Preconcentration and determination of Dithiocarbamate esticides (DTCs) using 1-(2-pyridylazo)-2-naphthol (PAN) modified -cyclodextrin polymer

Pesticides are efficient tools for preventing crop losses due to plant pests and disease. Amongst the different classes of pesticides, dithiocarbamates (DTCs) present an important class of organosulfur pesticides, widely used in agriculture. Dithiocarbamates do not belong to the systemic fungicide but are protectant fungicide applied prior to fungus infection. They act upon damaging fungi chiefly by surface deposits [454]. They are characterized by a broad spectrum of activity against various types of plant pathogens, low acute mammal toxicity, and low production cost. Furthermore, DTCs are also used clinically for the treatment of chronic alcoholism and as anticancer and antitoxic drug agents [455-56]. Dithiocarbamates can be absorbed by the organism via the skin, mucous membranes, respiratory and the gastrointestinal tracts. Dithiocarbamates are known to have toxicological and mutational effects. They possess variety of applications in agriculture as fungicides, as well as, in the rubber industry as vulcanization accelerators and antioxidants. Determination of DTCs is required for toxicological evaluations since the DTCs and their metabolites differ greatly in their action mechanism [457]. Maneb [Mn(II) ethylene bis{dithiocarbamate}] is an agricultural dithiocarbamate fungicide used on a wide variety of plant fungi and diseases. It may be applied to the foliage of plants, but it is also used for soil or seed treatment. Maneb is used primarily for almonds and stone fruits (drupes). Zineb [Zn(II) ethylene bis{dithiocarbamate}] is used as a fungicide to prevent crop damage in the field and to protect harvested crops from deterioration in storage or transport. Zineb is a foliar fungicide and is used to control late blight of potatoes, early blight of tomatoes and downy mildew. Ziram [Zn(II) bis{dimethyldithiocarbamate}] is a vegetable fungicide and is particularly used against anthracnose of tomatoes. Ferbam [Fe(III) tris{dimethyldithiocarbamate}] is an agricultural dithiocarbamate pesticide and is used as protective fungicide. The predominant methods for determining DTCs and metabolites are based on their decomposition to CS2, H2S or amines in an acidic medium, followed mainly by spectrometry [458] and head space gas chromatography [459] determination. One of the earliest methods for Zineb determination was based on the colorimetry [460]. Crnogorac have provided insight into the various methods for the residues analysis of dithiocarbamate pesticides [461]. HPLC and AAS were used to distinguish Propineb, Zineb, Maneb and Mancozeb fungicides [462]. GC-MS based method was devised and used for the determination of DTCs in foodstuffs [463]. Dithiocarbamates can also be determined by methods, like iodometry [464], polarography [465], biosensors [466], enzyme linked immune sorbent assay [467], indirect complexometry [468], FTIR spectrometry [469], microwave-assisted extraction [470] and derivative spectrophotometry [471]. Liquid chromatography (LC) coupled capillary electrophoresis (CE) with UV and /or electrochemical detection are the techniques most frequently used to discriminate and determine the different DTCs subclasses [472-75]. -Cyclodextrin (-CD) is a very stable oligosaccharide that is composed of seven glucose units linked with each other by -(1,4)-glycosidic linkage. It can form supramolecular complexes with several organic compounds by incorporating them into their hydrophobic cavities. When two or more -Cyclodextrins are covalently linked with each other they are known as the polymers. These -cyclodextrin polymers have been used for the preconcentration of various analytes [476-78]. Here is presented a method that is based on the preconcentration of dithiocarbamate pesticides such as Ziram, Zineb, Ferbam and Maneb using 1-(2-Pyridylazo)-2-naphthol (PAN) modified -Cyclodextrin polymers. Metallic part of the Dithiocarbamate pesticides form chelated complex with the PAN included in the cavity of -CD polymer. Various analytical parameters such as effect of pH, sorbent dose, shaking time, sample volume, eluent conc. and eluent volume were optimized using batch extraction procedure. The effect of the foreign ions (anions, cations and other dithiocarbamate pesticides) is also studied on individual DTC pesticide. The preconcentration factor has also been studied for each of the pesticides. The preconcentration factor is determined as the ratio of the largest sample volume to smallest eluent volume. The developed method is applied for the preconcentration/determination of pesticides in different water and food samples. The following reaction between the metallic part of the pesticides and the PAN loaded on to -Cyclodextrin polymer forms the basis of the determination of pesticide by above method.

Mn+-[ethylene bis(dithiocarbamate)] + PAN = PAN- Mn+-[ethylene bis(dithiocarbamate)] (EBDC pesticide) (Colored complex)For Mn+: Zn(II), Mn(II)EBDC: Ethylene bis (dithiocarbamate) pesticide

5.1 Preconcentration of Zineb using -CDP-PAN polymerThe present work describes preconcentration of Zineb using -CDP-PAN polymer5.1.1 Materials5.1.1.1 EquipmentA Shimadzu UV-1800 spectrophotometer (Shimadzu Ltd., Japan) equipped with the matched 10-mm quartz cells was used to measure absorbance. All pH measurements were performed using Digital century pH-meter CP 901 with a combined glass electrode. A thermostatic shaking water bath (Perfit India Ltd.) was used to carry out all the inclusive procedures.5.1.1.2. ReagentsAll reagents used were of analytical reagent grade. Double distilled water was used throughout the experiment.1 10-2M Zineb was prepared as given in literature [459]. Its stock solution was prepared in dimethyl sulphoxide (DMSO). Further dilutions were made as and when required. 4 10-6M solution of the PAN reagent was prepared by dissolving an appropriate amount of PAN (Fluka Chemical Company) in N,N-dimethylformamide (DMF) solvent. 1,4-Butanediol diglycidyl ether was obtained from sigma Aldrich chemical company (U.S.A.). -Cyclodextrin was obtained from SD fine chemical India private limited (Mumbai). Buffer solution used were hydrochloric acid/ sodium acetate for pH 2.0-3.5, sodium acetate/acetic acid for pH 4.0-6.5, ammonia/ammonium chloride for pH 8-11. Glass wares were washed with chromic acid and soaked in 5% nitric acid and rinsed with double distilled water. 5.1.2 Procedure5.1.2.1 Batch Extraction ProcedureAt room temperature, -CDP-PAN (500mg) and 10.0 ml of buffer solution (pH 9.5) were added to a 100-ml Stoppard conical flask. The mixture was allowed to stand for approximately 15 min so that -CDP-PAN could swell sufficiently. 60g of Zineb were added and made up to 100ml with double distilled water. After the mixture was shaken in the thermostatic shaking water bath for 45 min, 5.0ml of the supernatant solution was transferred into a 10ml volumetric flask and the absorbance was measured using standard spectrophotometric method [394]. Zineb retained on -CDP-PAN polymer was eluted using 5.0 mL of 2M HCl.5.1.3 Optimization of various parameters5.1.3.1 Effect of pH on % uptake of ZinebThe uptake of an analyte on a with PAN on the chelating polymer is dependent on the pH of sample solution due to the competitive reaction between chelate forming groups and hydrogen ions in the solutions. 60g. of Zineb were added to a 100 mL Stoppard conical flask. The pH of this solution was adjusted in the range of 2.5 to 10.5 using different buffer system and then the preconcentration procedure as described was applied. Quantitative uptake ( 95%) was obtained at pH 9.5 and above (Fig. 5.1.1). Therefore, 9.5 was chosen as the working pH for the subsequent studies. 5.1.3.2 Effect of the amount of polymer The amount of the polymer is another important parameter that affects the uptake of an analyte. A quantitative uptake ( 95%) cannot be achieved when the polymer is less than the optimum amount. On the other hand, an excess amount of polymer prevents the quantitative elution of the retained analyte by a small volume of the eluent. In order to optimize the smallest amount, 100-800 mg. of the polymer were added to the same volume of the sample containing 60g. of Zineb and preconcentrated by the general procedure. The quantitative uptake ( 95%) was obtained for and above 500 mg of polymer (Fig. 5.1.2). Therefore, 500 mg of polymer has been used for subsequent studies.5.1.3.3 Effect of shaking timeShaking time is an important factor in determining the possibility of application of the -CDP-PAN polymer for the selective uptake of an analyte. Different shaking time (ranging from 15 to 75min) were studied for the % uptake of Zineb by -CDP-PAN polymer. The percentage uptake reached maximum (above 95%) at 45 min (Fig. 5.1.3). Therefore, the shaking time of 45 min. was selected as the uptake equilibrium time.5.1.3.4 Effect of the sample volume on % uptake of ZinebIn order to explore the possibility of enriching low concentration of analytes from large sample volume, the effect of sample volume on the uptake of Zineb was also investigated. For this purpose, 25, 50, 100, 150, 200, 250, 300, 350, 400 mL of sample containing 60 g. of Zineb were taken. Quantitative uptakes ( 95%) were obtained for sample volume of 350 mL (Fig. 5.1.4). But for convenience of handling, 100 mL of sample volume was adopted for the preconcentration of analyte.5.1.3.5 Effect of shaking speed (r.p.m.) on % uptake of ZinebThe % uptake of Zineb increased gradually with a rise in the shaking speed (Fig. 5.1.5). The batch extraction technique is based on the choice of shaking speed that helps to improve the mass transfer. Shaking speed here acts as a driving force. The central dogma is that the increasing driving force could help in mass transfer and facilitate the concentration gradient between the sample solution and the polymer. Therefore, the present study suggests that the shaking effect is high-flying parameter for the maximum % uptake of an analyte ion. 5.1.3.6 Effect of Nature of Eluent on % uptake of ZinebIn order to choose the best eluent for the Zineb retained on -CDP-PAN polymer, various eluent were used. Among the eluents studied, the acids provided higher recovery efficiency than the organic solvents. From the results, it is obvious that higher uptake was obtained with Hydrochloric acid (HCl). 5.1.3.7 Effect of eluent conc. Since the uptake of Zineb at pH 2 is quite low, one can expect that elution will be favored in the acidic medium. Therefore, effect of the eluent concentration on the % uptake of Zineb was also examined. Different concentrations of HCl ranging from (0.5-4M) were tested in order to strip the Zineb from polymer. The uptake of Zineb increased, as HCl concentration increased up to 2.0M and it decreased above this concentration. Therefore an HCl concentration of 2.0 M was selected for subsequent studies (Fig. 5.1.6).5.1.3.8 Effect of Eluent volumeIn order to choose proper volume of the eluent, the retained complex was stripped with different volumes (16 mL) of 2.0 M HCl. It is clear that 5 mL would not be suitable because it gave a smaller preconcentration factor and 3mL was not sufficient for the elution (Fig. 5.1.7). Hence, 5 mL of 2.0 M HCl was chosen for elution of Zineb. The preconcentration factor is calculated by the ratio of the highest sample volume (350 mL) and the lowest eluent volume (5 mL). Thus, the preconcentration factor found was 70.

Fig. 5.1.1 Effect of pH on % uptake of Zineb

Fig. 5.1.2 Effect of amount of polymer on % uptake of Zineb

Fig. 5.1.3 Effect of shaking time on % uptake of Zineb

Fig. 5.1.4 Effect of sample volume on % uptake of Zineb

Fig. 5.1.5 Effect of shaking speed (r.p.m.) on % uptake of Zineb

Fig. 5.1.6 Effect of eluent concentration on % uptake of Zineb

Fig. 5.1.7 Effect of eluent volume on the % uptake of Zineb

5.1.4 Effect of Foreign A sample containing 60g. of Zineb, various foreign ions (both anions and cations) and other dithiocarbamate pesticides were prepared and general procedure was followed. The tolerance limit was defined as the amount of foreign ions causing a change less than 5% in the uptake of Zineb (Table 5.1.1).

Table 5.1.1 Effect of foreign ions on the determination of 60g. of ZinebForeign ionsTolerance limit[WForeign ion/WZineb]

NO3-, SO42-, HPO42-, SCN-, NO2-, PO43- >1000

Na+, K+, Mg2+, Ba2+, Al3+, Rb+, Cs+, Ag+1000

Sb3+, Ca2+, Zr4+, Ti500

Th4+, Sn2+, As3+200

aFerbam, bNi2+, cCu2+, dCo2+10

eHg2+, eCd2+, fPb2+, gFe2+, hManeb1

EDTA, Br-, F-, CN-, citrate1

a-masked with 1.0 mL of 5.0% ammonium oxalate solution; b-masked with 1.0 mL of 2.0% dimethylglyoxime; c-masked with 1.0 mL of 3.0% sodium thiosulphate; d-masked with 1.0 mL of 10.0% -benzilmonoxime; e-masked with 5.0 mL of 2.0% sodium thioglycollate solution; f- masked with 2.0 mL 0f 1.0% sodium sulphate solution; g-masked with 1.0 mL of 2.0% 1,10-phenenthroline; h-masked with 2.0 mL 3.0% sodium hexametaphosphate solution.

5.1.5 Validation of the methodThe validity of the method was checked by applying it for the determination of Zineb in water and vegetable samples. 5.1.5.1 Determination of Zineb in water samplesWater samples were collected from Punjabi University, Patiala. The water samples were immediately filtered through cellulose membrane filter (0.45 m pore size), and stored in precleaned polyethylene bottles. After that, pH of the sample was adjusted to 9.5 and the preconcentration procedure as described above was applied (Table 5.1.2).5.1.5.2 Determination of Zineb in vegetables/cropsThe method was applied for the determination of Zineb in crops and vegetable samples. A known amount of Zineb in dimethyl sulphoxide (DMSO) was crushed with 10 g. of crops and vegetable samples with the help of a pestle and mortar. The mixture was then stirred with magnetic stirrer for 1h to provide complete dissolution of Zineb and then filtered to separate the food residue from the solution containing Zineb. The residue was washed with DMSO to provide complete extraction of Zineb to the solution. Filtrate and washings were combined and evaporated to 20.0 mL on a water bath, diluted to 100 mL with DMSO and determined by the developed method. The results are depicted in (Table 5.1.3).

Table 5.1.2 Determination of Zineb in different spiked water samples (n=3) Sample Spiked(g.) Found(g.) % Relative % Recovery Error R.S.D.(%)

^Tap Water 0.0 N.D. ------- ------- 40.0 39.6 1.0 99.0 1.0 45.0 44.5 1.1 99.0 1.0 #Mineral Water 0.0 N.D. ------- ------- 25.0 24.6 1.6 98.4 1.6 20.0 19.6 2.0 98.0 1.3$Tap Water 0.0 N.D ------ ------- 30.0 29.5 1.7 98.3 1.2 50.0 49.5 1.0 99.1 1.0

N.D. not detected *n is the average of three replicate determinations ^Tap water samples were collected from Punjabi University, Patiala #Bottled Mineral water (Bisleri) locally available in market $Tap water samples collected from Urban Estate Phase-II Patiala

Table 5.1.3 Determination of Zineb in different vegetables/crops (n=3) ^Sample Spiked(g.) Found(g.)a % Relative % Recovery Error R.S.D.(%)

Wheat 0.0 N.D. ------ -------- 20.0 19.4 3.0 97.0 2.2 40.0 38.9 2.8 97.2 1.8 Rice 0.0 N.D. -------- -------- 15.0 14.6 2.7 97.3 2.0 35.0 34.0 2.8 97.1 2.0 Potato 0.0 N.D. -------- -------- 60.0 58.3 2.8 97.2 2.0 65.0 63.2 2.8 97.2 2.1

N.D. (not detected) *n is the average of three replicate determinations ^Samples were collected from local market in vicinity of Punjabi University, Patiala

5.1.6 Accuracy of the methodThe accuracy of the described preconcentration method was tested in the recovery studies by adding known amounts of Zineb to the water and food sample. The results obtained from the analysis of water and food samples were satisfactory. These results confirm the validity of the proposed method.

Table 5.1.4 Various parameters studied for the preconcentration of Zineb using -CDP-PAN polymer as extractant.Parameters Studied Range Selected Value pH 2.5-10.5 9.5Volume of buffer(mL) 6-18 10Shaking Time(min) 15-75 45Adsorbent Dose 100-700 500Sample Volume(mL) 50-450 100 Eluent Concentration(M) 0.5-4 2Eluent Volume(mL) 1-6 5Preconcentration factor -------- 70

5.2. Preconcentration of Ferbam using -CDP-PAN polymerThe present work describes the preconcentration of Ferbam using -CDP-PAN polymer.5.2.1. Materials5.2.1.1 EquipmentsEquipments are same as described in section 5.1.1.1.5.2.1.2 ReagentsFerbam sample was prepared as given in literature [459]. Its stock solution was prepared in dimethyl sulphoxide (DMSO). Further dilutions were made as and when required. 5.2.2 Procedure5.2.2.1 Batch Extraction procedureAt room temperature, -CDP-PAN (400mg) and 10.0 ml of buffer solution (pH 5.5) were added to a 100 mL Stoppard conical flask. The mixture was allowed to stand for approximately 15 min so that -CDP-PAN could swell sufficiently. 75g of Ferbam were added and made up to 100ml with double distilled water. After the mixture was shaken in the thermostatic shaking water bath for 40 min, 5.0ml of the supernatant solution was transferred into a 10ml volumetric flask and the absorbance was measured using standard spectrophotometric method [394]. Ferbam retained on -CDP-PAN polymer was eluted using 4.0 mL of 3M HCl.5.2.3 Optimization of various parameters5.2.3.1. Effect of pH on % uptake of FerbamThe uptake of an analyte on the chelating polymer is dependent on the pH of sample solution due to the competitive reaction between chelate forming groups and hydrogen ions in the solutions [461]. 75 g of Ferbam were added to a 100 mL Stoppard flask. The pH of this solution was adjusted in the range of 2.5 to 10.5 using different buffer system and then the preconcentration procedure as described was applied. Quantitative uptake ( 95%) was obtained at pH 5.5 (Fig. 5.2.1). Therefore, the working pH was chosen as 5.5 for the subsequent studies.

5.2.3.2. Effect of the amount of polymer on % uptake of FerbamThe amount of the polymer is another important parameter that affects the uptake of the analyte. A quantitative uptake ( 95%) cannot be achieved when the polymer is less than the optimum amount. On the other hand, an excess amount of polymer prevents the quantitative elution of the retained analyte chelate by a small volume of the eluent. In order to optimize the smallest amount, 100, 200, 300, 400, 500 mg. of the polymer were added to the same volume of the sample solution containing 75 g. of Ferbam and preconcentrated by the general procedure. The quantitative recoveries were obtained for and above 400 mg of polymer (Fig. 5.2.2). Therefore, 400 mg of the polymer has been used for subsequent experiments.5.2.3.3. Effect of shaking time on % uptake of FerbamShaking time is an important factor in determining the possibility of application of the -CDP-PAN polymer for the selective uptake of Ferbam. Different shaking time (ranging from 10 to 50 min.) were studied for the % uptake of Ferbam by -CDP-PAN polymer. The results of % uptake of Ferbam vs. the shaking time show that the percentage uptake reach maximum (above 95%) at 40 min (Fig. 5.2.3). Therefore, the shaking time of 40 min. was selected as the adsorption equilibrium time.5.2.3.4. Effect of the sample volume on % uptake of FerbamIn order to explore the possibility of enriching low concentration of analytes from large volume of solution, the effect of sample volume on the uptake of Ferbam was also investigated. For this purpose, 25, 50, 100, 150, 200, 250, 300, 350, 400 mL of sample containing 75 g of Ferbam were taken. Quantitative uptakes ( 95%) were obtained for sample volume of 300 mL (Fig. 5.2.4). But for convenience, 100 mL of sample volume was adopted for the preconcentration of analyte. 5.2.3.5 Effect of shaking speed (r.p.m.) on % uptake of FerbamThe % uptake of Ferbam increased gradually with a rise in the shaking speed (Fig. 5.2.5). The batch extraction technique is based on the choice of shaking speed that helps to improve the mass transfer. Shaking speed here acts as a driving force. The central dogma is that the increasing driving force could help in mass transfer and facilitate the concentration gradient between the sample solution and the polymer. Therefore, the present study suggests that the shaking effect is an outstanding parameter for the maximum % uptake of an analyte. 5.2.3.6 Effect of Nature of Eluent on % uptake of FerbamIn order to choose the best eluent for the Ferbam retained on -CDP-PAN polymer, various eluent were used. Among the eluents studied, the acids provided higher recovery efficiency than the organic solvents. Amongst acids studied, HCl provided higher recovery value5.2.3.7 Effect of eluent concentration on % uptake of FerbamThe effect of eluent concentration on the uptake of Ferbam was also examined. Different concentrations of HCl ranging from (0.5-4M) were tested in order to strip the Ferbam from polymer. The uptake of Ferbam increased, as HCl concentration increased up to 2.0M and it decreased above this concentration. Therefore an HCl concentration of 2.0 M was selected for subsequent studies (Fig. 5.2.6).5.2.3.8 Effect of Eluent volume on % uptake of FerbamIn order to choose proper volume of the eluent, the retained complex was stripped with different volumes (16 mL) of 2.0 M HCl. It is clear that 5 mL would not be suitable because it gave a smaller preconcentration factor and 3mL was not sufficient for the elution (Fig. 5.2.7). Hence, 4 mL of 2.0 M HCl was chosen for elution of the metal ion complexes. The preconcentration factor is calculated by the ratio of the highest sample volume (300 mL) and the lowest eluent volume (4 mL). Thus, the preconcentration factor obtained 75.

Fig. 5.2.1 Effect of pH on % uptake of Ferbam

Fig. 5.2.2 Effect of amount of polymer on % uptake of Ferbam

Fig. 5.2.3 Effect of shaking time on % uptake of Ferbam

Fig. 5.2.4 Effect of sample volume on % uptake of Ferbam

Fig. 5.2.5 Effect of shaking speed on % uptake of Ferbam

Fig. 5.2.6 Effect of eluent concentration on the % uptake of Ferbam

Fig. 5.2.7 Effect of eluent volume on the % uptake of Ferbam

5.2.3 Effect of foreign ionsSynthetic solutions containing 75 g of Ferbam and various types of foreign ions were prepared and general procedure was followed. The tolerance limit was defined as the amount of foreign ions causing a change less than 5% in the recovery of Ferbam (Table 5.2.1).

Table 5.2.1 Effect of foreign ions on the determination of 75 g of FerbamForeign ionsTolerance limit[WForeign ion/WFerbam]

NO3-, SO42-, HPO42-, SCN-, NO2-, PO43- >1500

Na+, K+, Mg2+, Ba2+, Al3+, Rb+, Cs+, Ag+, Dibam, Nabam, Vapam1000

Sb3+, Ca2+, Zr4+, Ti800

Th4+, Sn2+, As3+300

aNi2+, bCu2+, cCo2+10

dHg2+, dCd2+, ePb2+, fFe2+, gManeb, hZineb1

EDTA, Br-, F-, CN-, citrate1

a-masked with 1.0 mL of 2.0% dimethylglyoxime; b-masked with 1.0 mL of 3.0% sodium thiosulphate; c-masked with 1.0 mL of 10.0% -benzilmonoxime; d-masked with 5.0 mL of 2.0% sodium thioglycollate solution; e- masked with 2.0 mL 0f 1.0% sodium sulphate solution; f-masked with 1.0 mL of 2.0% 1,10-phenenthroline; g-masked with 2.0 mL 3.0% sodium hexametaphosphate solution; h-masked with 1.5 mL of 1% sodium thiocyanate.

5.2.4 Validation of the methodThe validity of the method was checked by applying it for the determination of Ferbam in water and vegetable samples. 5.2.4.1 Determination of Ferbam in water samplesWater samples were collected from the different parts of Patiala City. The water samples were immediately filtered through cellulose membrane filter (0.45 m pore size), and stored in precleaned polyethylene bottles. After that, pH of the sample was adjusted to 5.5 and the preconcentration procedure as described above was applied (Table 5.2.2).5.2.4.2 Determination of Ferbam in vegetables/cropsThe method was applied for the determination of Ferbam in crops and vegetable samples. A known amount of Ferbam in dimethyl sulphoxide (DMSO) was crushed with 10 g. of crops and vegetable samples with the help of a pestle and mortar. The mixture was then stirred with magnetic stirrer for 1h to provide complete dissolution of Ferbam and then filtered to separate the food residue from the solution containing Ferbam. The residue was washed with DMSO to provide complete extraction of Ferbam to the solution. Filtrate and washings were combined and evaporated to 20.0 mL on a water bath, diluted to 100 mL with DMSO and determined by the developed method (Table 5.2.3).

Table 5.2.2 Determination of Ferbam in different water samples (n=3)Sample Spiked(g.) Found(g.) % Relative % Recovery Error R.S.D.(%)

^Tap Water 0.0 N.D. ------- ------- 15.0 14.7 2.9 97.1 2.1 20.0 19.5 2.5 97.5 1.5$Tap Water 0.0 N.D. ------- ------- 35.0 34.0 1.0 99.0 1.0 40.0 38.7 1.7 98.3 1.2#R. O. Water 0.0 N.D ------ ------- 55.0 54.6 1.0 99.0 1.0 65.0 64.4 1.0 99.1 1.0

N.D. not detected; R.O. Reverse Osmosis *n is the average of three replicate determinations ^Tap water was collected from Punjabi University, Patiala $Tap water was collected from Urban Estate Phase-II, Patiala #R. O. Water was collected from Department of Chemistry, Punjabi University

Table 5.2.3 Determination of Ferbam in different vegetable samples (n=3)$Sample Spiked(g.) Found(g.) % Relative % Recovery Error R.S.D.(%)

Tomato 0.0 N.D. ------- -------- 20.0 19.6 2.0 98.0 1.3 40.0 39.6 1.0 99.0 1.0Potato 0.0 N.D. ------- -------- 50.0 49.5 1.0 99.0 1.0 55.0 54.6 1.0 99.0 1.0Cucumber 0.0 N.D. -------- -------- 70.0 69.4 1.0 99.1 1.0 75.0 74.3 1.0 99.1 1.0

N.D. not detected *n is the average of three replicate determinations $Samples were collected from Vegetable market near Urban Estate Phase-II, Patiala

5.2.6 Accuracy of the methodThe accuracy of the described preconcentration method was tested in the recovery studies by adding known amounts of Ferbam to the water and food sample. The results obtained from the analysis of water and food samples were satisfactory. These results confirm the validity of the proposed method.

Table 5.2.4 Various parameters studied for the preconcentration of Ferbam using -CDP-PAN polymer as solid phase extractant. Parameters Studied Range Selected range pH 2.5-10.0 5.5Volume of buffer(mL) 6-18 10Shaking Time(min) 10-50 40Adsorbent Dose 50-500 400Sample Volume(mL) 50-450 100 Eluent Concentration(M) 0.5-3 2Eluent Volume(mL) 1-6 4Preconcentration factor ------- 75

5.3 Preconcentration of Maneb using -CDP-PAN polymerThe present work describes preconcentration of Maneb using -CDP-PAN polymer.5.3.1 Materials5.3.1.1 EquipmentThe equipment are same as described in 5.1.1.1.5.3.2.1 ReagentsManeb was prepared as given in literature [27]. Its stock solution was prepared in dimethyl sulphoxide (DMSO). Further dilutions were made as and when required. 5.3.2 Procedure5.3.2.1 Batch Extraction procedureAt room temperature, -CDP-PAN (500mg) and 10.0 ml of buffer solution (pH 9.5) were added to a 100 mL Stoppard conical flask. The mixture was allowed to stand for approximately 15 min so that -CDP-PAN could swell sufficiently. 50g of Maneb were added and made up to 100 mL with double distilled water. After the mixture was shaken in the thermostatic shaking water bath for 45 min, 5.0 mL of the supernatant solution was transferred into a 10ml volumetric flask and the absorbance was measured. Maneb retained on -CDP-PAN polymer was eluted using 5.0 mL of 2M HCl.5.3.3 Optimization of various parameters5.3.3.1 Effect of pHThe uptake of an analyte on the chelating polymer is dependent on the pH of sample solution due to the competitive reaction between chelate forming groups and hydrogen ions in the solutions. 50g. of Maneb were added to a 100 mL of the sample, containing 500 mg. of polymer and shook for 45 min. The pH of this solution was adjusted in the range of 2.5 to 10.5 using different buffer system and then the preconcentration procedure as described was applied. Quantitative uptake ( 95%) was obtained at pH 9.5-10.5 0.01 (Figure 5.3.1). Therefore, 9.5 was chosen as the working pH for the subsequent studies.5.3.3.2 Effect of the amount of polymer on % uptake of ManebThe amount of the polymer is another important parameter that affects the uptake of an analyte. A quantitative uptake ( 95%) cannot be achieved when the polymer is less than the optimum amount. On the other hand, an excess amount of polymer prevents the quantitative elution of the retained analyte by a small volume of the eluent. In order to optimize the smallest amount, 100, 200, 300, 400, 500, 600 and 700 mg. of the polymer were added to the 100 mL of the sample solution containing 50 g of Maneb and preconcentrated by the general procedure. The quantitative recoveries were obtained for and above 500 mg of polymer (Fig. 5.3.2). Therefore, 500 mg of the polymer has been used for subsequent studies.5.3.3.3 Effect of shaking time on % uptake of ManebShaking time is an important factor in determining the possibility of application of the -CDP-PAN polymer for the uptake of Maneb. For studying the effect of shaking time on the % uptake, 500 mg. amount of polymer was shaken with 100 mL of sample containing 50 g of Maneb for different shaking time (ranging from 15, 30, 45, 60, 75) min. at optimum pH. The results of % uptake of Maneb vs. the shaking time show that the percentage uptake reached maximum (above 95%) at 45 min (Fig. 5.3.3). Therefore, the shaking time of 45 min. was selected as the adsorption equilibrium time. 5.3.3.4 Effect of the sample volumeIn order to explore the possibility of enriching low concentration of analytes from large volume of solution, the effect of sample volume on the retention of Maneb was also investigated. For this purpose, 50 g of Maneb were added into 25, 50, 100, 150, 200, 250, 300, 350, 400 mL of sample containing 500 mg. of polymer at optimum pH and shook for 45 min. Quantitative uptakes ( 95%) were obtained for sample volume of 350 mL (Fig. 5.3.4). But for convenience, 100 mL of sample solution was adopted for the preconcentration of Maneb. 5.3.3.5 Effect of shaking speed (r.p.m.) on % uptake of ManebThe % uptake of Maneb increased gradually with a rise in the shaking speed (Fig. 5.3.5). The batch extraction technique is based on the choice of shaking speed that helps to improve the mass transfer. Shaking speed here acts as a driving force. The central dogma is that the increasing driving force could help in mass transfer and facilitate the concentration gradient between the sample solution and the polymer. Therefore, the present study suggests that the shaking effect is an outstanding parameter for the maximum % uptake of an analyte.5.3.3.6 Effect of Nature of Eluent on % uptake of ManebIn order to choose the best eluent for the Maneb retained on -CDP-PAN polymer, various eluent were used. Among the eluents studied, the acids provided higher recovery efficiency than the organic solvents. Amongst acids studied, HCl provided higher recovery value5.3.3.7 Effect of eluent concentration on % uptake of ManebThe effect of eluent concentration on the uptake of Maneb was also examined. Different concentrations of HCl ranging from (0.5-4M) were tested in order to strip the Maneb from polymer. The uptake of Maneb increased, as HCl concentration increased up to 2.0M and it decreased above this concentration. Therefore an HCl concentration of 2.0 M was selected for subsequent studies (Fig. 5.3.6).5.3.3.8 Effect of Eluent volume on % uptake of ManebIn order to choose proper volume of the eluent, the retained complex was stripped with different volumes (16 mL) of 2.0 M HCl. It is clear that 5 mL would not be suitable because it gave a smaller preconcentration factor and 3mL was not sufficient for the elution (Fig. 5.3.7). Hence, 4 mL of 2.0 M HCl was chosen for elution of the metal ion complexes. The preconcentration factor is calculated by the ratio of the highest sample volume (350 mL) and the lowest eluent volume (5 mL). Thus, the preconcentration factor obtainable was 70.

Fig. 5.3.1 Effect of pH on % uptake of Maneb

Fig. 5.3.2 Effect of amount of polymer on % uptake of Maneb

Fig. 5.3.3 Effect of the contact time on % uptake of Maneb

Fig. 5.3.4 Effect of the sample volume on % uptake of Maneb

Fig. 5.3.5 Effect of shaking speed on % uptake of Maneb

Fig. 5.3.6 Effect of eluent concentration on % uptake of Maneb

Fig. 5.3.7 Effect of eluent volume on % uptake of Maneb

5.3.4 Effect of Foreign ionSamples containing 50 g. of Maneb and various amounts of foreign ions were prepared and general procedure was followed. The tolerance limit was defined as the amount of foreign ions causing a change less than 5% in the uptake of Maneb (Table 5.3.1).

Table 5.3.1 Effect of foreign ions on the determination of 50g. of ManebForeign ions Tolerance limit [WForeign ion/WManeb]

NO3-, SO42-, HPO42-, SCN-, NO2-, PO43->1000

Na+, K+, Mg2+, Ba2+, Al3+, Rb+, Cs+, Ag+, Dibam, Nabam, Vapam1000

Sb3+, Ca2+, Zr4+, Ti500

Th4+, Sn2+, As3+100

aFerbam, bNi2+, cCu2+, dCo2+, 10

eHg2+, eCd2+, fPb2+, gFe2+, Zineb, Ziram1

EDTA, Br-, F-, CN-, citrate1

a-masked with 1.0 mL of 5.0% ammonium oxalate solution; b-masked with 1.0 mL of 2.0% dimethylglyoxime; c-masked with 1.0 mL of 3.0% sodium thiosulphate; d-masked with 1.0 mL of 10.0% -benzilmonoxime; e-masked with 5.0 mL of 2.0% sodium thioglycollate solution; f- masked with 2.0 mL 0f 1.0% sodium sulphate solution; g-masked with 1.0 mL of 2.0% 1,10-phenenthroline; h-masked with 3.0 mL of 1% sodium thiocyanate solution.

5.3.4 Validation of the methodThe validity of the method was checked by applying it for the determination of Maneb in water and vegetable samples. 5.3.4.1 Determination of Maneb in water samplesWater samples were collected from the different parts of Patiala City. The water samples were immediately filtered through cellulose membrane filter (0.45 m pore size), and stored in precleaned polyethylene bottles. After that, pH of the sample was adjusted to 5.5 and the preconcentration procedure as described above was applied (Table 5.3.2).5.3.4.2 Determination of Maneb in vegetables/cropsThe method was applied for the determination of Maneb in crops and vegetable samples. A known amount of Maneb in dimethyl sulphoxide (DMSO) was crushed with 10 g. of crops and vegetable samples with the help of a pestle and mortar. The mixture was then stirred with magnetic stirrer for 1h to provide complete dissolution of Maneb and then filtered to separate the food residue from the solution containing Maneb. The residue was washed with DMSO to provide complete extraction of Maneb to the solution. Filtrate and washings were combined and evaporated to 20.0 mL on a water bath, diluted to 100 mL with DMSO and determined by the developed method (Table 5.3.3).

Table 5.3.2 Determination of Maneb in different water samples (n=3) Sample Spiked(g.) Found(g.) % Relative % Recovery Error R.S.D.(%)

Tap Water 0.0 N.D. ------- ------- 15.0 14.7 2.0 98.0 1.4 25.0 24.6 1.6 98.4 0.8 Bore Water 0.0 N.D. ------- ------- 40.0 39.6 1.0 99.0 1.0 30.0 29.5 1.7 98.3 1.2River Water 0.0 N.D ------ ------- 40.0 39.5 1.3 99.0 1.0 35.0 34.6 1.1 98.5 0.9

N.D. Not detected

Table 5.3.3 Determination of Maneb in vegetable samples (n=3) Sample Spiked(g.) Found(g.) % Relative % Recovery Error R.S.D.(%)

Tomato 0.0 N.D. ------- -------- 20.0 19.6 2.0 98.0 1.3 40.0 39.2 2.0 98.0 1.3 Potato 0.0 N.D. -------- -------- 50.0 49.5 1.0 99.0 1.0 45.0 44.4 1.3 98.6 0.8 Cucumber 0.0 N.D. -------- -------- 35.0 34.4 1.7 98.3 1.2 55.0 54.4 1.1 98.9 1.0

N.D. (not detected)

5.3.6 Accuracy of the methodThe accuracy of the described preconcentration method was tested in the recovery studies by adding known amounts of Maneb to the water and food sample. The results obtained from the analysis of water and vegetable samples are depicted in table respectively. The recovery values obtained from the water and vegetable samples were satisfactory. These results confirm the validity of the proposed method.

Table 5.3.5 Various parameters studied for the preconcentration of Maneb using -CDP-PAN polymer as solid phase extractantParameters Studied Range Selected Value pH 2.5-10.5 9.5Volume of buffer(mL) 6-18 10Shaking Time(min) 15-75 45Adsorbent Dose 100-700 500Sample Volume(mL) 50-450 100 Eluent Concentration(M) 0.5-4 2Eluent Volume(mL) 1-6 5Preconcentration factor -------- 70

.