dropping of sodium alginate into calcium chloride, leads to its solidi cation in the form beads.These beads, bearing multiple charged sites (COO -) available for electrostatic interaction with water soluble cations.In addition, the gel matrix formed with calcium, forms a good mesh for entrapping and adsorption of foreign materials in wastewater [10,11].
Chitosan is a derivative of chitin, a natural polymer, used in the form of akes, powder or hydrogel beads as a polymeric adsorbent.It is a linear polyelectrolyte that contains many (NH 3 + ) groups at acidic pH.Thus, chitosan forms electrostatic complexes with alginate to build up a physically cross-linked threedimensional matrix.Both, chitosan, and calcium-treated sodium alginate could be used alone or in combination with each other [11,12].
Molecularly imprinted polymers (MIPs) were incorporated in sample preparation and concentration techniques, solid phase extraction methods and electrochemical sensors, due to selective recognition.They were better used for adsorption of molecules in organic solvents.However, molecularly imprinted resins (MIRs) were hydrophilic in nature with large number of amino, imino and hydroxyl groups for molecular binding in aqueous media [13].
Chitosan (CHS) and sodium alginate (ALG) are good examples of natural polymers that may be used in removal of hazardous materials from wastewater [11].
In comparison, molecularly imprinted polymers (MIPs) and resins (MIRs) were synthesized to play the same role with expected higher selectivity and better adsorption-regeneration properties.
Resorcinol-formaldehyde resins (MIR) have a visible-light photocatalytic activity that help in generation of hydrogen peroxide from water molecules [14].Thus, they helped in chemical elimination of fungicides from wastewater.The addition of graphene oxide also helped more in catalytic photodegradation through enhancement of electron transfer [15].Iron-dopped metal halide 400 W visible light lamp to simulate sunlight in photocatalytic degradation procedures.

Preparation of calcium alginate beads (ALG)
Sodium alginate (1.5 g) was dissolved in 100 mL distilled water and dropped by a plastic dropper into a beaker of 10% calcium chloride solution.The formed beads were then washed with distilled water, dried with oven at 60°C till solidi cation, and collected for use, after FTIR characterization [11].

Preparation of chitosan/calcium alginate beads (CHS/ALG)
High molecular weight chitosan (CHS, 1.5 g) was mixed with 5 mL acetic acid, and then distilled water was added to 100 mL.Sodium alginate (ALG, 1.5 g) was dissolved in 100 mL distilled water.Both components were mixed in a ratio (4 CHS: 1 ALG) for 1 hr using a magnetic stirrer and left to stand for 1 hr to obtain a bubble-free mixture.With a plastic dropper, CHS/ALG mixture was dropped in 10% NaOH, washed several times with distilled water till neutral pH and nally the beads were collected and added to 10% CaCl 2 till solidi cation.Beads were ltered and washed with distilled water then dried with oven at 60°C till solidi cation, collected for FTIR characterization, and used for adsorption [11].

Synthesis of molecularly imprinted polymers (MIPs)
Three monomers were used in construction of imprinted cage to bind and eliminate AZX selectively and e ciently.Acrylamide and hydroxypropyl βcyclodextrin were mixed with methacrylic acid in equal ratio of (1.33: 1.33: 1.33 mM), in addition to 10 mM EGDMA cross linker and 0.15 mM benzoyl peroxide to initiate the polymerization reaction around the template of AZX 1 mM.Template, monomers, and cross linker were all mixed together in 5 mL porogenic solvent, acetonitrile, for 5 min under nitrogen, in a screw-capped glass test tube.In a 60°C thermostatically-controlled water bath, the sealed test tube was left over night for 24 hr, together with a blank test tube, without template, for synthesis of MIP and non-imprinted polymer (NIP), respectively [16].
Polymers (MIP, NIP) were collected, washed to remove unpolymerized materials and template, using a mixture of methanol and acetic acid (9: 1 v/v).The wash was scanned spectrophotometrically to ensure the cleanliness of the polymers.The powders were dried and characterized by FTIR and SEM.

Synthesis of molecularly imprinted resin (MIR)
Synthesis of superhydrophilic molecularly imprinted resin (MIR) was taken from Zhou et.al. [13] with some modi cations.Silica gel beads were used as a core of solid support for the imprinted composite resin.Using one-pot condensation method, 0.45 mL formaldehyde was mixed with 0.33 g resorcinol in 30 mL distilled water for 1 hr at 40°C, using a magnetic stirrer, to form mixture I.
In another beaker, 0.23 mL formaldehyde and 0.126 g urea were mixed in 10 mL distilled water at 80°C till dissolve.Then, 0.35 g silica gel was mixed with 0.1 mL formaldehyde and the mixture was added to the beaker to form mixture II.All these components were ultrasonicated, and then added to mixture I. Template (i.e., pyraclostrobin or tri oxystrobin) (0.04 M solution in acetonitrile), was added in 10 mL volume to the whole mixture (I + II), left at 40°C/450 rpm/40 min, and nally the mixture was maintained at 80°C for 16 hrs.The resin was collected and washed thoroughly with methanol/acetic acid/distilled water (3/1/1) mixture, till no peaks detected spectrophotometrically. Another procedure was done for synthesis of non-imprinted resin (NIR), and both MIR and NIR were dried, characterized by FTIR and SEM then kept for use.

Synthesis of graphene oxide (GO)
Graphene oxide was synthesized according to the method mentioned in [17].Three grams of graphite powder and 18 g KMnO 4 were added gradually and successively to a 9:1 mixture of concentrated H 2 SO 4 /H 3 PO 4 (360:40 mL) with stirring at room temperature in open-mouthed ask.Then, the temperature was raised to 50°C with stirring for 18 h.After cooling to room temperature, the mixture was poured onto 400 mL ice with addition of 30 mL H 2 O 2 and slightly stirred with glass bar.The mixture was centrifuged (5000 rpm for 5 min), and the sediment was collected and washed 7 times with 1 L 5% HCl solution each time, and further repeatedly with bi-distilled water (7 times, 1 L each time) until pH of the supernatant solution approached 7.After washing, the mixture was centrifuged (5000 rpm for 5 min), and the supernatant was discarded.The paste-like product was collected and dried at 60°C under constant vacuum for 2 days.The dried sample was ground, and sent for characterization using X-ray diffraction, FTIR and particle size measurement using TEM, to be used in other procedures.

Elimination procedures
A weighed amount of 0.1 g from CA, CHS/ALG and MIP adsorbents were added separately to bottle sets containing certain volume of AZX stock solution in acetonitrile diluted with distilled water of pH adjusted with NaOH and HCl.Another set of bottles were prepared in the same way using MIR as adsorbent and PYR as pesticide.The four sets were shaken at 150 rpm at room temperature.A sample was collected and ltered at zero time and after different time intervals to be measured spectrophotometrically for detection of AZX or PYR in solution, to evaluate the degree of elimination by each adsorbent [11].Blank experiment was performed to exclude the adsorption by glassware.
A group of three beakers were prepared as follows; beaker 1, TRI was added to distilled water together with 0.1 g MIR, beaker 2, 0.1 g GO was added to TRI and MIR, and beaker 3, TRI only as a control.These beakers were subjected to visible light (after 2 hrs stirring in dark for equilibrium) and stirred with magnetic stirrers to avoid coagulation of powders.A similar group of three beakers were kept in dark to cancel any other factors that may interfere with photodegradation.
The experiment was repeated three times to calculate the average amount eliminated per gram adsorbent under equilibrium conditions [11]: where (Q e ) is the capacity of elimination by adsorbent in (mg/g), C0 and Ce are the initial and nal concentration of AZX, PYR or TRI in (mg/L), V is the solution volume in (L) and m is the adsorbent mass in (g).
Another set of bottles were prepared as follows; AZX and NIP, AZX and MIR, PYR and NIR, PYR and MIP in the same way as mentioned above at pH 6. Capacity of elimination was calculated for each bottle to be used in evaluation of imprinting together with selectivity of MIP and MIR [18]: where (IF) is the imprinting factor that measure the magnitude of successful imprinting when its value is above unity.
where (α) is the selectivity factor that re ects the selectivity of imprints to the analyte of interest.

Evaluation procedures
Stock solutions of AZX, PYR and TRI were prepared in concentration 10 mg/ 100 mL acetonitrile and refrigerated for working standards preparation.Calibration curves were constructed by scanning a serial dilution set of AZX, PYR and TRI, spectrophotometrically, at wavelength λ max 245, 275 and 250 nm respectively.
This procedure helped in evaluation of washing step, in synthesis of MIP and MIR, and extent of elimination (Q e ).

Application on real wastewater samples
A volume of 5 mL Amistar® was diluted to 100 mL water, and the other fungicides were prepared by dissolving 10 g of Bellis® and Flint® in 100 mL water for each.All fungicides were applied on a eld (175 m 2 ) of cucumber in different intervals.After irrigation, different samples of wastewater drained were collected.Samples were ltered by lter paper and measured spectrophotometrically.Further dilutions may be needed to reach concentrations relevant to the linearity ranges of AZX (2.5-20 µg/mL), PYR (3-20 µg/mL) and TRI (3-25 µg/mL).Samples pH was measured to help in choosing the proper adsorbent.Adsorbents were added in 0.1 g for AZX and PYR.However, in case of TRI samples, 0.1 gm MIR was added to two beakers in addition to 0.1 gm GO in one of them.Zero readings were recorded, then elimination procedures were applied.Samples were measured spectrophotometrically to evaluate the elimination e ciency.

Characterization of adsorbents
Functional group analysis was studied by fourier transform infrared analysis (FTIR) for CA beads, CHS/ALG beads, MIPs, MIRs and their initial components, showed the disappearance of initials and formation of new product with different properties, because of functional group changes, gures (1, 2 and 3).It was noticed that both imprinted and nonimprinted polymers have similar peaks, with those of nonimprinted more intense due to the absence of any self-assembly and random distribution of functional monomers without any cavity formation as in imprinted ones [18].
Surface morphology was studied for both molecularly imprinted polymers and resins (together with their nonimprinted powders).

( )
Molecularly imprinted polymers ( gure 4) showed formation of cavities in the polymerization process in presence of AZX template ( gure 4, a), however, in absence of it, no cavities were constructed ( gure 4, b).
Molecularly imprinted resin SEM images showed the large particle size due to the presence of silica gel core.However, these particles were smaller in MIR ( gure 5, a) than in NIR ( gure 5, b), due to the imprinting reaction that allowed resin to be arranged around template molecules.

Optimization of elimination conditions
Time and pH for optimum adsorption and hence elimination of some strobilurins, were studied.Effect of pH on elimination was studied over the pH range 4-11 for both alginate and chitosan/alginate beads as they dissolve in highly acidic solutions.For alginate beads, it was noticed that after 4 hrs of shaking in room temperature at pH 10, the capacity of elimination was 210 mg/g for AZX.However, chitosan/alginate beads showed elimination capacity of 480 and 1140 mg/g at pH 4 and 10, respectively.After one day, adsorption capacity was decreased.
Capacity of MIP to eliminate AZX from aqueous solutions ranging from pH 2 to 11, was studied.A great improvement in elimination capacity (3290-3760 mg/g) was proved at pH 2-8, after reaching equilibrium.Few traces of AZX were detected after one day of shaking, however it was almost diminished after shaking for two days with MIP.After days, the substrate remained bounded to MIP adsorbent, thus elimination capacity was maintained over a large period.
Regarding MIR, its capacity of PYR elimination was comparable to that of MIP at pH 2-8.After equilibrium, it reached (3280-3320 mg/g) by shaking for one day and only few traces of PYR were detected after the second day.No decrease in elimination capacity by time.
Figure 7 shows the effect of pH on elimination of AZX from aqueous solution.Azoxystrobin solubility (6 mg/L) in water, together with its behavior in binding with different types of adsorbents studied, showed that the adsorption mechanism depends on Van der Waals forces without any ionic interaction [11].
Molecularly imprinted resin also showed a great elimination of PYR pesticide, ( gure 7), due to its superhydrophilic nature that helped much more in adsorption of PYR in aqueous medium.
For photocatalytic degradation, it was reported that MIR needed 18 hrs to activate photocatalysis of TRI (after equilibrium).This period was reduced to 12 hrs after addition of GO that accelerated the chemical degradation process induced by the same visible light source under the same conditions of stirring and room temperature.

Evaluation procedures
The developed method for spectrophotometric analysis used in evaluation was characterized and validated in table 1.
Table 2 showed the calculated capacity of elimination (Qe) for the four types of adsorbents at different pH ranges, and for MIR alone and with GO.Also, imprinting factor (IF) and selectivity (α) were calculated for both imprints (MIP and MIR).It showed that the addition of chitosan to alginate, increases the solidi cation of calcium alginate to form cavities ready for binding with adsorbate either in acidic or basic pH, in comparison with chitosan alone that works in basic pH only.In addition, more functional groups e.g.NH 2 were added to increase the binding capacity [11].This explains the better elimination capacity by chitosan/alginate beads rather than alginate alone.
Table 2 also showed values of imprinting factor (IF) for MIP and MIR, that re ected the success of imprinting procedures in synthesis of them.Selectivity coe cient (α) was calculated and explained the higher selectivity of both MIR and MIP.
In case of MIP, the addition of hydroxypropyl β-cyclodextrin to other monomers, built up a cup-like cavity that form multiple inclusion complexes with AZX, thus improving its elimination from wastewater greater than alginate and even chitosan/alginate beads.Also MIP could withstand different ranges of acidic pH, in addition to stability of adsorbate binding with time.However, the presence of silica core in MIR particles, made them arranged in a bed that offered a large adsorption surface area and was easily ltered or even may be packed in a column for wastewater puri cation, if needed.
Molecularly imprinted resins (MIR) under visible light, forms centers for charge carriers that serve as electron acceptors in photocatalysis [15].Addition of graphene oxide to MIR increased their ability for photocatalytic degradation of TRI in visible light, this was attributed to the enhancement of charge transfer driven by the honeycomb SP 2 network structure of GO [19].
All these advantages made the molecular imprinting technique superior in wastewater puri cation (physically or chemically) than natural adsorbents e.g., chitosan and alginate.

Application on real samples
As the measured pH of the real samples was neutral to slightly acidic, alginate and chitosan/alginate beads failed to eliminate AZX.However, MIP and MIR succeeded to eliminate AZX and PYR, with elimination capacity of 3600 mg/g and 3000 mg/g, respectively.
Molecularly imprinted resin helped in photodegradation of TRI in wastewater samples with elimination capacities of 5100.3 mg/g and 5200 mg/g after addition of GO, respectively.

Figure 1 FTIR
Figure 1 FTIR charts of MIP-AZX and its components together with NIP

Figure 2 FTIR
Figure 2 FTIR peaks of chitosan, alginate and their beads

Figure 3 FTIR
Figure 3 FTIR peaks of MIR and its components together with NIR

Figure 4 SEM
Figure 4 SEM images of MIP and NIP showing the difference in arrangement of formed cavities in MIP and solid surface in NIP

Figure 5 SEM
Figure 5 SEM images of MIR and NIR showing the difference in particle sizes as a result of arrangement of resin around template in MIR showing more cavities for binding Field Emission Gun) scanning electron microscope (SEM) (FEI Company, USA) was used for surface imaging of MIPs, NIPs, MIRs and NIRs (10.1 mm working distance, with in-lens detector, and excitation voltage 20 kV).

Table 2 .
Capacity of elimination of different adsorbents at different pH ranges for AZX and PYR, degree of photodegradation of TRI by MIR and MIR/GO at neutral pH, imprinting factor and selectivity of MIP and MIR at neutral pH