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FLOW INJECTION METHODS FOR THE DETERMINATION OF IN NATURAL WATER SAMPLES USING CHEMILUMINESCENCE / SPECTROPHOTOMETRIC DETECTION

BY MUHAMMAD ASGHAR

DEPARTMENT OF CHEMISTRY UNIVERSITY OF BALOCHISTAN, QUETTA PAKISTAN 2018

FLOW INJECTION METHODS FOR THE DETERMINATION OF PESTICIDES IN NATURAL WATER SAMPLES USING CHEMILUMINESCENCE / SPECTROPHOTOMETRIC DETECTION

Being a thesis submitted for the degree of

Doctor of Philosophy

In the University of Balochistan

By

Muhammad Asghar

Supervisor: Dr. Mohammad Yaqoob, Professor Co-Supervisor: Dr. Abdul Nabi, Professor

DEPARTMENT OF CHEMISTRY UNIVERSITY OF BALOCHISTAN, QUETTA PAKISTAN 2018 January 2018

Author’s Declaration

I Muhammad Asghar hereby state that my PhD thesis titled “Flow injection methods for the determination of pesticides in natural water samples using chemiluminescence / spectrophotometric detection” is my own work and has not been submitted previously by me for taking any degree from this university, UNIVERSITY OF BALOCHISTAN, QUETTA or anywhere else in the country/world. At any time if my statement is found to be incorrect even after my graduate the university has the right to withdraw my PhD degree.

Name of Student: Muhammad Asghar

Date: January 2018

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Plagiarism Undertaking

I solemnly declare that research work presented in the thesis titled “Flow injection methods for the determination of pesticides in natural water samples using chemiluminescence / spectrophotometric detection” is solely my research work with no significant contribution from any other person. Small contribution/help wherever taken has been duly acknowledged and that complete thesis has been written by me. I understand the zero-tolerance policy of the HEC and UNIVERSITY OF BALOCHISTAN, QUETTA towards plagiarism. Therefore, I as an author of the above titled thesis declare that no portion of my thesis has been plagiarized and any material used as reference is properly referred/cited. I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even after award of PhD degree and that HEC and the University has the right to publish my name of the HEC/University Website on which names of students are placed who submitted plagiarized thesis.

Student / Author Signature: ______Name: Muhammad Asghar

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Certificate of Approval

This is to certify that the research work presented in this thesis entitled “Flow injection methods for the determination of pesticides in natural water samples using chemiluminescence / spectrophotometric detection” was conducted by Muhammad Asghar under the supervision of Dr. Mohammad Yaqoob, Professor and Co-supervision of Dr. Abdul Nabi, Professor Department of Chemistry, University of Balochistan, Quetta. No part of this thesis has been submitted anywhere else for any other degree. This thesis is submitted to the Department of Chemistry, University of Balochistan, Quetta in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Field of Chemistry (Analytical / Environmental Chemistry), Department of Chemistry, UNIVERSITY OF BALOCHISTAN, QUETTA.

Student Name: Muhammad Asghar Signature ______

Dr. Mohammad Yaqoob, Professor Signature ______Supervisor

Chairperson Signature ______Department of Chemistry University of Balochistan

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UNIVERSITY OF BALOCHISTAN, QUETTA DEPARTMENT OF CHEMISTRY

CERTIFICATE

This is to certify that Muhammad Asghar was registered for PhD degree program (Registration No. 1998/UB–2014/R–274, dated May 31, 2014) in the Department of Chemistry, University of Balochistan. He has successfully completed 18 and 32-hour credit course work and laboratory/research work respectively on “Flow injection methods for the determination of pesticides in natural water samples using chemiluminescence / spectrophotometric detection”. He may be allowed to submit his thesis on the topic mentioned above to the University of Balochistan for the fulfilment of PhD degree.

Dr. Mohammad Yaqoob, Professor Dr. Abdul Nabi, Professor Supervisor Co-Supervisor

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ACKNOWLEDGEMENTS

I would like to thank my research supervisor Dr. Mohammad Yaqoob, Professor, and Co-supervisor, Dr. Abdul Nabi, Professor, Department of Chemistry, for their guidance, patience, assistance and encouraging me during my PhD studies at the University of Balochistan and it is real honor for me to work with them. I would like to acknowledge my appreciation to Dr. Manzoor Iqbal Khattak, Professor & Chairperson, and Dr. Masood Ahmed Siddiqui Professor and Dean Faculty of Basic Sciences for their support, interest and providing the facilities at the Department of Chemistry, University of Balochistan, Quetta. I gratefully acknowledge the financial support provided by the Higher Education Commission, Islamabad (Grant number 20–640/R&D/06) for the conduct of this research at the Department of Chemistry, University of Balochistan, Quetta, and the Directorate Higher Education and Technical, Govt. of Balochistan, for study leave and moral support. The author acknowledges Professor Paul J. Worsfold and his research group, School of Geography, Earth and Environmental Sciences, Plymouth University, Plymouth, PL4 8AA, United Kingdom for providing 8-hydroxyquinoline immobilized resin. I am also thankful to all of those mentioned above, encouraging me during the research work and in writing of this thesis and Department of Chemistry, University of Balochistan for awarding me a chance to pursue the PhD degree.

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Summary of the Thesis submitted for PhD degree by Muhammad Asghar On Flow injection methods for the determination of pesticides in natural water samples using chemiluminescence / spectrophotometric detection

In recent years, the increasing number of pollutants being detected in surface water has raised concern about the contamination of water resources. Pesticides contamination of soil, food and waters has become a serious problem. Pesticides are widely used for the control of insects, fungi, bacteria, weeds, nematodes, rodents and other pests. However, despite their many merits, pesticides are some of the most toxic, environmentally stable and mobile substances in the environment. Their excessive use has deleterious effects on humans and the environment; their presence in food is particularly dangerous. exposure can cause a variety of adverse health effects. These effects can range from simple irritation of the skin and eyes to more severe effects such as affecting the nervous system, mimicking hormones causing, reproductive problems and cancer. Strong evidence also exists for other negative outcomes from pesticide exposure including neurological defects, birth defects and neuro–developmental disorder. Pesticides are categorized into four main substituents: i) herbicides, ii) fungicides, iii) and iv) bactericides. In 2006 and 2008, the world used approximately 5.2 billion pounds of pesticides with herbicides constituting most of the world pesticide use at 40% followed by insecticides and fungicides with totals of 17 and 10% respectively. Therefore, the developments of simple and sensitive methods for the monitoring of pesticides are of great importance for the purpose. Chemiluminescence (CL) is defined as the electromagnetic radiation (ultraviolet, visible, or infrared) produced when a chemical reaction yields an electronically excited intermediate or product. The light intensity is directly related to the analyte concentration, thus allowing precise and sensitive quantitative analysis. The main attractions of CL for

vi biochemical analysis show excellent sensitivity over a wide linear range and low limits of detection. There are many inorganic and organic chemical reactions that produce CL in the liquid phase and some of these CL systems are luminol, lucigenin, morphine, codeine, pyrogallol, acridinium esters, acidic potassium permanganate and tris(2,2/-bipyridyl) / Ruthenium(II). Luminol, lucigenin, tris(2,2 –bipyridyl) Ruthenium(II), KMnO4 and cerium(IV) sulfate are the most commonly employed CL reagents and find wide applications in analytical chemistry and chemical analysis. Transition metals in uncommon oxidation states such as silver(III), copper(III) and nickel(IV) have been exploited in the CL systems as oxidizing agents. Flow injection analysis (FIA) is based on the injection of a small volume of liquid sample into a moving non-segmented continuous carrier stream of a suitable liquid. The injected sample becomes a part of a continuously moving stream and forms a zone. The processed sample carrying stream is then finally transported towards a detector that continuously records the absorbance, emission, electrode potential or other physical parameter. This thesis concerns research work in the area of FIA in conjunction with CL and UV-Visible spectrophotometric detectors for the determination of pesticides in natural water samples. The work presented in this thesis is divided in ten chapters. The first chapter is devoted to the knowledge on the pesticides, its classification, toxicology, handling of water samples, luminescence, CL and its reagents with their analytical applications related to pesticides analysis in natural water samples. The FIA with its basic components and analytical applications are also presented in tabulated form. The second chapter describes CL instrumentation including a brief introduction and characteristics of photomultiplier tube (PMT), continuous flow methods, glass spiral flow cells and a test method for the determination of manganese(II) using luminol– diperiodatoargentate(III) (DPA) system to examine the performance of PMT detector in FI-CL mode. The third chapter describes a FI-CL method for the determination of Mn2+, maneb and mancozeb fungicides based on the catalytic effect of Mn2+ on the oxidation of

vii lucigenin and dissolved oxygen in a basic solution. The Tween-20 surfactant has been reported for the first time to enhance lucigenin CL intensity in the presence of Mn2+ and maneb and mancozeb. The effect of thirty-two other pesticides (fungicides, herbicides and insecticides) was also investigated on this CL system. The fourth chapter describes the development of a simple FI-CL system for the determination of thiram and pesticides in natural water samples based on the 2+ strong enhancing effects of these pesticides on the Ru(bipy)3 –DPA CL system. Thiram could be determined in the presence of aminocarb using Triton X-100. The possible CL reaction mechanism is also discussed briefly. The fifth chapter describes a FI-CL method for the determination of thiabendazole (TBZ) fungicide based on its enhancement effect on diperiodatocuprate(III) (DPC) –

H2SO4 CL reaction. The method was successfully applied for the determination of TBZ in water samples using dispersive liquid–liquid micro-extraction (DLLME). The possible CL reaction mechanism for DPC–sulphuric acid–TBZ is also discussed. The sixth chapter describes a simple and sensitive CL procedure for the determination of (CYR) using FI technique. CYR has strong enhancing effect on the CL reaction of DPA in H2SO4 medium. Interference from chloride ions could be eliminated by using SPE procedure or incorporating an in-line ion exchange resin column

(IERC). The CL mechanism of DPA–H2SO4–CYR system was also discussed briefly. The seventh chapter describes a FI-CL method devised for the analysis of thiram in natural water samples. The CL intensity is enhanced when thiram is oxidized with potassium bromate under strong acidic conditions subsequently using quinine as a sensitizer. The proposed method was applied for thiram analysis in spiked natural water samples and results obtained were satisfactory with the previously reported HPLC method. A brief discussion on the possible CL reaction mechanism between thiram and potassium bromate enhanced by quinine has been elaborated. The eighth chapter describes a simple reversed FIA method for the determination of thiram and nabam fungicides in natural waters with spectrophotometric detection. It is based on the reduction of iron(III) in the presence of thiram/nabam in acidic medium at 60 oC and formation of iron(II)-ferricyanide complex with an absorbance maximum at 790 nm. The method was applied to determine thiram and nabam in water samples using

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Sep-Pak C18 cartridges for solid phase extraction procedure and the results obtained were not significantly different compared with a reported HPLC method. The ninth chapter describes another spectrophotometric method in conjunction with FIA technique for the quantitative analysis of 1-Napthylthiourea (antu). The reaction is based on the alkaline hydrolysis of antu to 1-naphthylamine at 30 C, coupled with diazotized sulphanilic acid, resulting in 4-(sulphophenylazo)-1-naphthylamine and was monitored at 495 nm. The analysis of antu in spiked water samples was extracted using Sep-Pak C18 cartridges. There was no significant difference between the proposed method and a reported HPLC method by applying F-test and paired Student t-test at 95% confidence level. In the tenth and final chapter, general conclusions and future trends are described followed by references, list of publications and title pages of articles published.

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CONTENTS Page No.

CHAPTER 1

INTRODUCTION

1 INTRODUCTION 01

1.1 PESTICIDES 01

1.1.1 Classification of pesticides 02

1.1.1.1 Herbicides 02

1.1.1.2 Insecticides 04

1.1.1.3 Fungicides 05

1.1.1.4 06

1.1.1.5 06

1.1.2 Pesticides toxicology 07

1.1.3 Preparation and handling of water samples for 12 pesticides analysis

1.1.3.1 Liquid-liquid extraction (LLE) 14

1.1.3.2 Solid phase extraction (SPE) 17

1.2 LUMINESCENCE 20

1.2.1 Chemiluminescence (CL) 21

1.2.1.1 CL reagents 21

1.3 FLOW INJECTION ANALYSIS (FIA) 41

1.3.1 Basic components of FIA 42

1.3.2 FI-CL/spectrophotometric applications for the 45 pesticides quantitative analysis in water samples

1.4 RESEARCH OBJECTIVES 47

1.4.1 Research objectives 47

1.4.2 Problem statement 47

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CHAPTER 2

CL INSTRUMENTATION AND DETERMINATION OF MANGANESE(II)

2 INTRODUCTION 49

2.1 PHOTOMULTIPLIER TUBES (PMTs) 49

2.1.1 Characteristics of PMT 50

2.2 BATCH LUMINOMETERS 51

2.3 CONTINUOUS FLOW METHODS 52

2.3.1 FI-CL instrument as a model 53

2.3.1.1 Flow cells 53

2.4 TESTING OF CL DETECTOR 55

FI-CL DETERMINATION OF Mn2+ USING LUMINOL–DPA CL DETECTION

2.4.1 EXPERIMENTAL 55

2.4.1.1 Reagents 55

2.4.1.2 Apparatus and procedure 57

2.4.1.3 Characterization of DPA 58

2.5.1 RESULTS AND DISCUSSION 58

2.5.1.1 FI manifold’s chemical and physical 58 parameters optimization

2.5.1.2 Analytical figures of merit 63

2.5.1.3 Possible CL reaction mechanism 64

2.6.1 CONCLUSIONS 64

CHAPTER 3

DETERMINATION OF MANGANESE AND MANGANESE CONTAINING FUNGICIDES WITH LUCIGENIN-TWEEN-20 ENHANCED CHEMILUMINESCENCE DETECTION

3 INTRODUCTION 66

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3.1 EXPERIMENTAL 70

3.1.1 Chemical reagents and solutions 70

3.1.2 FI manifold and procedure 72

3.2 RESULTS AND DISCUSSION 73

3.2.1 Chemical and physical parameters optimization 73

3.2.2 Analytical characteristics 77

3.2.3 Salinity effect on Mn2+ determination 78

3.2.4 Interference study 80

3.2.5 Mn2+ analysis in the reference materials and water 85 samples

3.2.6 Fungicides, herbicides and insecticides detection 86

3.2.7 Spectrophotometric studies 88

3.3 CONCLUSIONS 88

CHAPTER 4

DETERMINATION OF THIRAM AND AMINOCARB PESTICIDES IN NATURAL WATER SAMPLES USING FLOW INJECTION WITH TRIS(2,2/- BIPYRIDYL)RUTHENIUM(II)–DIPERIODATOARGENTATE(III) CHEMILUMINESCENCE DETECTION

4 INTRODUCTION 90

4.1 EXPERIMENTAL 92

4.1.1 Reagents and solutions 92

4.1.2 FI manifold and procedure 93

4.1.3 Sample collection and SPE procedure 94

4.2 RESULTS AND DISCUSSION 95

4.2.1 Kinetic curve of the CL reaction 95

4.2.2 Chemical and physical parameters optimization 96 studies

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4.2.3 Analytical characteristics 101

4.2.4 Interference study 102

4.2.5 Validation of the proposed method 103

4.2.6 CL reaction mechanism 104

4.3 CONCLUSIONS 105

CHAPER 5 FLOW INJECTION DETERMINATION OF THIABENDAZOLE FUNGICIDE IN WATER SAMPLES USING DIPERIODATOCUPRATE(III)–SULFURIC ACID–CHEMILUMINESCENCE SYSTEM 5 INTRODUCTION 106

5.1 EXPERIMENTAL 109

5.1.1 Reagents and solutions 109

5.1.2 FI manifold and procedure 110

5.2 RESULTS AND DISCUSSION 111

5.2.1 DPC-sulfuric acid CL system 111

Kinetic profile and CL spectrum of DPC–sulfuric 112 5.2.2 acid–TBZ

5.2.3 Optimization of key chemical and physical variables 112

5.2.4 Analytical figures of merit 116

5.2.5 Interference study 116

5.2.6 Detection of fungicides, herbicides and insecticides 117

5.2.7 Application to water samples 117

5.3.8 Possible CL reaction mechanism 119

5.3 CONCLUSIONS 121

CHAPTER 6

FLOW INJECTION DETERMINATION OF CYROMAZINE IN NATURAL WATER SAMPLES USING DIPERIODATOARGENTATE(III)-H2SO4-

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CHEMILUMINESCENCE SYSTEM

6 INTRODUCTION 123

6.1 EXPERIMENTAL 125

6.1.1 Reagents and materials 125

6.1.2 Preparation of DPA 125

6.1.3 Amberlite resin column 126

6.1.4 Water samples collection and SPE procedure 126

6.1.5 FI manifold and procedure 127

6.2 RESULTS AND DISCUSSION 128

6.2.1 Kinetic curve of the CL reaction 128

6.2.2 Chemical and physical parameters optimization 128

6.2.3 Analytical figures of merit 130

6.2.4 Interference study 132

6.2.5 Application to water samples 134

6.2.6 CL reaction mechanism 135

6.3 CONCLUSIONS 136

CHAPTER 7

POTASSIUM BROMATE-QUININE CHEMILUMINESCENCE DETECTION OF THIRAM IN WATER SAMPLES USING FLOW INJECTION ANALYSIS

7 INTRODUCTION 137

7.1 EXPERIMENTAL 139

7.1.1 Reagents and solutions 139

7.1.2 FI manifold and procedure 140

7.1.3 Analysis of natural water samples 141

7.2 RESULS AND DISCUSSION 141

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7.2.1 Kinetic curve of CL reaction 141

7.2.2 Chemical and physical parameters optimization 142

7.2.3 Analytical figures of merit 146

7.2.4 Interference study 147

7.2.5 Applications to water samples 147

7.2.6 Possible CL reaction mechanism 148

7.3 CONCLUSIONS 149

CHAPTER 8

REVERSE FLOW INJECTION SPECTROPHOTOMETRIC DETERMINATION OF THIRAM AND NABAM FUNGICIDES IN NATURAL WATERS

8 INTRODUCTION 150

8.1 EXPERIMENTAL 151

8.1.1 Reagents and solutions 151

8.1.2 FI manifold and procedure 152

8.2 RESULTS AND DISCUSSION 153

8.2.1 Chemical and physical parameters optimization 153

8.2.2 Analytical figures of merit 156

8.2.3 Interference study 158

8.2.4 Application to natural water samples 158

8.3 CONCLUSIONS 160

CHAPTER 9

DEVELOPMENT OF FLOW INJECTION SPECTROPHOTOMETRIC METHOD FOR ANTU USING SODIUM NITRITE AND SULPHANILIC ACID DIAZOTIZATION REACTION

9 INTRODUCTION 162

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9.1 EXPERIMENTAL 163

9.1.1 Reagents and solutions 163

9.1.2 FI manifold and procedure 164

9.1.3 Collection of water samples 165

9.1.4 HPLC method 166

9.2 RESULTS AND DISCUSSION 166

9.2.1 Optimization of physical and chemical parameters 166

9.2.2 Analytical figures of merit 171

9.2.3 Intrference study 162

9.2.4 Application to water samples 162

9.2.5 Possible reaction scheme 173

9.3 CONCLUSIONS 174

CHAPTER 10

10 GENERAL CONCLUSIONS AND FUTRURE TRENDS 175

10.1 GENERAL CONCLUSIONS 175

10.2 FUTURE TRENDS 176

REFERENCES 178

LIST OF PUBLICATIONS 218

PUBLICATIONS TITLE PAGES 221

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Page LIST OF TABLES No. Table 1.1 Organic major classes of herbicides with their solubility in water, 03 examples and basic structure. Table 1.2 Organic major classes of insecticides with their solubility in water, 04 examples and basic structure. Table 1.3 Organic major classes of fungicides with their solubility in water, 05 examples and basic structure. Table 1.4 Chemical structures of a few organic CL reagents. 22 Table 1.5 FI-CL methods for the determination of pesticides in natural water 35 samples. Table 1.6 FI-CL methods of DPA and DPC complexes for the determination 40 of various analytes. Table 1.7 FI-UV-Visible spectrophotometric methods for the determination 46 of pesticides in natural water samples. Table 2.1 PMT characteristics (Type 9798, Electron Tubes, Ruisilip, UK). 51 Table 2.2 Effect of physical parameters on the intensity of CL signals of 63 Mn2+ 0.01 µg mL–1 under optimized chemical parameters. Table 3.1 Physical parameters optimization for the quantitative analysis of 77 0.25 ppm of Mn2+ and 0.5 ppm of maneb. Table 3.2 Analytical characteristics of the developed FI-CL method for the 78 quantitative analysis of Mn2+ and it fungicides (maneb and mancozeb). Table 3.3 Metal cations interfering effect on blank and on Mn2+ quantitative 81 analysis (n = 3). Table 3.4 Maneb and mancozeb recovery test results from natural water 86 samples (n = 3). Table 3.5 Blank CL intensity (average of triplicate) on the proposed FI-CL 87 manifold of thirty-two (32) pesticides (2 ppm each) including different fungicides (serial number: 1 to 11), herbicides (serial number: 12 to 19) and insecticides (serial number: 20 to 32) with

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their trivial names, chemical formulas and physicochemical properties. Table 3.6 FI-CL developed method assessment by comparing with other 89 methods (batch or flow based) in terms of analytical figures of merit for the quantitative analysis of mancozeb and maneb. Table 4.1 Analytical characteristics of thiram and aminocarb pesticides. 101 Table 4.2 Thiram and aminocarb recovery results from spiked natural water 104 samples (n = 3). Table 5.1 Comparison of analytical characteristics of various analytical 108 approaches for the quantitative analysis of TBZ. Table 5.2 Physical parameters effect on the quantitative analysis of TBZ 115 (100 µg L–1) using FI–CL manifold. Table 5.3 Recovery study of TBZ in spiked water samples (n = 3). 118 Table 6.1 Analytical attributes of the proposed FI-CL method for the 130

quantitative analysis of CYR utilizing DPA–H2SO4 CL framework. Table 6.2 Evaluation of the analytical attributes of the proposed FI-CL 132 method with other techniques for the quantitative analysis of CYR. Table 6.3 Recovery study of CYR in spiked natural water samples, using an 134 in-line IERC (OH-form; 200 × 2.5 mm, i.d.) in the proposed FI-CL manifold and its comparison with a reported HPLC method. Table 6.4 Recovery study of CYR in spiked natural water samples using 135 SPE technique by the proposed FI-CL manifold without using IERC. Table 7.1. Evaluation of analytical attributes of the proposed flow-based 138 approach with the previously reported flow-based approaches for the quantitative analysis of thiram in water samples. Table 7.2. Optimization of physical parameters on the quantitative analysis 146 of thiram. Table 7.3. Recovery test of thiram in spiked water samples and its 148 comparison with a reported HPLC method (n = 4). Table 8.1. Chemical structures, trivial and IUPAC names and 150

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physicochemical properties of thiram and nabam fungicides. Table 8.2. Checking the effect of different physical parameters for the 158 quantitative analysis of 0.25 µg mL–1 (n = 4) of thiram and nabam each via the FIA–spectrophotometric manifold. Table 8.3. Results of recovery tests of thiram and nabam in natural water 160 samples (n = 4). Table 8.4. Summary of the proposed approach with previously reported 161 methods for the quantitative analysis of thiram fungicide. Table 9.1 Physical variables effect on the quantitative analysis of antu (n = 170 4). Table 9.2 Summary of the comparison of the analytical attributes of the 171 proposed analytical approach with the previously reported approaches for the analysis of antu. Table 9.3 Percent recovery of antu in spiked natural water samples (n = 4). 173

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Page LIST OF FIGURES No. Figure 1.1 Classification of pesticides extraction techniques from water 13 samples. Figure 1.2 Chemical structures of a = DPC, b = DPA and c = DPN. 37 Figure 1.3 A complete manifold for FIA, RCI and RC2 = Reaction coils 43 Figure 1.4 Two positions of six port rotary injection valve, A) loading position 44 of sample and B) carrying position of sample. Figure 1.5 (a) Simple FIA manifold (single channeled), (b) Chart recorder 45 peak showing detector output. Figure 2.1 PMT Cross sectional view 50 Figure 2.2 Basic design of conventional luminometer. 51 Figure 2.3 FI–CL manifold. 53 Figure 2.4 Picture for glass spiral Flow cells with variable capacity used in 54 FIA with CL detection Figure 2.5 Flat glass spiral flow cell with PMT casing. 54 Figure 2.6 Basic components of FI-CL system. 55 Figure 2.7 Proposed FI-CL manifold used for the quantitative analysis of 58 Mn2+. Figure 2.8 Optimization of (a) DPA, (b) KOH for DPA, (c) luminol and (d) 61 KOH for luminol concentrations on the determination of Mn2+. Figure 2.9 Calibration curve for Mn(II) under optimum conditions. 63 Figure 2.10 The possible luminol-DPA CL reaction mechanism. 64 Figure 3.1 Mancozeb and Maneb molecular structures 69 Figure 3.2 Proposed FI-CL manifold for Mn2+ and its fungicides (Maneb and 72 mancozeb) quantitative analysis. Figure 3.3 Optimization of chemical variables of FI-CL manifold for the 76 quantitative analysis of Mn2+ and its fungicides: (a), Lucigenin; (b), NaOH; and (c), Tween-20. Figure 3.4 Salinity effect over the range from 0 to 35 g mL–1 in Mn2+ absence 80 (blank) and in the Mn2+ presence of 0.01 ppm.

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Figure 3.5 HCl pH (eluent) effect on the intensity of CL. (a) Removal of 0.05 84 ppm Mn(II) and 0.1 ppm Fe(II); and (b) 0.05 ppm maneb and 0.01 ppm Fe(II) employing an in line 8-HQ column over 2.5 – 5.0 pH range Figure 3.6 Cations effect on the blank signal and on the quantitative analysis of 84 0.05 ppm each of maneb and mancozeb. Figure 4.1 Chemical structures of thiram and aminocarb. 90 Figure 4.2 FIA–CL manifold for the quantitative analysis of thiram and 94 aminocarb. 2+ Figure 4.3 CL kinetic curve of Ru(bipy)3 – DPA catalyzed by thiram. 95 Figure 4.4 Effect of chemical variables on the determination of thiram and 99

aminocarb: (a), effect of H2SO4 concentration of CL reagent stream; 2+ (b), effect of Ru(bipy)3 concentration prepared in 0.025 M H2SO4; (c),

effect of DPA concentration stream and (d) effect of KOH concentration of DPA stream. Figure 4.5 Effect of the Triton X–100 concentration on the CL intensity of 100 thiram (0.01 g mL–1, triangle) and aminocarb (0.2 g mL–1, circle) standard solutions. Figure 4.6 Calibration curves for (A) thiram and (B) aminocarb under 102 optimum conditions. Figure 5.1 Chemical structure of thiabendazole fungicide. 106 Figure 5.2 FI–CL manifold for the quantitative analysis of TBZ. 111 Figure 5.3 Effect of concentration of chemical variables on the variation of CL 114 signal intensity (a) effect of diperiodatocuprate(III) concentration (b) effect of potassium hydroxide concentration (c) effect of sulfuric acid concentration. Figure 5.4: Calibration curve for TBZ under optimum conditions. 116 Figure 5.5 UV–Vis absorption spectra. (a) DPC (0.25 mmol L−1 in KOH (5 120 mmol L–1)); (b) DPC + sulfuric acid (50 mmol L–1) and (c) DPC + sulfuric acid + TBZ (10000 µg L–1). Figure 5.6 The CL profile of DPC–sulfuric acid–TBZ system. (a) DPC: 0.25 122

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mmol L–1 in potassium hydroxide 5.0 mmol L–1; (b) DPC–sulfuric acid solution (50 mmol L–1) and (c) DPC–sulfuric acid–TBZ: (25 µg L–1); sample volume: 180 µL. Figure 6.1 Chemical structure of CYR. 123 Figure 6.2 FI-CL manifold for the quantitative analysis of CYR. 127

Figure 6.3 Kinetic curve of CYR–DPA–H2SO4 system. 128 Figure 6.4 CL signals obtained for a series of CYR standard solutions injected 131 in triplicate (inset shows calibration curve). Figure 6.5 Effect of chloride in CYR absence (on the blank) and on the 133 quantitative analysis of CYR 500 µg L–1. Figure 7.1 Chemical structure of thiram. 137 Figure 7.2 FI manifold for the CL based quantitative analysis of thiram in 140 water samples of natural origin.

Figure 7.3 Kinetic curve of KBrO3-quinine-H2SO4-thiram system. 142 Figure 7.4 Effect of chemical variables on the quantitative analysis of thiram: 145

(a) effect of KBrO3 concentration prepared in 0.1 M H2SO4; (b) effect

of quinine concentration prepared in 0.5 M H2SO4; (c) effect of H2SO4

concentration of KBrO3 stream and (d) quinine stream. Figure 7.5 Calibration curve for thiram under optimum conditions. 146 Figure 8.1 Reverse FI manifold for the quantitative analysis of thiram and 153 nabam. Figure 8.2 Variation of absorbance with; (a) ammonium iron(III) sulfate; (b) 155

HCl and (c) K3[Fe(CN)6] concentrations. Figure 8.3 Calibration curves for (A) thiram and (B) nabam under optimum 157 conditions. Figure 9.1 Chemical structure of ANTU (α-naphthylthiourea). 162 Figure 9.2 Optimized FI-spectrophotometric manifold for the quantitative 165 analysis of ANTU. Figure 9.3 Optimization of reagents concentrations. (A) Sulphanilic acid, (B) 169

sodium nitrite, (C) NaOH and (D) ethanol (% v/v).

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Figure 9.4 Traces of chart recorder of absorbance for ANTU different 172 standards.

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Acronyms and abbreviations

µ Micro 2,4,5-T 2,4,5-Trichlorophenoxyacetic acid 2,4-D 2,4-Dichlorophenoxyacetic acid 8-HQ 8-hydroxyquinoline A Peak area AChE AHS Agriculture Health Study ANTU α-naphthylthiourea ASV Adsorptive stripping voltammetry BL Bioluminescence bpy Bipyridine Bt Bacillus thuringiensis C18 Octyldecyl

C21H38ClN.H2O Hexadecylpyridinium chloride monohydrate C8 Octyl CE Capillary electrophoresis ChOx oxidase CTAB Cetyltrimethylammonium bromide CYR Cyromazine D Detector DAD Diode array detector DDAB Didodecyldimethylammoniumbromide DDD Dichlorodiphenyldichloroethane DDE Dichlorodiphenyldichloroethylene DDT Dichlorodiphenyltrichloroethane DLLME Dispersive liquid-liquid micro extraction DNA Deoxyribonucleic acid DNPO Bis(2,4-dinitrophenyl) oxalate DPA Diperiodatoargentate(III) DPC Diperiodatocuperate(III)

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DPN Diperiodatonickelate(IV) DPP Differential pulse polarography DTCs Dithiocarbamates DTNB 5,5/-dithio-bis(2-nitrobenzoic acid ECL Electrochemiluminescence EEC European Economic Community ELISA -linked immunosorbent assay EPA Environmental protection agency ESI Electrospray ionization ETU Ethylene thiourea EU European Union FI-CL Flow injection-chemiluminescence FL Fluorimetry FL Fluorescence GC Gas chromatography H Peak hight

H2O2 Hydrogen peroxide

H4P2O7 Pyrophosphoric acid HCHO Formaldehyde HCl Hydrochloric acid HDPE High-density polyethylene HPC Hexadecylpyridinium chloride HPLC High performance liquid chromatography HRP Horseradish peroxidase IAs Immunoassays IERC Ion exchange resin column – IO4 Periodate

K3Fe(CN)6 Potassium fericyanide

KMnO4 Potassium permanganate KOH Potassium hydroxide L Litre LD Lethal dose

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LLE Liquid-liquid extraction LNS Low nutrient sea water LOD Limit of detection LOQ Limit of quantification Luc•+ Lucigenin cation radical

LucO2 Lucigenin dioxetane m Milli M Molar MCL Maximum concentration level MCPA 4-chloro-2-methylphenoxyacetic acid MCPB 4-(4-chloro-o-tolyloxy) butyric acid MIPs Molecularly imprinted polymer membranes MLLE Micro liquid liquid extraction MPA Mono-periodatoargentate MS Mass spectrometry

Na2SO3 Sodium sulfite NaCl Sodium chloride N-dots Nitrogen-rich quantum dots NG Not given NMA N-methylacridone • O2 Superoxide radical OC Organochlorine OPA o-phthalaldehyde OPs Organophosphates OTP Organothiophosphorus PP Peristaltic pump

PbO2 Lead dioxide PCB Polychlorinated biphenyl PICL Photoinduced CL PIPs Plant incorporated protectants PMT Photomultiplier tube PO Peroxyoxalate

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PP Peristaltic pump ppb Parts per billion ppm Parts per million Pq2+ Paraquat cation PTFE Polytetrafluoroethylene R2 Coefficient of determination RC Reaction coil rFI Reverse flow injection Rh6G Rhodamine 6G RMM Relative molecular mass RP Reverse phase RSD Relative standard deviation RTP Room temperature phosphorescence 2+ / Ru(bpy)3 Tris(2,2 -bipyridyl) ruthenium(II) S Sample injection valve S/N Signal to noise ratio 2– S2O8 Peroxydisulfate SD Standard deviation SDME Single drop micro extraction SDS Sodium dodecylsulfate SIA Sequential injection analysis

SO2 Sulphur dioxide SPE Solid phase extraction SPME Solid phase micro extraction t Residence time tb Peak baseline width TBZ Thiabendazole TCNQ 7,7,8,8-tetracyanoquinodimethane TCPO Bis(2,4,6-trichlorophenyl) oxalate TMA 2,4,6-trimethylaniline TSD Thermionic specific detection UHP Ultra-high purity

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UV-Vis Ultraviolet visible V Volt W Peak width W Waste λmax Wavelength maximum є Molar absorptivity

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CHAPTER–I

INTRODUCTION

1 INTRODUCTION

1.1 PESTICIDES Pesticide is a class of biocide and can be defined as “any substance or mixture of substances intended for seducing, attracting, mitigation or destroying any pest” (US Environmental Protection Agency, 2007). It is a broad term and is used both for agricultural and non-agricultulral purposes. All those chemicals which have the activities and functions such as animal repellent, antimicrobial, avicide, bactericide, disinfectant (antimicrobial), fungicide, herbicide, , insect repellent, , molluscicide, nematicide, piscicide, predacide, , sanitizer and termiticide are included in the list of pesticide (Randall, 2013). Broad applications of pesticides include use in food production and storage, safety against diseases, agricultural , soil and seed treatment, homes gardening and many more. Instead of its wide applications, food products are lost world wide by more than an average of 35% (Corral, Delgado, Diez, & Soria, 2008; Janssen, 1997). Production in agriculture has been greatly improved via the effective usage of pesticides and has secured about one third of crop production globally (Eddleston, Buckley, Eyer, & Dawson, 2008). For example, the high growth of corn and wheat production in USA and UK respectively (Liu, Yuan, Yue, Zheng, & Tang, 2008), and the increase in crop production upto 126 million tons in China and 95 million tons in India in 2012 has been ascribed to some extent to the applications of pesticides (UN Food & Agriculture Organization Report, 2013). However, the uncontrolled use and accidental release of pesticides pollute different segments of the environment. As it is known, only one percent of applied pesticide reaches to the intended target during a spray, rest of the used and as well as accidental release from leaking pipes, spillage from underground storage tanks and waste dumps lead to the soil, air, water resources (ground and surface) pollution for a long period of time due to its persistent nature (Smith, & Gangolli, 2002; Knapton, Burnworth, Rowan, & Weder, 2006). In order to protect and secure public health and other forms of life, proper management of pesticides is inevitable. Thus, for accurate status assessment of the pesticides pollution in different segments of the environment, the demand for reliable, rapid and economical qualitative and quantitative determinations of pesticides is critical.

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1.1.1 Classification of pesticides Pesticides can be classified in many ways, and are broadly classified into two major groups and the first group of pesticides is termed as chemical pesticides and the second group is termed as biopesticides. Conventionally, the first group i.e. chemical pesticides are synthetic, which is employed for the direct killing or inactivation of a pest. Further classification of these pesticides is based on the type of target organism against which they are used. The main classes of synthetic pesticides are herbicides, insecticides and fungicides, which are used in order to eradicate the population of weeds, insects and fungi respectively. Other categories which are included in pesticides are nematicides, rodenticides, bactericides, acaricides and plant growth regulators (The Pesticide Manual 2000; Hornsby, Wauchope, & Herner, 1996; Sharma, 2006; Bhadekar, Pote, Tale, & Nirichan, 2011). The main classes of the pesticides are going to be described in the following paragraphs.

1.1.1.1 Herbicides These weeds controlling compounds can be classified in a number of ways. They can be foliage- or soil-applied herbicides based on their absorption by weeds through leaf tissues or roots, respectively. Herbicide can be selective or total based on the killing of selective weed or total vegetation, respectively. Due to their applications in different stages of crop, they can be categorized as pre- or post-emergence and presowing herbicides. In addition, chemical composition can also be used for the classification of herbicide. A few chemical families, to which an herbicide may belong, are triazine, thiocarbamate, sulfonylurea, phenylurea, phenylphenoxy, imidazolinone, dinitroaniline, chlorophenoxy, acetamide and pyridines. Table 1.1 shows these major organic classes of herbicides with aqueous solubility, common example of the individual class and their elementary organic chemical structures.

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Table 1.1 Organic major classes of herbicides with their solubility in water, examples and basic structure

Solubility in Class of Common H2O Basic chemical structure herbicide examples (g L–1)

Prometon, Triazine 0.001–10 ametryn, atrazine, N N simazine N

Asulam, butylate, O Thiocarbamate 0.001–1000 thiobencarb, S C N vernolate, Chlorimuron- O N ethyl, R Sulfonylurea 0.1–10 chlorsulfuron SO2 NH C N N

Diuron, linuron, Clx O Phenylurea 0.001–1 neburon, NH C N thidazuron Bifenox, Phenylphenoxy 1×10–4–1000 fomesafen, O fluazifop-butyl

CO2 Imazethapyr, N Imidazolinone 0.01–1000 imazapyr, imazaquin N

NO2 Dinitramine, Dinitroaniline 1×10–4–1×10–3 pendimethalin, N trifluralin,

NO2 Clx Silvex, MCPA, Chlorophenoxy 0.01–1000 O 2,4,5-T, 2,4-D

O

Alachlor, C Acetamide 0.1–10 metolachlor N

Diquat, Pyridines 500–1000 H C N+ N+ CH paraquat 3 3

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1.1.1.2 Insecticides Insecticides are commonly employed to kill pests especially insects and arthropods in crops. A number of other categories of pesticides such as rodenticides, nematicides and acaricides are also included in insecticides. Similarly, dinitrophenol and organotin, which belong to herbicides and fungicides, respectively, may have activities like insecticides. This class of pesticides may be applied to the soil for killing soil borne pests or to the aerial parts of the plants such as leaves, branches and stem. Table 1.2 shows the major chemical classes of insecticides (, organophosphorus, and organochlorine) with their examples, solubility and general chemical structures. Nearly all organochlorine (OC), except the less persistent ones ( and ), have been banned during 1970s in all the industrialized nations due to their high persistent nature. Currently, organophosphates (OPs) insecticides have more applications in agriculture or somewhere else. N- methylecarbamate insecticides also have the same application but the number of the compounds of this class is less than OPs. A few classes of insecticides such as nicotinyl, pyrethroid, rotenone and alkyloid have been synthetically derived from compounds occurring naturally. have less risk of toxicity to human in comparison to OPs. Table 1.2 Organic major classes of insecticides with their solubility in water, examples and basic structure

Solubility Class of in H2O Common examples Basic chemical structure insecticides (g L–1)

O C N O –6 –4 , , Pyrethroid 10 –10 O , O(S) , , Organophosphorus 10–4–1000 , P O(S)

DDE, , Organochlorine 10–6–10–3 Cl– containing insecticides endosulfan, O , , 0.01–100 , O C N

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1.1.1.3 Fungicides Those chemical compounds or biological organisms, which are employed as killers of fungi or their spores are termed as fungicides (Haverkate, Tempel, & Den Held, 1969). Fungicides may be either systematic, contact or translaminar. Systematic fungicides are absorbed and redistributed by xylem vessels of plants and get reached them to all parts of a plant upwardly or to the specific areas of a plant. Contact fungicides protect only those parts of a plant to which they come into contact by means of deposition, when they are sprayed. In case of translaminar fungicides, the redistribution is accomplished to the lower unsprayed leaves surfaces from the upper sprayed leaves surfaces (Daren, 2006). Fungicides can also be classified as pre- or post-harvest depends on their applications for the safety and protection of food commodities (vegetables, cereals and fruits) before or after harvest from fungal diseases respectively. The physicochemical properties of fungicides vary with the variation in their chemical structures. Various chemical classes of fungicides including phthalimide, imidazole, triazole, dithiocarbamates and miscellaneous have been shown in Table 1.3.

Table 1.3 Organic major classes of fungicides with their solubility in water, examples and basic structure

Solubility in Class of H2O Common examples Basic chemical structure fungicides (g L–1) O Captan, captafol, Phthalimide 10–3 folpet, procymidone N

O N Thiabendazole, Imidazole 10–3–10 imazalil, N

N Propiconazole, Triazole 10–1000 N myclobutanil N

S

Zineb, metriam, S C N Dithiocarbamates 0.1–100 maneb, mancozeb

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1.1.1.4 Rodenticides This class of pesticides is used to kill rodents (mice, rats and other rodents) (U.S. Environmental Protection Agency, 2004). Different types of chemicals act as rodenticides and are classified as (Brown, & Waddell, 2015), i) , ii) metal phosphides, iii) hypercalcemia and iv) others. i. Anticoagulants a) /4-hydroxycoumarins (, , , , and ), b) 1,3-indandiones (diphacinone, and ), c) 4-thiochromenones (), ii. Metal phosphides (phosphides of aluminium, calcium, magnesium and zinc), iii. Hypercalcemia ( (vitamin D3) and (vitamin D2), iv. Others (α-naphthylthiourea (antu), , , etc).

1.1.1.5 Biopesticides These pesticides can be classified into three major classes: i) plant- incorporated protectants, ii) microbial, and iii) biochemical. i. Plant incorporated protectants (PIPs) PIPs are those substances which are produced by plants and having pesticidal activity and they also include the genetic materials in the plant necessary to produce the pesticidal substances. In this regard, for example a gene from a bacterium (Bacillus thuringiensis), which produces toxic crystal protein (insecticide), is incorporated into a plant (cotton) genetic materials to produce genetically modified plant. Both the protein produced by the plant and the genetic materials are regulated as pesticides but not the plant (Tabashnik, Brevault, & Carriere, 2013). ii. Microbial pesticides This class of biopesticides includes active ingredients of microorganism related to bacteria, protozoa, viruses and fungi. Different kinds of the target pest(s) are controlled by the active ingredients of these pesticides with relative specificity. For example, certain fungi are used to kill certain weeds while other fungi are used to

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control specific insects. Bacterium Bacillus thuringiensis, or Bt, generates protein which selectively kills pests in a number of crops such as cabbage and potato. iii. Biochemical pesticides This class of biopesticides includes all those naturally occurring substances which exhibit non-toxic mode of action for the control of pests. These substances may interfere in mating of pests by attracting or repelling them using insect sex pheromones or effect their growth by using growth regulators (Gupta, & Dikshit, 2010). The advantages of biopesticides include sustainable environment and nontoxicity but the improper applications might reverse these advantages. However, the reasons for their limited applications in pest control may be ascribed to the less social awareness, less explored research field, low crop productivity and need for frequent usage in comparison to the synthetic pesticides (Bempah, Kwofie, Enimil, Blewu, & Martey, 2012).

1.1.2 Pesticides toxicology The extensive usage of pesticides in agriculture, homes and public areas for the prevention of unwanted creatures, has increased the direct and indirect exposure to human due to their stable and persistent nature. Millions of pesticides poisoning cases are reported annually worldwide, which include both acute and chronic toxic effects (Richter, 2002). The acute or shot term toxic effects of pesticides include nausea, diarrhea, abdominal pain, vomiting, headaches etc. Long term or chronic toxic effects of pesticides are neurological deficits, depression, cancer, diabetes, genetic disorders, skin diseases and even death. In the following paragraphs, various toxic effects of pesticides are described based on both direct and indirect exposures. i. Cancer Several studies have concluded that pesticides exposure significantly increases the risk of the cancer (Lynch, Mahajan, Freeman, Hoppin, & Alavanja, 2009; Freeman, et al., 2005; Alavanja, et al., 2003). The link between many types of cancers such as risk of leukemia, colon cancer, bladder cancer, thyroid cancer, brain cancer etc has been established with

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direct and indirect exposure to pesticides. The pesticides carcinogenicity can be understood with the collaborated studies and efforts at molecular biology, epidemiological studies and pesticides toxicology (Alavanja, Ross, & Bonner, 2013). It has been pointed out by epidemiological studies that many pesticides such as sulfallate, sulfate and organochlorine are carcinogenic, while and lindane cause tumor (Dich, Zahm, Hanberg, & Adami, 1997). Those consumption products, which have some pesticides residues (dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethane (DDD) and polychlorinated biphenyl (PCB)) such as seafood, fish, water and milk or other dairy products (Moon, Kim, Choi, Yu, & Choi, 2009; Li, et al., 2008; Buczynska, & Szadkowska, 2005; Pandit, & Sahu, 2002) have statistically significant association with cancer risks. In literature, the risk of following types of cancers has been made with pesticides exposure including a) childhood leukemia, b) bladder and colon cancer, c) thyroid cancer and d) brain cancer.

a) Childhood leukemia The abnormal production of white blood cells is a type of cancer termed as leukemia and it has been proved that its risks in children increase with the increase in exposure to pesticides (Ferreira, Couto, Oliveira, Koifman, & Brazilian Collaborative Study Group, 2013). The greater chances of leukemia risks in children are increased by three-fold if parents are exposed to pesticides and by more than 3.5-fold if children are exposed regularly to household pesticides (Mott, Fore, Curtis, Solomon, & Hanson, 1997). The cause of leukemia is the alteration in DNA of infants. It is found by researchers that infants less than 11 months, if exposed to pesticide like permethrin have seven times more chances of leukemia. The critical time for the risk of leukemia due to exposure to pesticides is from pregnancy upto 11 months of nursing (Ferreira, et al. 2013).

b) Bladder and colon cancer Pesticides containing aromatic amines and heterocyclic aromatic amines, such as imazethapyr and Imidazolinone, cause bladder cancer (Silverman, Devesa, Moore, & Rothman, 2006; Weisburger, 2002). The usage of imazethapyr, a heterocyclic

8

aromatic herbicide, has increased the risk of bladder and colon cancer by more than 137% and 78% respectively in exposed person (Koutros, et al., 2009).

c) Thyroid cancer The binding of receptor sites and destruction of thyroid gland by pesticides like organochlorine, which mimic thyroid hormones has been evidenced by researchers (Patrick, 2009). The possible link between the incidence of thyroid and other types of cancer in atrazine applicators was proposed by Agriculture Health Study (AHS) (Freeman, et al., 2011).

d) Brain cancer There are two types of brain tumors i.e. glioma and meningioma. The link of meningioma in females has been made clearly with pesticides exposure (Samanic, et al., 2008). The risk of brain cancer in children is twofold, whose mother has been exposed to pesticides before, during or after pregnancy, especially to termite killers. Brain cancer risk has been ascribed upto 50% by termite killer and 30% by the usage of other pesticides (Greenop, et al., 2013). ii. Depression and neurological deficits Chronic or acute exposure to carbamate, organophosphate, organochlorine or other pesticides during all stages of life (utero, childhood or adultness) may affect peripheral and central nervous system, and leads to Parkinson disease (chronic nervous disorder) (Keifer, & Firestone, 2007), depression (Beseler, et al., 2008) and decreased level of performance during farm work (Kamel, et al., 2003). Indirect exposure to pesticides also causes different neurological diseases such as decreased neurobehavioral development, depression, neurological damage of fetus, effect on children’s IQ scores and Parkinson’s disease (Eskenazi, et al., 2010; Bouchard, et al., 2011; Beseler, et al., 2006; Eskenazi, et al., 2007; Rauh, et al., 2011; Yesavage, et al., 2004). The toxicological effect of OPs pesticides such as chlopyrifos and has been presented to understand the neurological toxic effects of pesticides. In general, the OPs affect badly the axonal region of a neuron by inhibiting the esterase enzyme. This situation leads to a delayed polyneuropathy and overstimulation of

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postsynaptic receptors (Keifer, & Mahurin, 1996). Each year, 5000 poisoning cases are reported due to chlorpyrifos toxicity. Symptoms of these toxic effects in patients are changes in the velocity of nerve conduction, shaking and trembling of hand and arm, changes in vision and smell senses and vibrotactile sensitivity. It results in memory problems, fatigue, emotional changes, weakness of muscles and other neurobehavioral changes (Steenland, et al., 2000). In human body, cytochrome p-40 metabolizes parathion into , which is a powerful and potent inhibitor of cholinesterase. During the toxic effects of parathion, body shows various symptoms such as dizziness, headache, abdominal pain, limb pain, numbness and weakness, gait disturbance, fatigue, nausea, paresthesias, rhinorrhea or somnolence (Lee, London, Paulauskis, Myers, & Christiani, 2003). Among brain diseases caused by pesticides exposure, two famous brain diseases have been thoroughly investigated and are named as a) Alzheimer disease and b) Parkinson diseases.

a) Alzheimer disease Two concepts are proposed about the increase in dementia (decrease in brain capacity), both are related to pesticides exposure. Firstly, pesticide increases the dementia pathogenesis and secondly, Alzheimer disease is caused because pesticide affects neuron at molecular level and destroys its microtubules and hyperphosphorylation (Zaganas, et al., 2013). Those persons who are exposed to organochlorine and organophosphate at their late life, the risk of Alzheimer disease is high, because these pesticides affect acetylcholinesterase enzyme at synaptic junction in nervous system (Hayden, et al., 2010). Some herbicides like paraquat and rotenone affect the bioenergetic activities, redox reactions and oxygen of mitochondria, thus lead to Alzheimer disease (Thany, Reynier, & Lenaers, 2013).

b) Parkinson disease Lack of production of dopamine by substantia nigra neuron in brain is termed as Parkinson disease. In this disease muscles start trembling and loss its control, which results in lack of coordination. The production of dopamine is inhibited by two

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herbicides such as rotenone and paraquat (Qi, Miller, & Voit, 2014), and other pesticides and their metabolites affect modulate xenobiotic metabolism and mitochondria, which also develop Parkinson disease (Le Couteur, McLean, Taylor, Woodham, & Board, 1999). iii. Diabetes Various researchers have claimed an association between pesticides exposure and an increase risk of diabetes (Son, et al., 2010; Everett & Matheson, 2010;). The risk of prevalence of diabetes escalates with the handling and exposure of insecticides such as OC (Cox, Niskar, Narayan, & Marcus, 2007) and OPs (Montgomery, Kamel, Saldana, Alavanja, & Sandler, 2008). iv. Respiratory diseases A lot of reports are available in literature which associate and suggest the risk of respiratory diseases within the applicators and farmers with pesticides exposure. The list of these respiratory diseases includes rhinitis, asthma, bronchitis, farmer’s lung and wheeze (Slager, et al., 2009; Hoppin, et al., 2009; 2007a; 2007b; Hoppin, Umbach, London, Alavanja, & Sandler, 2002). v. Fertility Pesticides reduce male fertility, the association of impaired male fertility and exposure to 2,4-D and dibromochlorophane has been reviewed in literature (Sheiner, Sheiner, Hammel, Potashnik, & Carel, 2003). In addition to the reduction of male fertility, pesticides also cause alteration of hormone function and sperm genetics, reduction in the number of sperm and damage to germinal epithelium (Mortimer, et al., 2013). vi. Toxic effects of pesticides on fetus Various toxic effects of pesticides on fetus have been reported such as retardation of growth, congenital anomalies, weight loss, mutation and endocrine disruption. According to an estimation, 54% of pregnant women are exposed to pesticides during their pregnancy and among them 45% are due to bed room and 47% exposure is from pesticides used elsewhere in home. Fetus and children are more

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vulnerable to pesticides toxic effects because their immune systems are weak and unable to detoxify pesticides (Llop, et al., 2013). Various congenital anomalies such as orofacial clefts, conotruncal defects, limb anomalies and neural tube defects have been reported because of periconceptional pregnancy exposure to pesticides (Llop, et al., 2013) and accounts 54.4% of the total congenital malformation (Rojas, Ojeda, & Barraza, 2000). Growth of fetus is adversely affected by pesticides; weight loss of fetus is directly proportional to the exposure of pesticides (Wickerham, et al., 2012). Methylation of deoxyribonucleic acid (DNA) by pesticides results in epigenetic alteration of parent’s gametes before conception and exposure after conception causes; alteration of hormonal and immunological functions and mutation in somatic cell leads to brain and other types of cancers (Shim, Mlynarek, & van Wijngaarden, 2009). Overweight, obesity and failure to develop reproductive organs of children have also been linked with utero exposure to endocrine disrupting pesticides such as OC, atrazine azole etc (Wohlfahrt, et al., 2011; Bossi, Vinggaard, Taxvig, Boberg, & Jorgensen, 2013). vii. Other diseases Pesticides exposure results other diseases in human for example dyspnea and hepatitis, myocardial infarction, hearing loss, reduction in sperm quality, various thyroid diseases and an increase in the risk of suicides (Azmi, Naqvi, & Aslam, 2006; Dayton, et al., 2010; Mac Crawford, et al., 2008; Perry, et al., 2011; Goldner, et al., 2010; Faria, Fassa, & Meucci, 2014). Further, a number of evidences concerned to toxic effects of pesticides to other animals are available. For example, they cause toxicity to bees (Decourtye, & Devillers, 2010) and collapse their colonies (Watanabe, 2008), as well as cause sickness in wild birds and disturb their normal life functions (Mitra, Chatterjee, & Mandal, 2011). 1.1.3 PREPARATION AND HANDLING OF WATER SAMPLES FOR PESTICIDES ANALYSIS The paragraphs described in the previous section; give an idea about the risk of toxicity to humans and ecotoxicology of pesticides even at trace level in surface,

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underground and drinking waters. Growing concern about the water quality has led European Union (EU) to set a limit of 0.5 ppb for total pesticides and 0.1 ppb for individual pesticide in drinking water. However, when chronic and acute toxic effects and as well as persistency and bioaccumulation of pesticides are implied, the limits described so for must be lower than those for drinking water quality. To analyze pesticides in water samples at trace level, the requirement of sensitive and efficient techniques is inevitable. For this purpose, water samples must be reduced in size many times for the magnification of analyte concentration in final aliquot of sample employing phase transfers. There are two main types of phase transfers for pesticides extraction from water sample namely liquid-liquid extraction (LLE) and solid-phase extraction (SPE). These extraction methods for water samples preparation for the trace level quantitative analysis of pesticides can be further classified as given in Figure 1.1 (Hogendoorn, & Zoonen, 2000; Hatrik, & Tekel, 1996; van der Hoff, & Zoonen, 1999; Liska, & Slobodnik, 1996; Wan, & Wong, 1996).

Figure 1.1 Classification of pesticides extraction techniques from water samples

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1.1.3.1 Liquid-liquid extraction (LLE) There are two types of LLE for the extraction of pesticides from water (a) standard LLE and (b) micro LLE explained in the following paragraphs: a) Standard LLE LLE is the earliest and most widely used extraction technique for the extraction of pesticides from water samples and also include in EPA methods. In this technique two or more immiscible solvents are shaken vigorously for selective extraction of pesticides. Then the solvent liquid phase, rich in analyte concentration, is recovered and dried and reconstitute in mobile or stationary phase for analysis by suitable analytical method. Partition coefficient or equilibrium distribution between the donor and acceptor phase, determines the extraction efficiency and according to similarity principle depends on the matching of polarity of analytes and extraction solvents. For example, a standard EPA method (method # 8141) for OPs and carbamate residue from water sample is as: take spiked water sample (1 L) in a glass separatory funnel (2 L) and add NaCl (50 g), then add 100 mL of methylene chloride and shake vigorously (1-2 min). Wait for 10 min for complete separation of two phases and dry the extract after recovery from separatory funnel by passing through drying funnel containing anhydrous NaSO4 (50 g). Repeat the same procedure for two or more times and the volume of the pooled extract can be reduced by using vacuum evaporator or blowing down N2 gas. Advantages of LLE include compatibility with broad range of pesticides, simplicity, reliability, need of simple equipment and less skilled operator. LLE is also accompanied with a number of drawbacks such as consumption of large quantity of organic solvents, long extraction time, labor demanding, unfriend environmentally, and prolonged phase separation time when water sample containing large amount of organic content or suspended particles (runoff effluent). b) Micro-LLE Disadvantages in conventional LLE have been eliminated or minimized with the development of MLLE and miniaturization in standard LLE has been obtained only in the use of organic solvents during extraction. The principle of this technique is the extraction of large volume of water sample (donor phase) with a very small

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volume of organic solvent (acceptor phase) and the recovered organic phase can directly be analyzed. Extraction and preconcentration of analyte can be achieved simultaneously in MLLE without further treatment. For example, Zapf, Heyer and Stan, (1995), extracted 82 pesticides from spiked tap water sample by taking it (400 mL) in a narrow-necked bottle (500 mL) followed by addition of NaCl (150 g) and buffered to a pH of 6.5–7. After addition of toluene (0.5 mL), the sealed bottle was shaken vigorously for 20 min. The extracting solvent was brought to the bottle neck by the addition of NaCl saturated solution, recovered and analyzed with GC. The recoveries have been reported higher than 50%. Advantages of MLLE are simple equipment, low need of labor, time and cost, lower usage of organic solvent than LLE and thus is a green method. Disadvantages of the technique include low enrichment, non-automatable, can extract only non-polar pesticides, suitable for rapid screening and not for routine analysis. Two types of MLLE commonly employed for pesticides extraction from water samples, namely (i) dispersive liquid-liquid micro extraction (DLLME) and (ii) single drop micro extraction (SDME).

(i) DLLME This technique of liquid micro-extraction was first demostrated in 2006 (Rezaee et al., 2006) and soon became an efficient extraction technique for pesticides extraction especially from water samples. The advances in this technique focusing on the pesticides extraction from water samples have been thoroughly reviewed recently since 2006 (Primel, Caldas, Marube, & Escarrone, 2017). Principle of DLLME is based on ternary solvent system in which three solvents are involved i.e. disperser solvent, extractor solvent and aqueous phase being extracted. The disperser solvent (mL) must have miscibility with both the extractant (µL) and aqueous phase. Mixture of disperser and extractant is injected into aqueous phase forming cloudy solution due to small dispersed droplets of extractant in aqueous phase. This phenomenon results in a large surface area contact between aqueous phase and the extractant, thus equilibrium state is achieved quickly in a very short period of time. The mixture is then centrifuged, and the phase separation is accomplished due to the differences in their densities. If the extractant is denser, it will sediment to the bottom of the conical tube and can easily be collected and

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analyzed. Extractor solvent must not be miscible with the aqueous phase and should has great affinity to the analyte component. The most used organic extractor solvents having higher density than aqueous phase include chlorobenzene, chloroform, , tetrachloroethylene and dichloromethane. In contrary, lighter organic extractant solvents than water are also being used such as cyclohexane, 1-undecanol, 1-octanol, 1-hexanol, 1-dodecanol, 1-decanol, hexadecane, hexane and m-xylene. In addition, ionic liquids started to be used as extractant by various analysts to reduce toxicity from the exposure to the generated organic solvent residue (Primel, et al., 2017). The volume of the extractor solvent must be optimized, and various volumes of the solvents have been employed as optima from 5.2 to 685 µL for the extraction of pesticides and other analytes. The selection of the type of dispenser solvent depends on its miscibility with both the solvents and commonly used dispenser solvents include acetonitrile, and acetone for pesticides extraction from various environmental samples. In addition, other solvents such as surfactants, ionic liquids, propanols and tetrahydrofuran have also been rarely used. The volumes of these dispenser solvents have been employed from 7.8 to 2000 µL. Other factors which should be checked for enhanced recovery and enrichment factor include pH, salt concentration, centrifugation speed and time. DLLME has many applications for pesticides extraction from various environmental samples especially water (Primel, et al., 2017). This technique is associated with many advantages in relation to the conventional LLE such as high enrichment factor, low cost, speed, operation simplicity, easy to manipulate, environmentally green, less waste generator, organic extractant’s reduced volume and less laborious. The main disadvantage is the limited number of extractor solvents which have immiscibility with water, form cloudiness when mixed with aqueous phase and have high affinity for analyte (Primel et al., 2017).

(ii) Single drop micro extraction (SDME) Jeannot and Cantwell (1996) introduced SDME for the first time (Jeannot, & Cantwell, 1996). The principle of the technique is to expose a drop of the extracting solvent for a certain time to a sample and then to collecte and analyse it. The usual

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procedure is to expel a micro-drop of extracting solvent carefully and set at the needle tip of a microsyringe and then immersed in the solution of sample. Distribution equilibrium between micro-drop of extracting solvent and the sample solution is brought about by magnetic stirring for a certain time period. After retraction back of micro-drop into the micro syringe followed by analysis. This extraction technique can be sub classified as direct immersion SDME (DI-SDME), headspace-SDME (HS- SDME) and solidified floating organic drop microextraction (SFOD-ME). In DI- SDME a drop of extracting solvent is directly immersed in the sample solution, while in HS-SDME the drop is exposed to the headspace above the sample solution. Alternatively, in SFOD-ME the extraction solvent lighter than water is dropped on the surface of aqueous phase while stirring for a certain period (Zanjani, Yamini, Shariati, & Jonsson, 2007). The collection of this drop is difficult with micro-syringe; therefore, solidification of the extraction solvent is performed by cooling and is collected with spatula for analysis. Furthermore, the criteria for extraction solvent to be used as micro-drop is immiscibility with water and high boiling point (Garbi, Sakkas, Fiamegos, Stalikas, & Albanis, 2010). Advantages of SDME are one step extraction process, negligible consumption of toxic organic solvent and low operation time (Amvrazi, & Tsiropoulos, 2009).

1.1.3.2 Solid phase extraction (SPE) SPE is the extraction method which uses liquid and solid phase for the isolation of varieties of analytes from solution phase. It has also been commonly used as cleanup of sample before analysis (Das, & Kumar, 2014). The basic principle of SPE relies on the partitioning of analyte (pesticide) between liquid phase of sample matrix and solid phase (sorbent). In the first step, the analyte is adsorbed on the stationary phase, which can be reverse phase (non-polar e.g. C18) or normal phase (polar e.g. alumina or silica), and as a result retention of analyte takes place on it. After washing the components of matrix, the desorption or elution of the analyte is accomplished with a suitable solvent in the second step for further process and analysis (Ferenc, & Biziuk, 2006). The list of the most popular sorbents commonly used in standard SPE include graphitized carbon black, styrene-divinylbenzene copolymers, octyl-silica (C8) and octyldecyl (C18) (Soriano, Jimenez, Font, & Molto, 2001).

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Depending on the approach of system, SPE can be either in on-line or off-line configuration mode. In the former mode, the column of SPE is connected directly to the analytical column of chromatographic technique and separation and elution are achieved in single step. While in the off-line mode both functions are performed in two separate steps (Liska, 1993). However, the manual offline extraction mode is dominantly used for the extraction. Different types of SPE have been used for pesticides extraction from water samples and can be categorized generally in four types such as: (i) SPE cartridges, (ii) SPE disks, (iii) solid phase micro-extraction (SPME) and (iv) on column SPE. i) SPE cartridges In this technique, the sorbent is to be packed in polypropylene cartridge between two fritted disks. Liquid sample and other solvent can be passed through it either by positive pressure (pressure from syringe or by gas pressure or by gravity) or by suction. The objective of SPE can be the removal of interfering components of the sample matrix and pre-concentration (100-400-fold) of analyte when large volume of water sample (1-2 liters) is extracted and elution is performed with small volume of solvent (5-10 mL). The general procedure for using SPE cartridge include the following steps. First of all, the cartridge is prepared by washing with a small aliquot of non-polar solvent (e.g. acetone, ethyl acetate) followed by a polar solvent (water, methanol). Water sample is then passed through the undried cartridge at a relatively fast flow rate and if suspended solids are present in the sample, then it must be filtered before loading. The cartridge is washed with a small aliquot of water after the sample is loaded through it followed by drying by passing air. Elution of the analyte from the cartridge is brought with reverse order of the same solvents used in the preparation step. The eluate is dried under the gentle stream of nitrogen gas and reconstitute in the solvent used as mobile phase or carrier for analysis (Baez, Lastra, & Rodriguez, 1996). SPE has several advantages over conventional LLE such as short analysis time, need of less amount of organic solvents, lower cost, easy to transport from laboratory to filed and vice versa, for storage of unstable analytes, has potential for semiautomated or automated extraction. But the technique is also accompanied with some drawbacks, which are variable recovery for pesticides and need of prefiltration

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for pesticides surface water sample as the suspended solids present in it block the SPE cartridge (Baez, et al., 1996). ii) Solid phase extraction disks (SPE-disk) In SPE disks the sorbent such as C18 or C8 is bonded to a solid support and the configuration of 0.5 mm thick membrane is made like a disk. It allows water samples to be passed through it at a higher flow rate than SPE cartridge and a very large sample volume with trace quantity of pesticides can be passed (Riley, et al. 2005). Other advantages and disadvantages are same as of SPE-cartridges. The general sample extraction procedure for Empore disk is described in the following lines (Lambropoulou, Konstantinou, & Albanis, 2000). Before use, the SPE disk is conditioned by soaking into organic solvent such as acetone followed by incorporation in extraction manifold and passage of water sample. During the passage of sample the disk should not be dried. Elution of pesticide from the disk is performed by passage of small volume of organic solvent (e.g. ethyl acetate-dichloromethane mixture) or mixing with organic solvent in a closed vessel. Reduce the volume of organic solvent or dry it by evaporation and re-dissolve the pesticide residue in mobile phase or carrier.

iii) Solid phase micro-extraction (SPME) SPME was first demostrated in 1990 (Arthur, & Pawliszyn, 1990; Zhang, Yang, & Pawliszyn, 1994) and is a very simple procedure to prepare water sample and all the extraction steps (extraction, concentration, elution etc) have been integrated in just one device and one step. The device consists on a fused silica fiber (1–2 cm long), coated with suitable stationary phase (e.g. polydimethylsiloxane (PDMS)) and a modified syringe. The fiber is retractable and is housed in the solid metallic needle of the syringe for protection. By depressing the plunger of syringe, a measured length of the fiber can be exposed to sample. The coated polymer on the fiber can act like a sponge and the sample absorption or adsorption for extraction and concentrating takes place on the principle of chromatography (liquid-liquid or gas- liquid partitioning) (Ulrich, 2000). As a result, extraction on SPME fiber consists of two mode i.e. direct immersion mode and head space mode (HS-SPME). In the former mode, the analyte is extracted on fiber from liquid phase and in later mode the

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needle is inserted through a septum into the headspace of a liquid sample in a vial for extraction (Sakamoto, & Tsutsumi, 2004). Direct immersion mode (DI-SPME) is more sensitive, while HS-SPME is cleaner thus avoiding high background (Kataoka, Lord, & Pawliszyn, 2000). After extraction and sampling, the fiber is protected mechanically by retraction into metallic needle and withdrawn from the sample container. The next step is the desorption of analyte from fiber. For this purpose, the fiber is directly introduced into the GC hot inlet for the desorption of analyte thermally into the GC column or the elution is accomplished with mobile phase which is analysed by LC. The most common fibers which are coated on fused silicon are PDMS and polyacrylate (PA). In addition, other types of solid sorbents, which are employed for the coating of fibers include carboxen-PDMS, PDMS-divinylbenzene (PDMS-DVB), DVB-carbxen PDMS, carbowex-templated resin (CW-TR) and carbowax-DVB (CW-DVB) (Goncalves, & Alpendurada, 2002). Special types of fibers having characteristics of bipolarity is used for simultaneous pesticides analysis having different polarities in water samples. This is a solvent free extraction technique and fiber is reusable and can be used more than 100 injections. Further advantages are consuming less sample size and is highly well-suited with GC and LC. Main disadvantage of the technique is performance loss with increased usage. In addition, manual SPME needs expert operator for precise control of all variables. Disadvantages related to manual SPME are overcome by using in-capillary column SPME (Takino, Daishima, & Nakahara, 2001).

1.2 LUMINESCENCE Luminescence (lumen (Latin) = light) is the emission of light from an electronically energized compound specie coming back to its ground electronic state (Barnett, & Francis, 2005). There are three modes of luminescence, which are classified according to the processes through which the excited electronic state of the light emitter is generated. The three modes of luminescence include photoluminescence (excited state of emitter is generated by means of absorption of light), chemiluminescence (CL) (emitter’s excited mode is generated by a chemical reaction) and electrochemiluminescence (ECL) (electrochemical reaction is responsible for the generation of electronically excited emitter).

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Photoluminescence can be further classified into fluorescence and phosphorescence. This classification mainly depends on the spin of electron in higher energy i.e. in excited state. Electron, in the excited state having singlet state when transits to ground singlet state, is termed as spin allowed luminescent transition and the light emission is called as fluorescence. In contrary, if the spin of electron converts into triplet excited state, also called intersystem crossing, then transits to ground triplet state, the resultant light emission is termed as phosphorescence (Lakowicz, 1999). Bioluminescence (BL) is a naturally occurring CL in living creatures such as firefly, starfish etc (Girotti, Ferri, Bolelli, Sermasi, & Fini, 2001).

1.2.1. Chemiluminescence (CL) CL is the sub branch of luminescence, in which emission of light, in either ultraviolet, visible or infrared region of electromagnetic spectrum, results from a chemical reaction. CL reactions can be categorized as either direct (Dodeigne, Thunus, & Lejeune, 2000) or indirect (Townshend, 1990) CL reaction as depicted in reaction schemes given below. In the former type, an electronically excited intermediate product is formed during a chemical reaction and on the de-excitation of that excited product, emission of light takes place. Whereas in the later type of CL reactions, if the excited intermediate product is an inefficient emitter, the energy might be transferred to either a fluorophore or a sensitizer molecule having high quantum efficiency than intermediate excited product and the reaction is given as under:

In the above general CL reaction, the chemical reagents A and B are reactants, C is the intermediate product and * represents excited state.

1.2.1.1 CL reagents The most commonly used typical CL reagents include luminol, lucigenin, / 2+ Tris(2,2 -bipyridyl) ruthenium(II) (Ru(bpy)3 ), potassium permanganate (KMnO4),

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cerium(IV) (Ce(IV)), potassium fericyanide (K3[Fe(CN)6]), peroxyoxalate (PO), diperiodatoargentate(III) (DPA), diperiodatocuperate(III) (DPC) and diperiodatonickelate(IV) (DPN). The chemical structures of a few organic CL reagents are shown in the Table 1.4. The analytical applications of CL reagents have been thoroughly reviewed (Timofeeva, Vakh, Bulatov, & Worsfold, 2018; Adcock, Barnett, Barrow, & Francis, 2014; Waseem, Yaqoob, & Nabi, 2013; Iranifam, 2013; Perez, Gonzalez, Rodriguez, Lara, & Campana, 2016; Lara, Campana, & Aaron, 2010; Su, Chen, Wang, & Lv, 2007; Campana, Lara, Gracia, & Perez, 2009; Lara, Rodriguez, Gonzalez, Perez, & Campana, 2016; Gorman, Francis, & Barnett, 2006; Zhao, Sun, & Chu, 2009; Fletcher, Andrew, Calokerinos, Forbes, & Worsfold, 2001; Campana, & Lara, 2007; Dodeigne, Thunus, & Lejeune, 2000; Brown, Francis, Adcock, Lim, & Barnett, 2008; Kricka, 2003; Gracia, Campana, Chinchilla, Perez, & Casado, 2005; Mestre, Zamora, & Calatayud, 2001).

Table 1.4 Chemical structures of a few organic CL reagents.

In the following paragraphs, the CL reaction mechanisms of some CL reagents used in the proposed study with their analytical applications coupled with flow injection analysis (FIA) for various pesticides estimation in water samples are described.

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i. Luminol Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) was synthesized in 1908 and is one of the most widely employed CL reagent (Schmitz, 1913; Thornton, & Maloney, 1985). The CL reaction scheme for the oxidation of luminol (pH 10 – 11, quantum yield ≈ 0.01) (Schulman, 1983; Roswell, & White, 1978; White, & Roswell, 1985) is shown as under:

– Luminol CL reaction has been oxidized by many oxidants such as H2O2, IO4 , – – OCl , MnO4 , DPA and DPC (Lopez, et al., 2001; Yang, Zhang, & Wang, 2010). The reaction of luminol in the presence of H2O2 has been catalyzed by some transition metal ions such as Co2+, Cu2+, Fe2+, Cr2+, Mn4+ and Ni2+ and complexes e.g. heamoglobin and peroxidases (Marquette, & Blum, 2006). A number of articles are present in literature using FIA–CL (Mestre, Zamora, & Calatayud, 2001) for the simple, sensitive and selective analysis of pesticides, metal ions, amino acids, toxicants and pollutants (Perez, Alegria, Hernando, & Sierra, 2005; Song, Yue, & Wang, 2006; Satienperakul, Cardwell, Kolev, Lenehan, & Barnett, 2005). Luminol CL reactions coupled with different oxidizing agents in continuous flow systems have many applications for the quantitative analysis of pesticides in various samples matrices including water. In alkaline medium, the luminol CL reaction with KMnO4 has been employed for the estimation of various pesticides in water matrices. Yang, & Zhang (2013) have used this reaction for the quantitative analysis of azoxystrobin in ground, lake and tap waters. This reaction also has applications for picoxystrobin quantitative analysis in river, ground and tap waters (Zhang, Yang, Zhu, & Dong, 2012), carbofuran, and in vegetable and water samples by post HPLC column CL (Perez, & Campana, 2008), carbofuran in lettuce and water samples (Perez, Gracia, Campana, Casado, & Vidal, 2005) and

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carbaryl in vegetal food and natural waters (Perez, Campana, Casado, & Gracia, 2004a; Perez, Campana, Gracia, Casado, & del Olmo Iruela, 2004b). Some researchers have attempted to establish simple FI-CL methods for the quantitative analysis of pesticides based on the photoinduced CL (PICL) procedures. In this technique pesticides have been hydrolysed in a photoreactor in the presence or absence of an oxidant and the resulted photoproducts have been used to oxidze luminol. The photodegradation of three dithiocarbamate fungicides such as maneb, nabam and thiram has been studied under alkaline conditions and the resulted photoproducts have oxidized luminol (Waseem, Yaqoob, & Nabi, 2009). This CL reaction has paved a way for the quantitative estimation of these pesticides in various water samples of natural origion. The same group has established one more FI-CL approach for simetryn quantitative analysis in water samples of natural origin based on its hydrolysis under alkaline conditions into its photoproducts which resulted in luminol oxidation when an oxidant or a catalyst was not present during the CL reaction (Waseem, Yaqoob, & Nabi, 2008). The CL reaction of luminol and H2O2 coupled with sodium chloride (NaCl) as an enhancer has been used for the quantitative analysis of (Liu, Li, Wu, & Lu, 2007) and

(Du, Liu, & Lu, 2003) in water samples. The oxidizing property of H2O2 has been used for alkaline luminol oxidation and a CL method in FI mode has been developed for carbendazim quantitative analysis in the samples of tap water (Liao, & Xie, 2006).

Horseradish peroxidase (HRP)-luminol-H2O2 CL system has been employed for quantitative analysis of asulam in tap water (Sanchez, Diaz, Bracho, Aguilar, & Algarra, 2009), in tap water (Diaz, Bracho, Algarra, & Sanchez, 2008) and atrazine (Chouhan, Rana, Suri, Thampi, & Thakur, 2010) and these pesticides enhanced the CL emission of this reaction. Using the same CL reaction, quenchometric methods have been established for the quantitative analysis of asulam in pure and raw water samples (Sanchez, Diaz, Tellez, & Algarra, 2008). To increase the selectivity of luminol-H2O2 CL system, various researchers have used on-line molecularly imprinted polymer membranes (MIPs) for pesticides selective extraction followed by their quantitative analysis. Using this technique, pesticides such as thifensulfuron-methyl has been determined in tap, sea, reservoir and well water samples (Xie, Gao, Zhou, & Li, 2011). Table 1.5 presents the analytical charcteristics

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of all the aforementioned luminol FI-CL methods for the quantification of pesticides in water samples.

ii. Lucigenin Lucigenin (N,N′-dimethyl-9,9′-diacridinium nitrate: Luc2+ was introduced in 1935 (Gleu, & Petsch, 1935), this organic and classical CL reagent exhibits weak CL emission in basic solutions (Maskiewicz, Sogah, & Bruice, 1979a), and intense CL • emission in the presence of H2O2 or superoxide radical (O2 ) (Maskiewicz, Sogah, & Bruice, 1979b; Vladimirov, & Proskurnina, 2009). The CL reaction mechanism for lucigenin involves the one-electron reduction of Luc2+ generating the cation radical of •+ • lucigenin (Luc ), which further reacts with O2 generating dioxetane of lucigenin

(LucO2). This dioxetane ring containing lucigenin is highly unstable and promptly decomposes to excited N-methylacridone (NMA) which produces intense green light emission (λmax ≈ 440 nm) due to its active oxidized intermediate product N- methylacridone (quantum yield in the range of 0.01 – 0.02) (Kruk, 1998; Rauhut, Sheehan, Clarke, & Semsel, 1965a; Rauhut, Sheehan, Clarke, Roberts, & Semsel, 1965b). The general CL reaction mechanism for lucigenin is given below:

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This CL reagent has been widely employed in analytical science for the quantitative analysis of organic and inorganic reductants, superoxide radicals and inorganic anions in diverse samples both using batch and flow injection (FI) CL techniques (Dong, Wang, Peng, Chu, & Wang, 2017; He, & Cui, 2013; Asgher, Yaqoob, Waseem, & Nabi, 2011; Wabaidur, Alam, Alothman, & Eldesoky, 2014; Khajvand, Alijanpour, Chaichi, Vafaeezadeh, & Hashemi, 2015; Wang, et al., 2016; Zhidkova, Proskurnina, Parfenov, & Vladimirov, 2011; Li, & Lee, 2005; Du, Li, & Guan, 2007; Malejko, Zylkiewicz, & Kojlo, 2010; Attiq-ur-Rehman, Yaqoob, Waseem, Nabi, & Khan, 2010). Despite extensive literature research, we did not find any flow or batch method to estimate pesticides in water samples using lucigenin or its derivatives as a CL reagent

′ 2+ iii. Tris(2,2 -bipyridyl) ruthenium(II) [Ru(bpy)3] 2+ [Ru(bpy)3] is a coordination compound and commonly found in a red colored hexahydrated salt of chloride with a chemical formula of

C30H24N6Cl2Ru.6H2O. Orange colored CL radiation is generated, having wavelength 2+ 3+ maximum of 620 nm, from the excited state of [Ru(bpy)3] when [Ru(bpy)3] is reduced during the chemical reaction under acidic conditions as given:

2+ 3+ Before use, [Ru(bpy)3] must be oxidized to [Ru(bpy)3] (Lee, & Nieman, 1996) by using diverse oxidation methods, such as chemical (with various oxidizing agents e.g. KMnO4, Ce(IV) and PbO2), electrochemical and photochemical (UV– irradiation in the presence of peroxodisulphate) oxidation. This chemical specie can be reduced by various analyte compounds, for example, oxalate, aliphatic amines and 2+ aminoacids and thus generating electronically excited [Ru(bpy)3] * molecule, which on radiative relaxation and deexcitation to electronically ground state emits light. 2+ The analytical applications of [Ru(bipy)3] CL employing various analytical approaches (FIA, batch CL, miniaturized ECL flow through cell, HPLC post column

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2+ CL, capillary electrophoresis (CE), immobilized [Ru(bipy)3] CL or ECL and ECL inhibition) for the quantitative analysis of various analytes, which may be organic or inorganic, have been extensively and critically reviewed (Perez et al., 2016; Lara et al., 2016; Waseem et al., 2013; Iranifam, 2013; Lara et al., 2010; Compana et al., 2009; Su et al., 2007; Gorman et al., 2006). 3+ FIA coupled with [Ru(bipy)3] CL has many applications for the estimation of various pesticides in diverse samples including water samples of natural origin. An herbicide related to triazine class, namely atrazine has been quantified in water sample (Beale, Porter, & Roddick, 2009) and in this method the carrier and sample 3+ streams were propelled by a peristatic pump, while the Ru[(bipy)3] oxidized by 3+ PbO2 was injected into the proposed FICL manifold and the [Ru(bipy)3] was reduced to the excited lower oxidation state emitter by atrazine. HPLC is a very powerful tool for different pesticides separation from each other in diverse samples and it has many applications for the quantitative analysis of pesticides with lower LODs when coupled with some post column CL reactions. 2+ Thus, using an HPLC post column CL reaction of [Ru(bipy)3] , a procedure has been devised for the quantitative analysis of propoxur, , and carbaryl (N-methylcarbamate pesticides) (Ruiz, Lozano, & Garcia, 2007) in fruit and water samples. The mechanistic approach of the method was the on-line generation of N- methylamine from N-methylcarbamate pesticides when they were irradiated in a UV- 3+ light photoreactor after separation by HPLC column and reuduction of [Ru(bipy)3] 3+ by these N-methylamine subsequently. In this method, the [Ru(bipy)3] had also been 2+ produced online photochemically by [Ru(bipy)3] oxidation in the presence of peroxydisulfate. The same mechanistic approach has also been used for the quantitative analysis of carbaryl (Ruiz, Lozano, Tomas, & Martin, 2003) in various samples including water, and carbofuran and promecarb (Ruiz, Lozano, Tomas, & Martin, 2002) in water, soil and grains samples using FI-CL techniques. The analytical figures of merit, concerned to the flow based methods and using this 2+ coordination compound [Ru(bipy)3] as a CL reagent for pesticides quantitative analysis in natural water samples are given in Table 1.5.

iv. Potassium permanganate (KMnO4)

In 1920, the CL application of KMnO4 was reported for the first time, when

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pyrogallol was oxidized by acidic KMnO4 in the presence of H2O2 (Grinberg, 1920).

Since 1975, the extensive use of KMnO4 as a CL reagent has been started when a method was deviced and used for the quantitative analysis of sulphur dioxide (SO2) in atmospheric samples (Stauff, & Jaeschke, 1975).

The CL reaction mechanism for KMnO4 CL reaction has been explored by various researchers and the generalized light producing pathways given in the reactions schemes (Slezak, et al., 2011). According to one type of CL reaction mechanism, the multistep reduction of Mn(VII) takes place by analyte and produces Mn3+ and analyte radicals as intermediates (Scheme-I). This Mn3+ can be stabilized under strong acidic conditions or by using complexing agents such as polyphosphate. Furthermore, the intermediate analyte radicals reduce the Mn3+ and generate the excited Mn2+ (Scheme-II) (Hindson, Francis, Hanson, Adcock, & Barnett, 2010) as a red-light emitter with λmax. of 734 ± 5 nm or 689 ± 5 nm in polyphosphates (Scheme- III) when comes to ground state (Adcock, Francis, Smith, & Barnett, 2008). According to a second type of CL reaction mechanism, the pool of Mn3+ can also be generated with reducing agents other than analyte such as thiosulfate, formic acid or formaldehyde (HCHO) (Scheme-IV). Further reduction of Mn3+ takes place with analyte, generating light emitting excited Mn2+ (Scheme-V) which on deexcitation generates CL emission (Scheme-VI) (Slezak, et al., 2011; Francis, et al., 2011).

Mn7+ + Analyte Mn3+ + Intermediate radicals (I) Mn3+ + Intermediate radicals Mn2+* + Other products (II) Mn2+* Mn2+ + hv (734±5 nm) (III)

OR

Mn7+ + Reducing agent (HCOOH or HCHO) Mn3+ + Oxidized products (IV) Mn3+ + Analyte (reducing agent) Mn2+* + Oxidized products (V) 2+* 2+ Mn Mn + hv (734±5 nm) (VI)

The analytical applications of KMnO4 are reflected from the comprehensive review articles covering the quantitative analysis of diverse analytes including different organic and inorganic species in environmental, pharmaceutical, forensic, biological, consumer products, clinical and agricultural samples (Adcock, et al., 2014; Hindson, & Barnett, 2001; Adcock, Francis, & Barnett, 2007; Iranifam, 2016; Perez,

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et al., 2016; Waseem, et al., 2013).

Many FI-CL methods using KMnO4 as a CL reagent in acidic medium have been established for pesticides quantitative analysis in water samples. A FI- photoinduced (PI)-CL procedure for the direct quantitative analysis of a carbamate insecticide namly carbaryl in natural water samples has been reported without using any CL enhancer (Tsogas, Giokas, Nikolakopoulos, Vlessidis, & Evmiridis, 2006). Two carbamate pesticides such as carbaryl and carbofuran have also been analysed quantitatively in water samples of natural origin using FI acidic KMnO4–sodium sulfite (Na2SO3) CL reaction system. This method can also be used for the quantitative analysis of OPs pesticides such as dinoseb, diazinon and (Waseem, Yaqoob, & Nabi, 2007). The photolysis of a nitrile herbicide: bromoxynil, was performed under alkaline condition, sensitized by ethanol and the photofragments were quantified by FI direct CL reaction of KMnO4 in polyphosphoric acid medium. Utilization of the technique is for the assurance of this herbicide in water samples and pesticide formulation (Pawlicova, et al., 2006). Stop FI–CL method has been devised for antu quantitative analysis (rodenticide) in samples of river water, oat grain, barley and wheat. The method is based on the reaction of antu with acidic KMnO4 using HCHO as a sensitizer and three analytical parameters related to light, such as maximum intensity, rate of light decay process and total emitting area, were measured (Murillo, Garcia, & Rubio, 2005). To reduce the generation of waste and consumption of reagents, analytically green PICL coupled with the multicommutation flow based methods have been developed for the analysis of pesticides quantitatively in water samples, using acidic

KMnO4 CL reaction. Employing this technique, the quantitative analysis of three imidazolinone pesticides such as imazapyr, imazaquin and imazamethabenz-methyl has been performed in tap and mineral waters (Ricart, & Mateo, 2013). Herbicides related to buminafos family have enhanced this CL system and a method has been developed for the quantification of buminafos in spiked waters and soil samples (Malo, & Calatayud, 2008). The alkaline photolysis of karbutilate herbicide and subsequent chemiluminometric reaction with KMnO4 in polyphosphoric aicd medium, using continuous flow multicommutated assembly, led its quantitative analysis in water and human urine samples (Amorim, Garcia, Montenegro, Araujo, &

Calatayud, 2007). Using the same technique in H2SO4 acidic medium, the pesticides

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such as propanil (Garcia, Icardo, & Calatayud, 2006), chlorsulfuron (Mervartova, Calatayud, & Icardo, 2005) and asulam (Chivulescu, Icardo, Mateo, & Calatayud, 2004), have been determined in water samples. The analytical figures of merit and other characteristics for all FI-CL methods using KMnO4 as a CL reagent for the quantitative analysis of different pesticides in natural water samples are given in Table 1.5.

v. Cerium(IV) sulfate (Ce(SO4)2) Ce(IV) is a strong oxidizing agent and highly stable in an acidic medium

(generally H2SO4) and generates weak CL emission when reduced by reducing agents such as sulfites, thiosulfate, dithionite and rhodamine B (RhB) or rhodamine 6G (Rh6G) dyes (Waseem, et al., 2013). Generally, the type of emitting species has not been established statisfactorilly during the CL reactions of Ce(IV) and five chemical species have been claimed to be the CL emitters: such as Ce(III) (emission λmax. = 355 * nm), excited SO2 (emission spectrum = 300 – 450 nm), fluorophore, oxidized and excited products of analyte and singlet oxygen dimer. The intermediate oxidized products of some analytes with Ce(IV) act as excited fluorophore and results in the 2– enhancement of CL emission. If sulfite (SO3 ) is oxidized with Ce(IV) in acidic medium, excited SO2 is formed as a final product. The continuum of the CL emission * spectrum of electronically excited sulfur dioxide (SO2 ), which is generated during 2− the CL reaction of Ce(IV)–SO3 , extends over the range of 450 – 600 nm. The * –1 energy of SO2 was calculated to be 75.9 kcal mol , with the zero-point vibrational * effects. The CL emission intensity is further enhanced, when energy from SO2 is transferred to the flourophores acting as analytes, through the phenomenon of resonance energy transfer or highly strong fluorescent compounds acting as enhancers (rhodamine B, quinine or lanthanide ions) (Kaczmarek, 2015). RhB and Rh6G compounds have also been used with Ce(IV) as sensitizer and the CL light emission at λmax. 425 nm is ascribed to the formation of excited complex between Ce(IV) and RhB, while light emission at 555 and 446 nm is due to the excited unoxidized and oxidized Rh6G compounds respectively (Waseem, et al., 2013). The CL mechanisms 2– of Ce(IV) with SO3 and Rh6G (Mervartova, Polasek, & Calatayud, 2007) are elaborated in the following reaction schemes I and II respectively.

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Scheme–I

Scheme–II

The CL reactions of Ce(IV) in flow modes have many applications for different pesticides quantification in natural water samples. For the analysis of eight pesticides belongs to the carbamate family in water samples of natural origin, an HPLC–PICL analytical procedure using the CL reaction of Ce(IV)–quinine has been reported. These carbamate pesticides include thiophenate-methyl, , thiodicarb, , , , methiocarb and aldicarb. The method was based on the off-line SPE, separation of pesticides using HPLC column and photodegradation, followed by Ce(IV) CL reaction. High selectivity of this method has been achieved for those carbamate pesticides which have sulphur in the backbone of their chemical structures (Icardo, Lloret, & Cartas, 2016). Quinmerac: an auxin herbicide, has been quantitatively analysed in water samples using FI-PICL-

Ce(IV)-Na2SO3 reaction in H2SO4 medium (Icardo, Paz, & Perez, 2015). Using Ce(IV)-quinine CL reaction, an FI-PICL procedure has been developed to determine N-methylcarbamate pesticides qunatitatively in natural water sample, these pesticides include methomyl, aldicarb and butocarboxim. The pesticides were first photodegraded followed by oxidation with Ce(IV) (Paz, Icardo, & Sanchez, 2014). A similar approach has been used for the analysis of zinc containing dithiocarbamate fungicides: zineb and ziram, with photodegradation (Paz, & Icardo, 2008), and thiram

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without photodegradation (Waseem, Yaqoob, & Nabi, 2010) in spiked natural water samples. Nine organothiophosphorus (OTP) pesticides have been estimated in marine and fresh water samples and these OTP pesticides include chlorpyrifos, diazinon, , -sulfoxide, malathion, , , and pirimiphos-methyl. Initially, pesticides were separated using an HPLC C18 column, followed by post column alkaline hydrolysis and CL reaction with Ce(IV) enhanced by hexadecylpyridinium chloride (HPC) (Icardo, Zamora, Cartas, & Lloret, 2014). The response of Ce(IV) CL reaction coupled with HPLC and FI has been employed for dimethoate quantitative analysis in water samples from natural origin after hydrolysis in strong alkaline conditions (Icardo, Paz, Baron, & Badena, 2012). An herbicide related to the auxin plant growth hormone class namely, 3-indolyl acetic acid, has been analysed quantitatively in water samples using its enhancement effect on the CL reaction of Ce(IV)–β-cyclodextrine in HNO3 acidic medium (Neves, Garcia, & Calatayud, 2007).

The CL reaction of Ce(IV) in HNO3 acidic medium sensitized by Rh6G, has been used for carbaryl quantitative analysis in grain, soil and water samples. Three CL emitters have been proposed in this reaction such as excited Ce(III) ion, excited Ce(III)-carbaryl complex and excited Rh6G (Pulgarin, Molina, & Lopez, 2006). The

CL reaction of Ce(IV)-Na2SO3 in H2SO4 acidic medium has been enhanced by carbofuran and thus a method was devised for its quantitative analysis in cabbage samples (Xie, Ouyang, Guo, Lin, & Chen, 2005). The analytical figures of merit of these FI–CL methods based on the Ce(IV) CL reactions are presented in Table 1.5. In addition, the analytical applications of CL reactions utilizing cerium(IV) sulfate as CL reagent have also been thoroughly and critically reviewed (Waseem, et al., 2013; Kaczmarek, 2015; Chen, & Fang, 2007).

vi. Potassium ferricyanide (K3[Fe(CN)6])

The oxidizing power of K3[Fe(CN)6] has been exploited in various CL reactions under alkaline medium. The CL emitting species may be the oxidized intermediate product of an analyte (Lv, Zhao, Zhu, Xu, & Zhang, 2005), singlet oxygen (Han, Zhang, Kang, Tang, & Zhang, 2012) or singlet oxygen dimer (Ishii, Tsukino, Sekine, & Nakata, 2009), and further enhancement of the CL emission has been reported by employing various sensitizers such as Rh6G (λmax. emission = 560

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nm) (Lv, et al., 2005), oxidized 2-phenyl-4, 5-di (2-furyl) imidazole (λmax. emission =

525 nm) (Han, et al., 2012), and K3[Fe(CN)6]–gallic acid complex (λmax. emission = 530 and 700 nm) (Ishii, et al., 2009) respectively.

A number of pesticides have been determined by employing K3[Fe(CN)6] CL reaction systems coupled with FI. Two different FI-CL and HPLC–CL methods have been deviced for the quantitative analysis of 4–(4–chloro–o–tolyloxy) butyric acid (MCPB) in water samples of natural origin. Both methods are based on the CL reaction of photodegraded products of the pesticide with alkaline ferricyanide. The sensitivities of the methods have been enhanced by appling off-line SPE pre- concentration technique and HPLC-CL method has shown more selectivity (Lloret, Cartas, Icardo, & Benito, 2016). This pesticide has additionally been analysed quantitatively in water samples, utilizing the CL response of the same CL reaction, i.e. the photodegraded products of MCPB have been reacted with alkaline ferricyanide in a FI mode. The sensitivity of the method has been increased from 0.5 ppb to 0.13 ppb, employing SPE preconcentration by factor of 4 (Cartas, Benito, & Lloret, 2012). A PICL method has been established in FI mode for imazalil quantitative analysis in water samples. The method was based on the photodegration of imazalil under basic medium, followed by CL reaction with alkaline ferricyanide. Sensitivity and selectivity have been increased by employing SPE procedure, and preconcetration factor of 50 has been selected for the purpose (Lloret, Cartas, & Benito, 2010). PICL in flow system of multicommutation mode for asulam herbicide’s quantitative analysis in water sample has been proposed and two CL reactions viz acidic KMnO4 and alkaline K3[Fe(CN)6] were compared in terms of selectivity and sensitivity and the former one was found better than the latter one (Chivulescu, et al., 2004). PICL coupled with continuous flow multicommutation and alkaline ferricyanide has been utilized for the chemiluminometric quantitative analysis of benfuresate herbicide in water samples (Garcia, & Calatayud, 2008). The analytical figures of merit and other characteristics for all these FI-CL methods employing ferricyanide as an oxidant for pesticides in water samples are given in Table 1.5. vii. Peroxyoxalate derivatives The CL of peroxyoxalate (PO) was first introduced in 1963 (Chandross, 1963)

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and analytical methods of POCL are based on the indirect CL reactions. The mechanism is based on the aryl oxalate ester oxidation such as bis(2,4,6- trichlorophenyl) oxalate (TCPO) and bis(2,4-dinitrophenyl) oxalate (DNPO) in the presence of an oxidant e.g. H2O2, forming 1,2-dioxetanedione as an intermediate. This intermediate chemical specie makes a charge transfer complex with a fluorophore by donating an electron from the fluorophore to the intermediate. The fluorophore gets excited electronically when the electron is transfered back to it from the intermediate product as illustrated in reaction scheme given below. The emitted CL wavelength is the characteristic of the excited fluorophore (Schuster, 1979; Bos et al., 2004; Kazemi, Abedirad, Vaezi, & Ganjali, 2012).

POCL analytical applications for the quantitative analysis of different analytes have been thoroughly reviewed (Iranifam, 2016; Perez et al., 2016; Waseem et al., 2013; Campana et al., 2009; Tsunoda, & Imai, 2005). The off-line photodegraded products of carbaryl i.e. methylamine has been derivitized with o-phthalaldehyde (OPA) for the generation of a fluorescent compound, which was then estimated by employing the FI-CL reaction of TCPO in sodium dodecylsulfate (SDS) medium coupled with H2O2 as an oxidant and imidazole as a fluorophore. Carbaryl analysis in water and vegetal samples has been performed using this FI method (Chinchilla, Campana, Gracia, Rodrıiguez, & Vidal, 2004) and the related analytical characteristics are reported in Table 1.5.

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Table 1.5 FI-CL methods for the pesticides quantitative analysis in natural water samples.

Linear range LOD Samples Analyte Reaction R2 Ref. (ppm) (ppm) h–1 Luminol –4 Azoxystrobin Luminol–KMnO4 0.001–0.1 1.3×10 NR 0.998 (Yang, & Zhang, 2013) –4 Picoxystrobin Luminol–KMnO4 0.002–0.150 2.7×10 NR 0.9992 (Zhang, et al., 2012) –4 –4 Thifensulfuron- Luminol–H2O2 3.9×10 –19.4 3.2×10 11 0.9912 (Xie, et al., methyl 2011) –4 –4 Asulam Luminol–H2O2–HRP 3.6×10 –0.035 1.2×10 NR 0.990 (Sanchez, et al., 2009) Maneb Luminol–DTCs 0.01–4.0 0.01 100 0.9987 (Waseem, et Nabam photoproducts 0.01–4.0 0.008 0.9998 al., 2009) Thiram 0.05–1.0 0.005 0.9987 –6 –5 –7 Asulam Luminol–H2O2–HRP 1.2×10 –1.38×10 3.5×10 NR 0.9993 (Sanchez, et al., 2008) –5 –3 –5 Carbofuran, Luminol–KMnO4 3.86×10 –1.650×10 3.86×10 NR 0.9890 (Perez, & Carbaryl 6.4×10–6–1.65×10–4 6.4×10–6 0.9910 Campana, Methiocarb 5.83×10–5–1.650×10–3 5.83×10–5 0.9931 2008) –4 Pirimicarb Luminol–H2O2–HRP 0.00425–0.03075 1.2×10 NR 0.997 (Diaz, et al., 2008) Simetryn Luminol 0.01–2 0.0075 100 0.9997 (Waseem, et al., 2008)

Fenitrothion Luminol–H2O2–NaCl 0.01–2.0 0.004 NR 0.9993 (Liu, et al., 2007)

Carbendazim Luminol–H2O2 0.02–2.00 0.00724 15 0:9952 (Liao, & Xie 2006)

Carbofuran Luminol–KMnO4 0.06–0.5 0.02 NR 0.9984 (Perez, et al., 2005)

Carbaryl Luminol–KMnO4 0.005–0.1 0.0049 NR 0.9979 (Perez, et al., 2004a)

Carbaryl Luminol–KMnO4 0.005–0.1 0.0049 NR 0.9979 (Perez, et al., 2004b)

Monocrotophos Luminol–H2O2–NaCl 0.02–1.0 0.007 NR 0.9991 (Du, et al., 2003) 2– –4 –4 Chlorpyrifos Luminol–IO4 4.8×10 –0.484 1.8×10 NR 0.9969 (Song, et al., (inhibition) 2002) Paraoxon Luminol-AChE– 0–0.075 7.5×10–4 15 0.995 (Roda, et al., Aldicarb ChOx–HRP 0–0.1 0.004 0.997 1994) Tris(2,2/–bipyridyl) ruthenium(II) 2+ Atrazine Ru(bpy)3 –H2SO4 0.00215–2.150 0.0013 NR 0.9795 (Beale, et al., 2009) 2+ 2– Bendiocarb Ru(bpy)3 –S2O8 0.1–25 0.004 NR 0.9992 (Ruiz, et al., Carbaryl 0.1–25 0.018 0.9995 2007) Promecarb 0.1–25 0.042 0.9990 Propoxur 0.1–25 0.020 0.9990 2+ 2– Carbaryl Ru(bpy)3 –S2O8 0.04–4.0 0.012 200 0.9995 (Ruiz, et al., 2003) 2+ 2– Carbofuran Ru(bpy)3 –S2O8 0.22–11.2 0.053 200 0.9998 (Ruiz, et al., Promecarb 0.41–16.6 0.085 0.9997 2002) Potassium permanaganate

Imazapyr KMnO4–H2SO4 0.05–10 0.05 12 0.992 (Ricart, & Imazaquin 0.1–10 0.1 0.996 Mateo, 2013) Imazamethaben 0.01–10 0.01 0.993

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z-methyl

Buminafos KMnO4–H2SO4 0.01–1.0 0.005 48 0.9999 (Malo, & Calatayud, 2008)

Carbaryl KMnO4–H2SO4– 0.1–1.0 0.01 180 0.9996 (Waseem, et Carbofuran Na2SO3 0.1–1.0 0.05 0.9993 al., 2007) Dinoseb 0.2–2.0 0.1 0.9977 Diazinon 0.3–1.5 0.1 0.9988 Malathion 0.6–1.8 0.3 0.9966

Karbutilate KMnO4–H4P2O7 0.02–20 0.01 17 0.9998 (Amorim, et al., 2007)

Bromoxynil KMnO4–H3PO4 0.005–1.00 0.005 134 0.9997 (Pawlicova, et al., 2006)

Carbaryl KMnO4–H2SO4 0.01–1.0 0.0148 NR 0.9998 (Tsogas, et al., 2006)

Propanil KMnO4–H2SO4 0.01–5 0.008 20 0.9999 (Garcia, et al., 2006)

Antu KMnO4–H2SO4– 0.05–3.00 0.005–0.01 NR 0.998 (Pulgarian, et HCHO al., 2005)

Chlorsulfuron KMnO4–H2SO4 0.1–1.3 0.06 25 0.992 (Mervartova, et al., 2005)

Asulam KMnO4–H2SO4 0.04–5 0.04 30 0.9990 (Chivulescu, et al., 2004) Cerium(IV) sulfate –4 –5 Aldicarb Ce(IV)–H2SO4– 1×10 –0.0025 9×10 5 0.992 (Icardo, et Butocarboxim Quinine 1×10–4–0.0025 9×10–5 0.994 al., 2016) Ethiofencarb 7×10–4–0.0035 2.3×10–4 0.993 Methomyl 1×10–4–0.003 6×10–5 0.998 Methiocarb 7×10–4–0.0035 2.6×10–4 0.991 Thiodicarb 1×10–4–0.0025 6×10–5 0.998 Thiofanox 1×10–4–0.0035 6×10–5 0.999 Thiophanate–methyl 7×10–4–0.0035 2.7×10–4 0.992 –5 Quinmerac Ce(IV)–H2SO4– 0.002–0.6 8×10 144 0.9990 (Icardo, et Na2SO3 al., 2015) –6 Methomyl Ce(IV)–H2SO4– 0.002–0.08 5×10 123 NR (Paz, et al., Aldicarb quinine 0.005–0.05 5×10–6 NR 2014) Butocarboxim 0.002–0.06 5×10–6 NR Oxamyl 0.002–0.06 1×10–5 NR

Omethoate, Ce(IV)–H2SO4– 0.006–0.1 0.0018 5 0.992 (Icardoa, et Dimethoate, C21H38ClN.H2O 0.02–0.15 0.006 0.992 al., 2014) Disulfoton sulfoxide, 0.004–0.1 0.0012 0.992 Methidathion, 0.004–0.1 0.0012 0.992 Phosmet, 0.0027–0.1 0.0008 0.992 Malathion, 0.01–0.3 0.003 0.992 Diazinon, 0.004–0.1 0.0012 0.992 Pirimiphos-methyl, 0.01–0.3 0.003 0.992 Chlorpyrifos, 0.004–0.1 0.0012 0.992 Dimethoate Ce(IV)–HCl– 5×10–4–0.1 5×10–5 105 0.9992 (Icardo, et C21H38ClN.H2O al., 2012)

Thiram Ce(IV)–H2SO4– 0.0075–2.5 0.0075 120 0.9988 (Waseem, et quinine al., 2010)

Ziram Ce(IV)–H2SO4– 0.005–1 0.001 121 NR (Paz, & Zineb quinine 0.005–0.2 0.001 101 NR Icardo, 2008) –4 3-indolyl acetic acid Ce(IV)–HNO3– 0.5–15.0 1×10 159 0.9989 (Neves, et β-cyclodextrine al., 2007)

Carbaryl Ce(IV)–HNO3– 0.05–2.0 0.0287 NR 0.999 (Pulgarin, et Rh6G al., 2006) Potassium ferricyanide MCPB Alkaline 0.005–0.1 0.0024 60 0.990 (Lloret, et ferricyanide al., 2016)

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MCPA Alkaline 0.0015–0.6 5×10–4 90 NR (Cartas, et ferricyanide al., 2012) Imazalil Alkaline 0.75–5 and 0.015–0.1 0.171 and 12 NR (Lloret, et ferricyanide 0.0034 al., 2010) Benfuresate Alkaline 0.001–4 1×10–4 22 0.9999 (Garcia, & ferricyanide Calatayud, 2008) Asulam Alkaline 2–4 0.5 30 0.998 (Chivulescu, ferricyanide et al., 2004) Peroxyoxalate

Carbaryl PO–H2O2– 0.026−0.65 0.009 NR 0.9959 (Chinchilla, imidazole–SDS et al., 2004)

HRP = horseradish peroxidase, DTCs = dithiocarbamates, Rh6G = rhodamine 6G, 2– AChE = acetylcholinesterase, ChOx = choline oxidase, S2O8 = peroxydisulfate,

H4P2O7 = pyrophosphoric acid, HCHO = formaldehyde, C21H38ClN.H2O = hexadecylpyridinium chloride monohydrate, MCPB = 4–(4–chloro–o–tolyloxy) butyric acid, MCPA = 4–chloro–2–methylphenoxyactic acid, SDS = sodium dodecylsulfate, NR = not reported. viii. Ag(III), Cu(III) and Ni(IV) complexes Transition metals such as Ag and Cu in trivalent states and Ni in tetravalent state have been used as oxidizing agents in different CL reaction systems. Polydentate – ligand such as periodate (IO4 ) are employed for the stabilization of these transition metals in uncommon higher oxidation states by chelating with these transition metals (Balikungeri, & Pelletier, 1978; Murthy, Sethuram, & Rao, 1986). These oxidants have found analytical applications in alkaline and or acidic conditions in biological, environmental and pharmaceutical analyses. The chemical structures of DPA, DPC and DPN are shown in Figure 1.2.

Figure 1.2 Chemical structures of a = DPA, b = DPC and c = DPN

Without using any CL reagent, these oxidants have been used for the quantitative analysis of various analytes. For example, in H2SO4 acidic solution, the DPC dissociates and produces superoxide anion radicals. These radicals on recombination generate an energy rich dimer of oxygen molecule, which on de- excitation produces CL emission at 492 nm. The energy from this dimer can be

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transferred to a fluorophore which may be an analyte being determined. The excited fluorophore emits light at its characteristic wavelength (Sun, Wang, Chen, & Wang, 2011). 5– The CL reaction mechanism of DPA [Ag(HIO6)2] ] in acidic medium (Sun, Chen, & Wang, 2010; Shen, Shi, & Sun, 2007; Sun, Chen, & Wang, 2009; Lin, & Yamada, 1999; Zhao, Zhao, & Xiong, 2013) is as under:

5– While, the CL reaction of DPC [Cu(HIO6)2] (Sun, et al. 2011) in acidic solution is:

+ 2+ [Cu(H2IO6)(H2O)] + H3O Cu + H5IO6 + O2

O2 + O2 (O2)2*

(O2)2* 2O2 + Light (491 nm)

(O2)2* + Flourophore 2O2 + Flourophore*

Flourophore* Flourophore + Light

In alkaline medium, the DPA and DPC have also been employed for the direct quantitative analysis of various analytes. The DPA in alkaline condition makes an excited complex with analyte which promptly emit light while the oxidized analyte product is liberated slowly (Yang, & Zhang, 2010). The CL reaction of DPC is based on generation of excited oxidized product of analyte and further enhancement of the CL emission has been achieved with strong reducing agent such as ascorbic acid (Hu, Zhang, & Li, 2010). The CL reaction of DPA in alkaline medium is depicted in the scheme given below:

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- [Ag(H3IO6)2] + Analyte Complex* Complex* Complex + Light

Complex Analyte (Oxidized) + Ag(I)

And the CL reaction of DPC in alkaline medium is given below:

5- 3- - Cu(HIO6)2] + 2H2O [Cu(OH)2(H3IO6)2] + 2OH

Analyte + Cu(III) Analyte (oxidized)* + Cu(II)

Analyte (oxidized)* Slow Analyte (oxidized) + Light

Analyte (oxidized)* + Ascorbic acid Analyte (oxidized) + Ascorbic acid* Fast Ascorbic acid* Ascorbic acid + Light

In addition, DPA and DPC reagents have been used as oxidants coupled with luminol CL reagent, while the analyte acts as either CL emission enhancer or inhibitor. The CL emission wavelength (425 nm) has suggested that the 3- aminopthalate is the CL emitter (Yang, Zhang, & Wang, 2010a; Yang, Zhang, & Wang, 2010b) as given below:

Various analytical applications of DPA (Sun, Chen, Wang, & Wen, 2009; Sun, et al., 2009; Chen, & Sun, 2010; Sun, et al., 2010; Fu, Li, & Hu, 2015; Sun, Shi, Wang, & Chen, 2012; Zhao, et al., 2013; Fu, Ke, & Fei, 2015; Yang, & Zhang, 2010; Zhao, & Si, 2013; Fu, Li, & Hu, 2016; Shi, et al., 2009; Yang, et al., 2010a; Sun, Chen, Shi, & Li, 2011; Yang, & Zhang, 2012; Wang, Dong, Chen, Su, & Sun, 2015;) and DPC oxidants (Sun, et al., 2011; Hu, et al., 2010: Sun, Wang, & Wang, 2013; Hu,

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& Li, 2013; Yang, et al., 2010b; Hu, Zhang, & Yang, 2008a; Hu, Zhang, & Yang, 2007; Hu, Li, & Zhang, 2011; Hu, Li, & Zhang, 2013; Yao, Zhang, Zeng, Zeng, & Zhang, 2014; Hu, & Zhang, 2008; Hu, Zhang, & Yang, 2008b) based on FI-CL technique are reported in Table 1.6.

Table 1.6 FI-CL methods of DPA and DPC complexes for the quantitative analysis of various analytes.

Linear range LOD Analyte Sample matrix Reaction R2 Ref. (ppm) (ppm) Diperiodatoargentate (DPA) –3 Lomefloxacin Pharmaceuticals, DPA-H2SO4 0.02994–3.68 9.1×10 0.9992 (Sun, et al., Enrofloxacin urine, serum, milk 0.4–3.0l 3.1×10–3 0.9987 2009) Pefloxacin 0.154–2.764l 4.4×10–3 0.9977 –3 Norfloxacin Capsule, serum, DPA-H2SO4 0.0134–5.4 3.1×10 0.9982 (Sun, et al., urine 2009) –2 Enoxacin Pharmaceuticals, DPA-H2SO4 0.066–3.3 2.0×10 0.998 (Chen, & serum, urine Sun, 2010) –3 Ofloxacin and Pharmaceuticals, DPA-H2SO4 0.0224–4.48 5.3×10 0.999 (Sun, et al., Levofloxacin biological fluids 0.0195–4.88 8.3×10–3 2010) –4 –5 Indole-3-acetic acid Urine, mung bean DPA-H2SO4 1.0×10 –0.01 7.7×10 NG (Fu, et al., sprouts, soil 2015) –2 Cefazolin sodium Injectable powder, DPA-Rh6G-H2SO4 0.04–4.0 1.73×10 0.9902 (Sun, et al., human urine 2012) –3 –4 Gemifloxacin Pharmaceutical, DPA-H2SO4- 1.0×10 –0.37 7.3×10 0.9939 (Zhao, et biological fluids CTAB al., 2013) –4 –4 Hydrocortisone Pharmaceuticals, DPA-H2SO4 3.0×10 –0.1 2.2×10 0.9991 (Fu, et al., serum 2015) Uric acid Serum DPA-KOH 0.067–33.62 2.02×10–2 0.9993 (Yang, & Zhang, 2010) Isoxicam Pharmaceuticals, DPA-Flourescein- 3.0×10–4 –1.0 7.0×10–5 0.9991 (Zhao, & biological fluids CTAB Si, 2013) Ferulic acid Medicinal herbs DPA-N-Dots 0.3–10 8.0×10–2 NG (Fu, et al., 2016) Cortisol Human blood DPA-luminol 1.8×10–5– 7.25×10–6 0.9992 (Shi, et al., serum 2.72×10–3 2009) Amikacin sulfate Serum DPA-luminol 0.0399–3.91 1.58×10–2 0.994 (Yang, et al., 2010a) 10-hydroxycamptothecin Injections, urine, DPA-luminol 0.02–1.0 6.5×10–3 0.9978 (Sun, et al., serum 2011) –3 – H2O2 Rainwater, DPA-luminol 1.36×10 –0.136 3.751×10 0.9939 (Yang, & artificial lake 4 Zhang, water 2012) Cyromazine Fresh milk, milk DPA-luminol- 0.02–70 6.0×10–3 0.0997 (Wang, et powder CTAB al., 2015)

Diperiodatocuprate (DPC) −3 Ofloxacin Pharmaceutical DPC-Na2SO3– 0.01–2.0 8.7×10 0.9989 (Sun, et al., Levofloxacin preparations and H2SO4 9.2×10−3 0.9992 2011) human urine Ergometrine Serum sample and DPC-Ascorbic 4.0×10−3–0.4 1.1×10−3 NG (Hu, et al., Maleate pharmaceutical acid-KOH 2010) injections Cefazolin sodium Injectable powder DPC-luminol 0.02–2.0 4.58×10–3 0.9978 (Sun, et al., and urine 2013)

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Ractopamine Urine DPC-luminol 1.0×10–3–1.0 3.4×10-4 NG (Hu, & Li, 2013) Dihydralazine sulfate Serum DPC-luminol 7.0×10−3–0.86 2.1×10−3 0.9933 (Yang, et al., 2010b) –6 –6 H2O2 Exhaled Breath DPC-luminol 3.4×10 –0.034 1.39×10 0.9943 (Hu, et al., condensate 2008a) –6 –6 H2O2 Cigarette smoke DPC-luminol 3.4×10 –0.034 1.39×10 0.9943 (Hu, et al., condensate 2007) Lincomycin Serum DPC-luminol 0.01–5.0 3.5×10–3 NG (Hu, et al., 2011) Amikacin sulfate (AKS) Serum DPC-luminol 4.0×10–3–4.0 1.2×10–3 0.9974 (Hu, et al., 2013) Mitoxantrone (MTX) Pharmaceutical DPC-luminol 5.0×10–3–0.1 1.1×10–3 0.9991 (Yao, et al., preparations and 2014) serum samples –3 Cholesterol Serum DPC-luminol-H2O2 0.019–0.19 2.25×10 0.9972 (Hu, & Zhang 2008) –4 –7 Hydroxyl radicals Ultrasonic DPC-luminol-H2O2 1.7×10 –0.017 7×10 0.9957 (Hu, et al., aqueous solution 2008b)

Rh6G = rhodamine 6G, CTAB = cetyltrimethylammonium bromide, N-dots = nitrogen-rich quantum dots.

1.3 FLOW INJECTION ANALYSIS FIA technique was first demostrated in 1975 (Ruzicka, & Hansen, 1975) and soon became a useful technique in the field of analytical chemistry and chemical analysis and about more than 20,000 original research articles published in the scientific journals since its development. The history and review of these achievements is found in a large number of research articles, monographs and books published (Ruzicka, & Hansen, 1981; Ueno, & Kina, 1983; Karlberg, & Pacey, 1989; Burguera, 1989; Fan, 1993, 1995; Valcarcel, & Luque de Castro, 1995; Calatayud, 1996, Trojanowicz, 2000, 2008, Kolev, & Mckelvie, 2008, Biurrun, 2009; Zagatto, et al., 20011; Capillas, & Nollet, 2016). In FIA, a small known volume of liquid sample is injected into an unsegmebted carrier stream moving through narrow bore tube. The injected sample zone is gradually dispersed in the carrier stream by diffusion and convection and merges with other reagents at predetermined confluence points to produce detectable species that can be measured by a number of flow through detectors. The output peak- shaped signal of the detector can be used to quantitate the relative analyte concentration (Ruzicka, & Hansen, 1981). Three principles such as samples injection, reproducible timing and controlled dispersion in association with each other constitute the total principle of FIA. The

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objective of sample injection is to introduce sample zone’s reproducible volume into the unsegmented and continuously moving stream without disturbing system’s flow. Reproducible timing for sample injection and residence time in the detector plays a pivotal role for the reproducible peak to peak separation at baseline and peaks height and to avoid erroneous results. Time of 10 – 30 s is the most suitable, but to enhance the sensitivity, the residence time in detector is increased. For this reason, the precise combination of residence time and dispersion is crucial to closely suit a specific chemical reaction. FIA’s most important aspects also include controlled dispersion, which must suit for a particular FIA system. As the sample zone travels along the tube having narrow bore (1 mm id.), thus dilution or dispersion of the sample takes place. The design of the flow system depends on different variables including dispersion coefficient, residence time and dispersion factor. According to Ruzicka and Hansen, the coefficient of dispersion (D) is the ratio of the sample before and after the dispersion process, that is the ratio of the steady state signal (Co) and to the analytical peak height (Cmax.) i.e.

D = Co/Cmax

Where, Co and Cmax are the concentration of analyte at the peak maximum for the undiluted and diluted analyte respectively. The reasonable design of an FIA system depends on the degree of dilution of the original sample when it reaches to the detector and the time difference between the sample injection and the readout. Sample dilution, when it moves along the carrier, increases with an increase in dispersion. For the ease, the categorization of sample dispersion can be as: limited (D = 1 – 3), medium (D = 3 – 10) and large (D > 10) (Ruzicka, & Hansen, 1981). Among them, medium dispersion is the most useful from analytical point of view and makes up the most FIA applications.

1.3.1 Basic components of FIA The basic components of an FIA system include a peristaltic pump, an injection valve, flexible plastic tubes to connect the injection valve to the detector equipped with a flow through cell and a chart recorder. A complete transport system includes carrier and reagent streams, pump, sample injector, reaction/mixing coils and detector also called FI manifold as depicted in Figure 1.3.

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Figure 1.3 A complete manifold for FIA, RC1 and RC2 = Reaction coils.

The most commonly used pumps for the propulsion of carrier and reagent streams are the peristaltic pumps. Commercially, several types of these pumps are available such as Ismatec Reglo 100 (Glattbrugg, Switzerland), Minipuls 3 (Gilson, France) and many more. These pumps have the capacity to accommodate 2 – 8 streams (channels), can propel the solution from 0.0005 to 40 mL min–1 at a steady constant flow rate and the most common flow rate which is sufficient to meet the needs of FIA is from 0.5 to 3.0 mL min–1. The injection of a reproducible and measured volume of sample in continuous flow of carrier stream is performed with an injection valve. For this purpose, a six- port rotary injection valve is commonly employed. Sample loops of variable volumes, typically range from 10 to 300 µL, can be incorporated into sample valve and filled by samples with peristaltic pump or syringe. Figure 1.4 shows two positions of six port rotary injection valve, in position A, the sample is filled in sample loop, and in position B, the measured volume of the sample is injected into the sample carrier stream.

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Figure 1.4 Two positions of six port rotary injection valve, A) loading position of sample and B) carrying position of sample.

Flexible tygon or silicon tubes of uniform inner diameters make the manifold. The reaction or mixing coils consist on tubes of suitable lengths winded around methacrylic cylinders. Broad applicability of FIA for wet chemical reactions is reflected from its flexibility to be coupled with many types of flow through detector. Electrochemical and optical detectors are the commonly used detectors with FIA. The broad range of detectors used with FIA include UV-visible spectrophotometry (Saurina, & Cassou, 2001; Tzanavaras, & Themelis, 2007;), CL (Fletcher, et al., 2001; Chen, & Fang, 2007; Waseem, et al., 2013; Iranifam, 2013;), ECL (Yin, Dong, & Wang, 2004; Bard, 2004; Richterm, 2004), fluorimertry, atomic absorption spectroscopy, electrochemical detectors, diode array detector and others (Hansen, 2004; Medina, Martinez, Cordova, & Barrales, 2017). The chart recorder peak to peak measurement has been shown in Figure 1.5 and the peak hight is commonly measured for the evaluation of detector signal followed by peak area measurement (Hansen, 2004). For automated operation of pump, sample injection valve and detector, a computer may also be employed.

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Figure 1.5 (a) Simple FIA manifold (single channeled): P; peristaltic pump, S; sample injection valve, D; detector and W; waste, (b) Chart recorder peak showing detector output: S; recording starts at the injection time, H; hight of peak, W; width of peak, A; area of peak, t; residence time corresponds to the measurement of peak height and tb; peak baseline width (Ruzicka, Hansen, & Mosbaek, 1977).

1.3.2 FI-CL/spectrophotometric applications for the pesticides quantitative analysis in water samples A number of FI–CL (Table 1.5) and FI-UV-Visible spectrophotometric methods (Table 1.7) (Maya, Estela, & Cerda, 2011; Portela, Pereira, Matos, & Vaz, 2003; Danet, Bucur, Cheregi, Badea, & Serban, 2003; Cassella, Garrigues, Santelli, & Guardia, 2000; Rodriguez, Romero, & Tena, 1999; Ferre, Boque, Band, Larrechi, Rius, 1997; Shi, & Stein, 1996; Guardia, Khalaf, Carbonell, & Rubio, 1995; Agudo, Rios, & Valcarcel, 1993; Khalaf, Rubio, & Guardia, 1993a; Khalaf, Saneenon, & Guardia, 1993; Khalaf, Sancenon, & Guardia, 1993b; Khalaf, Sancenon, & Guardia, 1992; Sedeno, Nova, & Diez, 1988) have been reported for the quantitative analysis of pesticides in natural waters with analytical characteristics. In addition, pesticides analysis have been reported in comprehensive reviews and books by means of HPLC and gas liquid chromatrophy coupled with different

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detectors (mass spectrometry, tendum mass spectrometry, ultraviolet spectrophotometry, flurescence, electron-capture detector, nitrogen phosphorus detector, flame photometry and atomic emission spectroscopy) (Grimalt, & Dehouck, 2016; Masia, Varela, Gonzalez, & Pico, 2016; LeDoux, 2011; Tuzimski, & Sherma, 2015; Tadeo, 2008; Barbosa, et al., 2017; Liu, et al., 2017; Lei, et al., 2017; Ji, et al., 2017), capillary electrophoresis (CE) and immunoassays (IAs) (Tadeo, 2008; Chui et al., 2017; Wuethrich et al., 2015; Hua et al., 2017; Liu et al., 2016) techniques in food and environmental samples.

Table 1.7 FI-UV-Visible spectrophotometric methods for the quantitative analysis of pesticides in natural water samples.

Linear range LOD Sample Analyte Reaction and wavelength R2 Ref. (ppm) (ppm) h–1

2+ Paraquat Ascorbic acid–KIO3–Pq in 0.005–0.250 0.0007 34 0.9995 (Maya, et basic medium, λmax. 610 and 720 al., 2011) nm. Carbaryl Alkaline hydrolysis of pesticide 2.01–301.8 0.2 60 0.9992 (Portela, et. −phenylhydrazine al., 2003) hydrochloride, λmax. 495 nm. Paraoxon Acetylthiocholine–AChE– 0.002–0.08 0.0013 3 0.9706 (Danet, et thiocholine–DTNB, λmax. 405 al., 2003) nm, (inhibition)

Carbaryl UV analysis, λmax. 280 nm. 0.1–1.0 12 10 0.999 (Cassella, et al., 2000) Bendiocarb Alkaline hydrolysis of 2.5–160 2.5 60 >0.99 (Rodrigue, Benfuracarb pesticides–TMA–SDS, λmax. 3.1–266 3.1 >0.99 et al., 1999) Carbaryl 390, 380, 520, 390, 390, 380 0.30–122 0.30 >0.99 Carbofuran and 380 nm respectively 1.6–162 1.6 >0.99 Methiocarb Promecarb 2.5–57.6 2.5 >0.99 Propoxur 1.4–73.2 1.4 >0.99 0.40–125 0.40 >0.99 Carbaryl Alkaline hydrolysis of 2–8 0.08 NG 0.9847 (Ferre, et Carbofurane pesticides–sulfanilic acid– 2–8 1.04 0.9849 al., 1997) Propoxur nitrous acid, λmax. 340-650 nm 2–8 0.75 0.9921 Isoprocarb (multivariate calibration) 2–8 0.71 0.8600 Paraoxon Acetylthiocholine iodide–with 5×10–5–5×10–4 NG 2 0.998 (Shi, & AChE–DTNB, λmax. 412 nm Stein, (inhibition) 1996) Propoxur Alkaline hydrolysis of 5–100 0.12 80 0.9999 (Guardia, et pesticide–p–aminophenol– al., 1995) KIO4, λmax. 600 nm Paraquat Reduction of pesticide with 4×10–4–0.0055 1.1×10–4 NG NG (Agudo, et dithionite ion, λmax. 605 nm al., 1993) Carbaryl Alkaline hydrolysis of 1.2–6 0.0265 110 0.9999 (Khalaf, et pesticide–p–aminophenol– al., 1993c) KIO4, λmax. 596 nm Ethiofencarb Alkaline hydrolysis of 2–50 0.033 NG 0.9999 (Khalaf, et pesticide–p–aminophenol– al., 1993a) KIO4, λmax. 638 nm Alkaline hydrolysis of 0.05–17 0.05 120 0.9998 (Khalaf, et pesticide–p–aminophenol– al., 1993b) KIO4, λmax. 576 nm

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Propoxur Alkaline hydrolysis of NG 0.1 80 0.9998 (Khalaf, et pesticide–p–aminophenol– al., 1992) KIO4, λmax. 600 nm Carbaryl Alkaline hydrolysis of 10.0–40.0 0.08 27 0.9993 (Sedeno, et pesticide–sulfanilic acid, λmax. 1.0–10.0 0.9996 al., 1988) 515 nm 0.1–1.0 0.9992

DTNB = 5,5/-dithio-bis(2-nitrobenzoic acid, TMA = 2,4,6-trimethylaniline, SDS = sodium dodecyl sulfate, NG = not given

1.4 RESEARCH OBJECTIVES

1.4.1 Research objectives i. To develop sensitive and selective flow techniques based on chemiluminescence and spectrophotometric detectors for pesticides analysis. ii. To optimized various chemical and physical variables concerned to the FI-chemiluminescence and spectrophotometric detectors for the analysis of pesticides in natural water samples. iii. To extract pesticides from natural waters using solid and liquid phase extraction techniques. iv. To check / examine the accuracy of the established methods with standard / reported methods. v. To publish articles in national and international refereed analytical / environmental chemistry journals having impact factors.

1.4.2 Problem statement The un-controlled use and accidental release of pesticides pollute different segments of the environment such as air, soil and especially surface and ground water resources due to its persistent nature. This could render a possible threat towards human health. Thus, for accurate status assessment of the pesticides pollution in different segments of the environment, the demand for reliable, rapid and economical qualitative and quantitative determinations of pesticides is critical. Millions of pesticides poisoning cases are reported annually worldwide, which include both acute (abdominal pain, diarrhea, headaches, nausea, vomiting etc) and chronic (cancer, depression, diabetes, genetic disorders, neurological deficits, skin diseases and even

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death) toxic effects. The risk of toxicity to humans and ecotoxicology of pesticides even at trace level in surface, underground and drinking waters has become a problem worldwide. Growing concern about the water quality has led EU to set a limit for the maximum allowable concentration of pesticides in drinking water. According to this water quality, the concentration of total pesticides in drinking water must not exceed than 0.5 ppb and of individual pesticide must not exceed than 0.1 ppb. However, when chronic and acute toxic effects and as well as persistency and bioaccumulation of pesticides are implied, the limits described so for must be lower than those for drinking water quality. To analyze pesticides in water samples at trace level, the requirement of sensitive and efficient techniques is inevitable. Various analytical approaches have been established for the quantitative estimation of pesticides in water samples including HPLC, GC, voltammetry, IAs, CE and many more. These methods are accurate and sensitive, but some of these instruments are not widely used in general laboratories due to large consumption of reagents, lengthy procedures, low sample throughputs and availability of expertise. Therefore, techniques having attributes of simplicity, easy to operate, sensitivity, fast, requirement of small sample size and applicability to a wide concentration range of pesticides, need to be developed. FIA coupled with CL and UV-Visible spectrophotometry was selected to establish fast, sensitive, and simple procedures for pesticides quantitative analysis in water sample of natural origin.

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CHAPTER–II CL INSTRUMENTATION AND DETERMINATION OF MANGANESE(II)

2 INTRODUCTION Among UV-Visible spectroscopic analytical techniques, CL is the most sensitive and selective in comparison to UV-Visible and fluorescence spectroscopy. This is the reason which recommends and directs its applications in most of the analytical laboratories. The exploitation and utilization of analytical potentials of CL systems need the quantification of low light level with efficiency and accuracy. Several devices can be employed for the monitoring of CL such as imaging devices, photographic films, photocells, multichannel detectors and photomultiplier tubes (Nabi, 1991; Nabi, 1992).

2.1 PHOTOMULTIPLIER TUBES (PMTs) The photomultiplier tube acts as a detector of radiant energy of electromagnetic spectrum over the regions of ultraviolet, visible and infra red wavelengths. It is a very sensitive and versatile etector over these ranges. The most suitable and frequently used detection devices with high gains for low light level are photomultiplier tubes (PMTs) (Ingle, & Crouch, 1988; Van, 1985). To keep the consistent total gain, the PMTs must be operated with the consistent and stable power supplies. Basically, there are two varieties of PMT, for example, side-on and end-on PMTs, bearing its own advantages. The cheapness, economical prices and need of less space make the former one superior, but the later one is better than the side-on versions and models in terms of large photocathode area, uniformity of responses and light collection (Drozdowicz, 1999). The cross-sectional view of a PMT shown in Figure 2.1, is an evacuated and sealed transparent glass or quartz made envelope. Inside the envelop various components of PMT, such as, a photo emissive cathode, many additional electrodes termed as dynodes and an anode, are arranged in a regular manner. The photons of the incoming light strike the photo emissive cathode, which is a metal coated on with an alkali metal (Na, K, Cs or Sb) or mixture of elements (Ga/As), and eject electrons. These electrons are then attracted by a dynode, kept at positive voltage with respect to the cathode and ejection of more electrons takes place. The process is repeated at the second and other dynodes and multiplication of electron number takes place. In this way, a cascade of electrons is received in the end by the anode. The number of electrons reached to the collector is in direct proportion with the intensity of the

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incident radiation first falling on the surface of photo emissive cathode of the PMT (Robinson, Frame, & Frame, 2005; Skoog, Holler, & Crouch, 2006). An amplifier is employed in current-to-voltage configuration for the conversion of photocurrent into voltage and then further amplified. The readout signal must be in digital domain and for this purpose signal from analog transducers is converted into digital domain. Signal in digital domain can be treated very accurately with digital methods or with software in a microcomputer (Campana, & Baeyens, 2001).

Figure 2.1 PMT cross sectional view.

2.1.1 Characteristics of PMT Commercially, a variety of PMT tubes are available and its type can be selected for use based on the requirement. The ultimate sensitivity which is necessary to be achieved, relies on the characterisitics of PMT. Practically, for the normal achievement of light over the entire front of the tube, the end window configuration of the phototube is efficient. The quantum efficiency of the photocathode (number of emitted electrons per incident photon) depends on the range of wavelength of light. Thus, the selection of the type of photocathodes is dependent on the spectral range of interest. In addition, other factors which must be considered for the selection of PMT are: window materials of the photocathode and number of dynodes; as the PMT with larger number of dynodes gives greater number of secondary electrons at a given voltage. The selection of power supply depends on its high accuracy and excellent resolutional adjustment.

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The features and characteristics for PMT (Type 9798B), which was used as a detector for CL detection during experiments has been shown in Table 2.1. Table 2.1 PMT characteristics (Type 9798B, Electron Tubes, Ruislip, UK).

Type of phototube: End window Spectral range: 280-900 nm Active diameter: 29 mm Dynodes: 11 Photocathode: KCs Dark current at 20 oC: 2.0 nA

2.2 BATCH LUMINOMETERS Various lumniometers / photometers are commercially available which are used for the measurement of CL and BL based on PMT detector. These devices have been thoroughly reviewed (Stanley, 1982). Luminometer’s essential components include a system for the control of temperature, detector usually PMT, chamber of detector, device for reagent mixing and a cuvette (Figure 2.2). The basic principle of batch luminometer is based on injecting reagents into a sample containing cuvette / reaction vessel positioned in front of the PMT. The amount of emitted light is in direct proportion with the concentration of analyte.

Figure 2.2 Basic design of conventional luminometer.

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There are many applications of batch luminometers and they have been commonly employed for the analysis of metabolites and (Ovesen, et al., 2002; Andreotti, & Berthold, 1999). However, these devices are associated with several disadvantages for analytical purposes such as high relative standard deviation (RSD) ( 5%), improper mixing of reagent and sample, limited dynamic range, low sensitivity and slow photon counting rate. There are a number of commercially available luminometers for analytical purposes such as i) LKB 1250 and 1251 lumniometers from LKB, Wallac OY, Turku 10, Finland, ii) Packard 6100 Pico-Lite luminoleter from Packard Instrument Company, Illinois, USA, iii) AutoLumat Multi-Tube LB 953 Luminometer and Biolumate LB 9505 from Berthold Wirdbad, Germany, iv) Luminescence analyzer EnSpectr L405 from Enhanced Spectrometry Inc. CA 94040, USA and others (Nabi, 1991).

2.3 CONTINUOUS FLOW METHODS This technique is called continuous flow, because the analyte and other reagents such as CL reagent are propelled uninterruptedly in separate streams or channels before they mix or merge at a confluence point to make a mixed or a merged stream. After the confluence point the observation of the emitted CL is made and the data is collected for further processing. Both the rate of CL reaction and intensity of the CL emission be contingent on the flow rate of the streams and volume of the injected analyte. Flow rates of carrier and reagent streams must be set according to the reaction rate, slow reaction rate needs a slow continuous flow of streams and vice versa. The components of the instrumentation for the CL quantitative analysis of an analyte in a continuous flow include a flow through cell, photo-detector such as PMT, mixing chamber for reagents and a data acquisition and processing device. The analytical community have also used commercially available FIA instruments including i) FIAlab2005 analyser from FIAlab Inc., USA, ii) FIA2000 multichannel analyzer from Burkard, UK iii) QuikChem 8500 series FI analyzer from Lachat, USA and iv) FIA system from MLE company, Dresden, Germany (Capillas, & Nollet, 2016).

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2.3.1 FI-CL instrument as a model A purpose built FI manifold with CL detector has been shown in Figure 2.3. This basic FIA-CL system arrangement illustrates the injection of a sample into a carrier stream at a suitable pH, continuous propulsion and circulation of reagents in separate streams, the combination and mixing of separate stream at a confluence point for a CL reaction and its detection. After merging of reagent solutions with the analyte at a confluence point, mixing occurs in a glass made flow cell and the CL reaction takes place directly in front of the PMT window. The emitted radiations in CL reaction are converted into photocurrent by PMT attached with a high voltage power supply, amplified, digitized and recorded with suitable and appropriate data acquisition electronic device such as a chart recorder. This arrangement works well for CL reaction with a fast reaction kinetics.

Figure 2.3 FI–CL manifold.

2.3.1.1 Flow cells The flow cell is the most vital and significant component of FI manifold for CL measurement. The configuration of flow cell should ensure the generation of maximum light during the mixing of chemical reagents in front of the PMT. Different varieties of flow cells can be used in FI-CL manifold, but the most important and widely used flow cell is termed as flat spiral flow cell made of glass tube. This spiral flow cell can be constructed easily due to its simple design and was first introduced in 1980 (Burguera, Townshend, & Greenfield, 1980). It can be incorporated and fixed very close to the light sensitive area i.e. photo emissive

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cathode of the PMT to ensure maximum light acquisition by the cathode. Different FI- CL flat spiral flow glass cells are shown in Figure 2.4.

Figure 2.4 Pictures for glass spiral flow cells with variable capacity used in FIA with CL detection.

Figure 2.5 shows the aluminum housing for PMT and glass flat spiral flow cell. During the proposed studies, the spiral flow cell used with an internal diameter and volume of 1.5 mm and 300 µL, respectively, and located in a straight line in front of an end window PMT (Electron Tubes, UK, Type = QL30F/RFI,). The Y-piece for merging the reagent streams to become a single stream, glass coil for CL reaction and PMT as a photodetector were surrounded in a light tight aluminum housing. The distance between the confluence point i.e. Y piece and spiral glass flow cell was fixed by 1.5 cm.

Figure 2.5 Flat glass spiral flow cell with PMT casing.

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For incorporation and positioning of glass coil in front of the PMT in the manifold, two holes were drilled in aluminum housing and the coil was threaded in the housing at a distance of 2 mm from photo emissive cathodic surface. The Y-piece was passed through and fitted in the central hole in the housing and connected with the inlet of glass spiral flow cell. For the discharge of solution mixture as a waste, the outlet of flow cell was connected with Tygon tubing and passed via the second hole in the housing. For the prevention of any loss of CL radiation, the flow cell and Y-piece were completely enclosed with an aluminum plate. This plate was so designed that it could easily flanged the housing of PMT and screwed on. The basic components of an FI-CL system are shown in Figure 2.6.

Figure 2.6 Basic components of FI-CL system.

2.4 TESTING OF CL DETECTOR

FI-CL QUANTITATIVE ANALYSIS OF Mn2+ USING LUMINOL–DPA CL DETECTION

2.4.1 EXPERIMENTAL

2.4.1.1 Reagents

The reagents and chemicals used during the proposed study were of analytical grade and deionized water (Elga, Purelab Option, High Wycombe, Bucks, UK) was

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employed for the arrangement of solutions used in these experiments and also used for the preparation of solutions in upcoming experiments described in other chapters of this dissertation. Plastic and glassware, which were used for the stock and running standard solutions arrangement or used elsewhere during the experimental studies, were kept for a week in 20% hydrochloric acid (HCl) for precleaning, thoroughly rinsed and cleaned with deionized water and stored and stockpiled in zip locked plastic bags. Luminol (3-aminophthalhydrazide, C8H7N3O2, RMM. 177.16) from

Fluka, Buchs, Switzerland, sodium periodate (NaIO4, RMM 213.89) from Fisher

Scientific UK, silver nitrate (AgNO3, RMM 169.87) from Acros Organic, New Jersey,

USA, Geel Belgium, potassium peroxidisulphate (K2S2O8, RMM 270.33), potassium hydroxide (KOH, RMM 56.11) and mangan(II)-sulfate-1-hydrat (MnSO4.H2O) from Merck, Damstadt, Germany, were obtained. The stock solution of luminol (1 mM) was prepared when 17.7 mg of its compound was dissolved in 100 mM of KOH followed by sonicating for 40 min. This solution was stable almost for at least one month storing at 4 oC when not in use. Luminol working standard solutions were arranged by diluting its stock solution aliquots with aqueous solution of KOH 8 mM. The stock solution of KOH (1 M) was arranged, when 5.611 g of KOH was dissolved in 100 mL of deionized water and for the preparation of its running standards, different required aliquots of the stock solution were diluted with deionized water. The Mn2+ stock solution (100 ppm) was prepared when 153.6 mg of

MnSO4.H2O was dissolved in 50 mL deionized water. Further serial dilutions of the stock solution were performed with deionized water for the preparation of series of working standard solutions. A reported scheme was followed accordingly for the synthesis of diperiodatoargentate(III) (DPA) complex (Balikungeri, Pelletier, & Monnier, 1977), the mixture of the chemical reagents containing 136 mg of AgNO3, 342 mg of NaIO4,

300 mg of K2S2O8, and 800 mg of KOH was dissolved 100 mL of UHP deionized water in a round bottom flask having capacity of 0.5 L and heated to the boiling while stirring. The mixture was kept boiling for about 20 min and a froth having color of orangish-yellow was achieved. This mixture was heated while stirring for another 20 minutes. Before filtration through Gooch crucible, it was brought to room temperature

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by means of cooling at room temperature. For the removal of potassium sulfate, the mixture was further cooled at an ice bath followed by filtration while cold. The filtrate was added 40 mL sodium nitrate (50% w/v) for the separation of DPA crystals after keeping them at room temperature for some time until it attained the room temperature. When the crystallization process was completed, the supernatant turned colorless. The crystals were washed with UHP water several times after filtration until the complex started dissolving. After drying, the synthesized complex was stored in brown colored glass bottles until to be used for further research activities. The working standards of the complex were arranged when the required quantity of DPA complex was dissolved in 8 mM of KOH and the concentration of DPA solutions were determined from the noted absorbance at 362 nm (є = 1.26 × 104 mol–1 L cm–1).

2.4.1.2 Apparatus and procedure The proposed FI-CL manifold for Mn2+ quantitative analysis is illustrated in Figure 2.7. The sample carrier (deionized water) and reagents were propelled by a four-channel peristaltic pump (Reglo 100, Glattbrugg-Zurich, Switzerland) at a flow rate of 1.5 mL min–1. Tygon tubes (Ismatec Switzerland) of 1.02 mm i.d. were used for the construction of whole manifold tubing. The DPA stream (10 μM in 8 mM KOH) merged with the luminol stream (0.6 μM in 8 mM KOH) at a confluence point. This merged stream was then mixed at a T piece with the stream of sample carrier. The carrier stream was injected with standards/samples of Mn2+ (60 µL) through a six-way rotary injection valve (Rheodyne 5020, Anachem, Luton, UK). The CL reaction took place in a flat glass spiral flow cell (1.5 mm i.d., 1.8 cm diameter) fixed in front of an end window PMT (9789B, Electron tube Ltd, Ruisilip, UK) supplied by 1000 V with a 2-kV power supply (Electron Tubes, PM20SN, UK). Light tight aluminum housing was used to enclose completely the T-piece, glass spiral flow cell and the PMT. The output of the PMT was recorded on a chart recorder (BD 40, Kipp & Zonen, Holland). The components of the manifold were connected with each other using PTFE tubing flow tubes (0.8 mm i.d., Fisher Scientific, Loughborough, UK). Almost the same instrumental components such as PTFE and Tygon tubings, peristaltic pump, six-way rotary injection valve, glass spiral flow cell, PMT, power supply and chart recorder have been employed for experimental studies throughout this dissertation.

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Figure 2.7 Proposed FI-CL manifold for the quantitative analysis of Mn2+. R1 = deionized water, R2 = DPA 10 µM in KOH (8 mM) and R3 = luminol 0.6 µM in KOH (8 mM).

2.4.1.3 Characterization of DPA A reported scheme was followed for the synthesis of DPA complex accordingly (Balikungeri, Pelletier, & Monnier, 1977) in the laboratory. The synthesized of DPA complex was characterized by using UV-Vis spectrophotometer (Model 6505, Jenway Ltd. Felsted, Dunmow, Essex, UK). The absorption spectrum for a fresh solution of the complex was scanned from 200 – 500 nm and two absorption maxima were obtained in the UV region at 253 and 362 nm as reported in literature (Balikungeri, et al., 1977; Asghar, Yaqoob, Haque, & Nabi, 2013).

2.5.1 RESULTS AND DISCUSSION 2.5.1.1 FI manifold’s chemical and physical parameters optimization For the selection of optimum experimental parameters for the quantitative analysis of Mn2+, various chemical and physical variables were investigated under univariate approach. Optimization of the chemical parameters includes checking the effect of concentrations of DPA, luminol and KOH for both solutions. The physical parameters optimized were flow rate and sample injection volume. A 0.01 ppm of Mn2+ and PMT voltage of 1000 V were used during all these optimization studies and other parameters used have been mentioned elsewhere.

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Strong oxidant in the form of DPA oxidizes luminol into 3-aminophthalate in excited state, which can be catalyzed by Mn2+. It has been demonstrated that the luminol CL reaction with DPA needs low luminol concentration with better results in 2+ comparison to the CL reaction of luminol with H2O2 catalyzed by metals such as Ni , Cu2+ and Cr3+. In addition to this, the DPA also undergoes strong redox reaction with luminol (Yang et al., 2010a). Thus, the concentration of DPA has great influence on the oxidation of luminol. Therefore, the DPA concentration effect on the CL signal intensity was explored from 1 to 60 µM. The CL signal intensity for Mn2+ was increased upto 10 µM (Figure 2.8a) and selected as an optimum for subsequent studies. By increasing further the DPA concentration, CL signals with quenched intensities were achieved, possibly because of DPA self-absorption at higher concentrations.

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Figure 2.8 Optimization of (a) DPA, (b) KOH for DPA, (c) luminol and (d) KOH for luminol concentrations on the quantitative analysis of Mn2+: Experimental conditions: sample loop volume, 60 µL; PMT voltage, 1000 V; flow rate, 1.3 mL min–1; Mn2+, 0.01 ppm. Each result was the mean of three measurements.

As it is known, the DPA complex is insoluble in concentrated alkaline solution (Balikungeri, et al., 1977) and low concentration of DPA is not stable in distilled water (Asghar, et al., 2013). Therefore, KOH concentration for DPA stream was optimized from 0.8 to 10 mM. An increase in CL signal intensity was detected with increase in KOH concentration and the strongest CL signals with the highest signal to noise ratios were obtained at 8 mM of KOH (Figure 2.8b), selected as an optimum for DPA stream and was used for further experiments. Further increase in the KOH concentration made the proposed CL system noisy by increasing the background CL intensity. The intensity of CL signals showed high dependence on the luminol concentration and the proposed FI-CL system requires lower luminol concentration as compared to other luminol CL systems using other oxidants. For this purpose, luminol concentration effect was checked from 0.1 to 10 µM. The intensities of CL signals

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were enhanced by increasing in the luminol concentration and maximum CL intensities with the highest signal to noise ratios were observed at 0.6 µM (Figure 2.8c) and was designated as an optimum for succeeding experimental work. Beyond the optimum concentration, the CL signal intensity and S/N ratio dropped to lower value. Luminol exhibits CL reactions in alkaline solution with different oxidants. The CL reaction of luminol depends on concentration of KOH. Therefore, the effect of the concentration of KOH in the luminol solution was studied over the range of 0.8 – 10 mM. The highest CL signals intensity with the highest S/N for Mn2+ was observed at 8 mM of KOH (Figure 2.8d), which was designated as an optimum for succeeding experimental work. More increase in KOH concentration caused in the increase of background CL intensity rather than increase in the CL signal intensity, and therefore signal to noise ratio was decreased. The kinetics of luminol-DPA CL reaction has been reported as very fast, the CL signal peak reaches to the peak maximum within 3 second and comes to the base- line within 10 s (Yang et al., 2010). As the reaction is very fast, therefore effect of flow rate is inevitable to be studied to increase the injections rate and sensitivity and to decrease the consumption of reagents. Thus, the flow rate effect was checked over the range of 0.5 – 3 mL min–1 and the highest CL signal intensity was detected at 1.5 mL min–1 as shown in Table 2.2 and was selected as an optimum. Further increase in flow rate diminished the CL signal intensity. The volume effect of sample injection loop was studied from 60 to 300 µL and increased CL signals detected with the increase in sample injection loop volume as shown in Table 2.2, but 60 µL was opted as an optimum to reduce the consumption of standards and sample solutions, to speed up the CL response and to increase the application of the proposed FI-CL approach to a wider linear range of Mn2+ concentration.

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Table 2.2 Effect of physical parameters on the intensity of CL signals of Mn2+ 0.01 ppm under optimized chemical parameters. For the optimization of each physical parameter all other parameters with their optimum conditions were used i.e., sample injection volume of 60 µL, flow rate of 2.0 mL min–1 and PMT voltage of 1000 V.

Flow rate (mL min–1) 0.5 1.0 1.5 2.0 2.5 3.0

CL signals (mV)* 70 870 1320 1165 1100 1040

Sample volume (μL) 60 120 180 240 300 360

CL signals (mV)* 1324 1533 1747 1803 1859 1894

*Mean of four injections

2.5.1.2 Analytical figures of merit The Mn2+ calibration curve was got under optimum conditions and was linear over the range of 0.1 – 100 ppb (R2 = 0.9998, n = 7) with regression equation of y = 132.6x + 7.1734, (y = CL intensity (mV), x = concentration (ppb) (Figure 2.9). The concentration of the analyte giving the CL signal intensity equal to three times of the peak-to-peak base line noise (S/N = 3) i.e. the LOD was 0.05 ppb with injection throughput of 125 h–1. The RSD was 2.5% for 10 replicates analyses of 20 ng mL–1 of Mn2+.

Figure 2.9 Calibration curve for Mn(II) under optimum conditions.

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2.5.1.3 Possible CL reaction mechanism DPA is a stable anionic complex of silver(III) with a chemical formula of [Ag 2− (H2IO6)(OH)2] , reacts with luminol in alkaline aqueous solution and results CL emission (Yang, et al., 2010; Shi, et al., 2009; Ma, et al., 2012). The CL emission intensity of this reaction is greatly enhanced by the catalytic activity of Mn2+ on luminol oxidation in DPA presence. The commonly oxidized product of luminol is the excited 3-amiophthalate, which on de-excitation usually emits the light centered at 425 nm (Shi, et al., 2009). The most possible CL reaction mechanism has been shown in Figure 2.10.

Figure 2.10 The possible luminol-DPA CL reaction mechanism.

2.6.1 CONCLUSIONS The developed FI-CL method is very simple, sensitive, highly precise and accurate for Mn2+ quantitative analysis, based on its catalytic activity on the alkaline DPA and luminol CL reaction. The advantage of the method is the economy of the consumption of the luminol solution. The sensitivity and rapidity of the method can

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be estimated easily from the limit of detection (0.05 ppb) and high injection throughput (120 h–1), respectively. The method could easily be employed for the Mn2+ quantitative analysis and its fungicides especially in natural water samples after sample preparation process because those pesticides which contain Mn in their chemical structure can easily liberate Mn(II) in strong alkaline solutions. Thus, the FI- CL instrumentation could be used for the development of novel and sensitive continuous flow-based CL methods for the quantitative analysis of pesticides in natural waters and other samples.

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CHAPTER–III DETERMINATION OF MANGANESE AND MANGANESE CONTAINING FUNGICIDES WITH LUCIGENIN-TWEEN-20 ENHANCED CHEMILUMINESCENCE DETECTION

3 INTRODUCTION Manganese (Mn) is present in the crust of earth upto about 0.1% and from abundance point of view in the earth crust, it is the twelfth most abundant element (Wedepohl, 1995). The daily nutritional requirement of Mn is approximately fifty micrograms per kilogram of body weight and therefore, is included in trace essential element (Metrohm, 2005). Various biological enzymes use Mn for different biological processes, for example, phytoplankton use Mn enzyme in photosystem II (PSII) for splitting of water to provide electrons for PSII’s different reactions (Sunda, Huntsman, & Harvey, 1983). Superoxide dismutase enzyme activity, present in marine diatoms, is highly dependent on Mn (Peers, & Price, 2004). This element exists in dissolved form in lower oxidation state (Mn2+), but when oxidized to higher oxidation state (Mn(IV)), turns into insoluble form in the form of MnO2. This oxide of Mn acts as a scavenger for other elements exists in trace amount. In large concentrations, this element has shown toxic effects both for plants and other organisms, for example, chronic poisonings to the central nervous system of human have been reported (United Nation Environment Program, 1991). Mn concentration in natural water systems is usually very low and its normal concentration generally falls in 100 – 1000 ppb range (Van Staden, & Kluever, 1997). Under slight reducing environment, the particulated form of Mn(IV) is diffused from sediments into the column of water. This process usually takes place in the anoxic sediments near a shore or in a layer having less dissolved oxygen. Various sources such as hydrothermal inputs, riverine, sediments release, coastal and Aeolian dust are responsible for the introduction of Mn into the oceans. Mn is a fantastic tracer of a wide assortment of marine bio-geochemical process, for example, an aeoline inputs (Shiller, 1997) and Fe contribution from mainland edges and aqueous vents (Klunder, Laan, Middag, Baar, & Bakker, 2012; Middag, De Baar, Laan, Cai, & Van Ooijen, 2011). Various batch and flow based quantitative measurement procedures and methods, using different techniques, for the evaluation of Mn status in diverse samples have been establish. These samples matrices include naturally occurring or certified materials, pharmaceuticals, food, plants, fertilizers, sediments, soil, waste and natural water samples. The analytical techniques which have been employed for the purpose include colorimetry / spectrophotometry (Basargin, Oskotskaya,

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Gribanov, & Kuznetsov, 2012; Micic, Simonovic, & Petkovic, 2006; Dogutan, Filik, & Apak, 2003; Islas, Resing, & Bruland, 2006; Su, Li, Ma, & Tao, 2004), electrothermal and flame AAS (Knap, Kilian, & Pyrzynska, 2007; Manzoori, Amjadi, & Abulhassani, 2009), ICP-MS (Amais, Donati, & Nobrega, 2011), potentiometry (Ohura, Ishibashi, Imato, & Yamasaki, 2003), and cathodic stripping voltammetry (Roitz, & Bruland, 1997). These techniques frequently experience the ill effects of an assortment of drawbacks: they could be complex, time consuming procedures or tedious, absence of enough affectability and sensitivity or require costly instruments. Light emission in VU, visible or IR region during a chemical reaction is known as CL (Campana, & Baeyens, 2001; Barnett, & Francis, 2005) and its attractive and advantageous features which make it as a sensitive technique for trace analyses of different elements and other essential compounds include low LOD, high sensitivity, applicability to a wide linear range of analyte concentration, simple and low costing instrumentation and high sample throughput (Wabaidur, Naushad, &

Alothman, 2012). Oxygen or H2O2 reacts with lucigenin in the presence of catalysts, such as a number of metal cations, and produces electronically excited intermediate product namely N-methylacridone, which on de-excitation, emits CL radiations centered at 440 nm. The CL reactions of lucigenin can also be catalyzed by those metals, which do not catalyze the luminol CL reactions. While, both luminol and lucigenin CL reactions can be employed for the quantitative estimation of reactive oxygen species (Hyrsl, Lojek, Ciz, & Kubala, 2004; Rost, Karge, & Klinger, 1998). FI coupled with fluorescence and spectrophotometric detection systems have been thoroughly reviewed by Worsfold and co-authors (2013) for the quantitative analysis of nutrients (micro and macro) in natural water samples. In this review, various attributes such as selectivity, high sample throughput, sensitivity and suitability for depth profiles and underway analysis of flow techniques coupled with spectrophotometric and luminometric detection have been emphasized. The 7,7,8,8- tetracyanoquinodimethane (TCNQ) oxidation has been catalyzed by Mn2+ in alkaline medium and thus a flow-based CL method was established for its quantitative analysis. A mini column of chelating resin namely 8-hydroxyquinoline was incorporated in the manifold and a LOD of 0.1 nM has been achieved (Chapin, Johnson, & Coale, 1991). Doi, Obata and Maruo (2004) developed a sensitive method for the quantitative estimation of Mn at picomolar level in seawater samples using its

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catalytic effect on luminol and H2O2 CL reaction. In this method, ammonium acetate buffer has been employed to buffer seawater sample to pH of 8.5 and subsequently pre-concentrated onto an 8-HQ chelating resin. The elution of Mn from the column has been performed with dilute HCl solution and passed through a reaction coil (3 m) o after mixing at 25 C with the solution of NH4OH and luminol. Recently, the same procedure has been employed by Middag, et al., 2011, in flow injection mode with only modification of changing ammonium acetate buffer with ammonium borate for buffering the sample in-line. The method’s LOD and RSD (n = 22) were obtained as less than 0.02 nM and 2.19% respectively. Mn concentration in SAFe deep samples and surface were found as 0.31 ± 0.01 nM and 0.73 ± 0.01 nM, respectively, which were in the census range of 0.74 ± 0.06 – 0.35 ± 0.06 nM. Mn quantification method in portable water has been described (Bowie, Fielden, Lowe, & Snook, 1995) and in this method no pre-concentration has been performed by using an in-line incorporated mini column in the manifold because the concentration of Mn was higher in the sample matrix than the method’s sensitivity. Further enhancement and sensitization of this CL reaction mechanism has been brought by using bilayer vesicles of didodecyldimethylammoniumbromide (DDAB) and eosin Y, respectively. Okamura et al. (1998) has described the establishment of flow based CL approach for Mn(II) quantitative analysis in sea water. The oxidizing power of H2O2 on luminol oxidation has been catalyzed by Mn2+ and thus a CL enhancement method was devised for the analysis of Mn2+ and method’s upper and lower LODs were obtained as 4 µM and 0.029 nM, respectively. The RSD for 10 nM of Mn2+ was achieved as 0.58%. Other specifications of the method included the incorporation of 8-HQ and MAF mini- column in the manifold for the removal of other metals interferent effect and using a long reaction coil of 6.1 m for obtaining a strong CL emission at flow cell. Maneb is an ethylenebisdithiocarbamate fungicide and this dithiocarbamate fungicides is commonly used for the protection of field crops, vegetables, orchards, greenhouses, variety of seeds, forestry and fruits from a wide spectrum of fungal diseases (Risks of mancozeb, & maneb, 2007). This fungicide poses relatively low toxic effects on human beings but its degraded product ethylene thiourea (EU) has been ascribed with strong toxic effects (Dearfield, 1994) such as teratogenicity and carcinogenicity (Ulland, Weisburger, Weisburger, Rice, & Cypher, 1972). This

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fungicide is also responsible for the induction of Parkinson like disease known as permanent extrapyramidal syndromes (Meco, Bonifati, Vanacore, & Fabrizio, 1994). Mancozeb is a broad-spectrum fungicide and is commonly used for the protection of field crops, nut, vegetables and many fruits from a large number of fungal diseases. These diseases include rust on roses, scab on pears and apples, leaf spot and potato blight. This fungicide is also commonly employed for the seed treatment of cereal grains, peanuts, cotton, corn, tomatoes, potaotoes, safflower, flax and sorghum (Extension Toxicology Network, 1996). Mancozeb has been proved to be a non-toxic fungicide to mammals when given orally. The oral LD50 of it has been reported and calculated when given to rat over the range of 5.0 – 11.2 g kg–1 of rat body weight or it may be greater than this (Edwards, Ferry, & Temple, 1991) and is one of the most widely used pesticides worldwide (Environmental Protection Agency, 2002). The maneb and mancozeb chemical structures have been shown in Figure 3.1.

S H * N S * S N Mn H S x

Maneb (C4H6MnN2S4)x, Mw. 265.3

S H * N S Zny S N Mn H S x

Mancozeb (C4H6MnN2S4)x (Zn)y, Mw. (265.3)x + (65.4)y

Figure 3.1 Mancozeb and Maneb molecular structures

In this chapter, a FI-CL method development for Mn2+ and its fungicides (maneb and mancozeb) quantitative analysis has been detailed based on their enhancement effect on lucigenin CL reaction in micellar medium. The LODs obtained

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were as 0.1 ppm for Mn2+ and 1 ppm for its fungicides. The sample throughputs for Mn2+ and its fungicides were achieved as 90 and 120 h–1, respectively. The interferent effect of other metals were removed by incorporating an 8-HQ mini-column and also employing SPE extraction procedure. Mn2+ was successfully quantified in certified reference materials (CRMs) of river water (SLRS-4 and SLRS-5) and its fungicides in water samples of natural origin. Effect of thirty other pesticides was also checked on the CL reaction.

3.1 EXPERIMENTAL 3.1.1 Chemical reagents and solutions All glass and plastic ware used for storing stocks and other reagent solution was precleaned with detergent and rinsed with UHP water. Then this ware was stored in HCl 10% (v/v) for a weak, followed by keeping them in HNO3 10% (v/v) for the same period (AnalaR, Merck Darmstadt, Germany) and rinsed several times with UHP water and this water was also employed for thorough rinsing of other labware. All the washed and cleaned lab wares were saved from contamination by storing them in zip locked plastic bags. Analytical grade chemicals, reagents and standards were used for the preparation of stocks and other running standard solutions in UHP water unless stated otherwise. Low nutrient seawater (LNS), having salinity of 35 was acquired from Ocean Scientific International (Wormly, UK). The stock solutions of 1000 ppm of Mn2+, 160 ppm of maneb and 100 ppm of mancozeb were prepared when 0.261, 0.008 and 0.005 g of Mn(NO3)2 (Merck, Darmstadt, Germany), maneb and mancozeb (Dr. Ehrenstorfer GmbH, Germany) were dissolved in 0.05 L of HCl (0.01 M), UHP water and ethanol, respectively. The working standard solutions of Mn(II) and its fungicides such as mancozeb and maneb were arranged when the required aliquots of their respective stock solutions were diluted with 0.1% and 0.01% (w/v) of Tween-20 solution. The stock solution of 1 mM of lucigenin was prepared when 0.051 g of lucigenin (Fluka, Buchs, Switzerland) was dissolved in 0.1 L of UHP water. This solution after sonication was stored in dark at 4 oC and was found to be stable for at least 30 days. The 75 µM working standard solution of lucigenin was arranged when 18.75 mL of its stock solution was diluted upto 0.250 L with UHP water.

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The stock solution of 5 M of NaOH was prepared when 20 g of it was dissolved in 0.1 L UHP water and 0.5 and 0.75 M working standard solutions of it were prepared when 25 and 37.5 mL of its stock solution were diluted upto 0.250 L with UHP water, respectively. The stock solutions of 10% w/v of various surfactants such as polyoxyethylene lauryl ether (Brij-35), cetyltrimethylammonium bromide (CTAB) (MP Biomedicals, Solon, OH, USA), polyoxyethylenesorbitan monolaurate (Tween- 20) and SDS (Bio-Rad Laboratories, Hercules, CA, USA) and polyethylene glycol tert-octylphenyl ether (Triton-X-100) (Research Organics, Cleveland, OH, USA) was prepared when 2.5 g of each surfactant was dissolved in UHP water (25 mL) in amber colored bottles and stored at room temperature in dark. The working standard solutions of these surfactants were organized by diluting the required aliquots of each in UHP water. For interferences study, the stock solutions of 1000 ppm of each cation such as Mg2+, Ca2+, Fe3+, Fe2+, Cd2+, Co2+, Cu2+, Pb2+, Cr3+, Ni3+, V3+ and Zn2+ was prepared when a required quantity from the respective nitrate salts or required volume from AAS standards (Merck, Darmstadt, Germany and Spectrosol, BDH, UK) were dissolved or diluted with HCl (0.01 M). The stock solutions of 1000 ppm of various 2– – – 3– 3– – – anions and organic compounds such as SO4 , NO3 , NO2 , PO4 , AsO4 , Cl , CO3 , – HCO3 , humic acid, ascorbic acid and phenol were arrangrd by dissolving the required quantities from their respective salts or organic compounds in UHP water. The working standard solutions of the chemical species were arranged by diluting them with Tween-20 of 0.1 or 0.01% (w/v). The stock solutions of 100 ppm of various pesticides, related to its various classes, such as tridemorph, thiram, thiobencarb, thiabendazol, , simetryn, , propanil, , permethrin, paraquat, nabam, molinate, malathion, fubridazol, fenarimol, dodemorph, dinoseb, diazinon, , carbofuran, carbendazim, benomyl, bendiocarb, atrazine, asulam, , aminocarb, and aldicarb (Dr. Ehrenstorfer, Augsburg, Germany, all the pesticides CRMs mentioned in this dissertation are obtained from this source) were prepared when required quantities of them were dissolved from their CRMs in absolute ethanol or UHP water based on their solubilities. The pesticides solutions were stored in dark brown colored

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bottles at –4 oC and series of their working standard solutions were arranged by diluting the required serial aliquots of their stock solutions with 0.01% Tween-20.

3.1.2 FI manifold and procedure Figure 3.2 shows the proposed FI-CL manifold employed for Mn2+ and its fungicides quantitative analyses. Carrier and reagent streams were propelled by a four-channeled peristaltic pump and a fixed volume of Mn2+ and its fungicides standards solution were injected into the carrier stream (Tween-20). The analytes containing carrier stream was allowed to merge with CL reagent stream (alkaline lucigenin) at a T-piece. This merged stream voyaged about 3.0 cm and then entered through a glass spiral reaction flow cell fitted and flanged in-front of window of an end window PMT. The T-piece, CL reaction glass spiral flow cell and PMT were encased and enclosed in an aluminum casing (light tight) to prevent any interference from environmental light. Power supply of 2.0 kV was connected and attached with the PMT for providing reproducible and constant supply of voltage to it. A flatbed chart recorder was also connected with the PMT to record its output. Tygon tubing of 1.02 mm id. was used to connect the whole manifold.

Figure 3.2 Proposed FI-CL manifold for Mn2+ and its fungicides (Maneb and mancozeb) quantitative analysis. R1 = Carrier stream; 0.1% (w/v) of Tween-20 for Mn2+ and 0.01% (w/v) for its fungicides, R2 = second stream; 0.75 M NaOH for Mn2+ and 0.5 M for its fungicides, R3 = CL reagent stream; 75 µM aqueous lucigenin, flow

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rate; 1.6 and 2.1 mL min–1 for Mn2+ and its fungicides, respectively, and PMT voltage; 1000  1 V.

3.2 RESULTS AND DISCUSSION 3.2.1 Chemical and physical parameters optimization The optimization studies were first performed to evaluate and establish the best experimental conditions for the quantitative analysis of Mn2+ and then the same experimental variables were re-optimized for Mn2+ containing fungicides (maneb and mancozeb). For the quantitative estimation of Mn2+, various chemical and physical variables were checked adopting a univariate approach and all measurements were performed in triplicate and the concentration of aqueous (UHP) Mn2+, sample loop volume and PMT voltage used throughout the optimization studies were 0.5 ppm, 180 µL and 1000 ± 1V. Results for the optimized chemical and physical variables are shown in Figure 3.3 and Table 3.1, respectively.

Lucigenin reacts with O2 or H2O2 in basic medium and produces an electronically excited N-methylacridone (NMA), which on de-excitation produces blue-green light and the continuum of its CL emission spectrum extends over the λ range of 420 – 560 nm with λmax. centered at 473 nm (Lee, Karim, Alam, & Lee, 2007). The concentration of lucigenin was, therefore, optimized over the range of 1.0 – 100 µM, because the CL intensity highly depends on the concentration of CL reagent. A linear increase in the intensity of CL signals was achieved as the lucigenin concentration increased upto 100 µM as shown in Figure 3.3a, but keeping in view the solubility of the CL reagent, background CL stability and the signal CL emission intensity, 75 µM was selected as an optimum for further experiments. In basic pH range, lucigenin generally reacts with oxidizing agents and optimum concentration of NaOH or KOH for this purpose may ranges from 0.1 to 1.0 M (Nieman, 1991). If NaOH and lucigenin are premixed, the lucigenin will dissociate and cause high CL background as noise within a few hours. Therefore, the solutions of these two reagents must be delivered in streams to reaction flow through cell separately. The optimization of NaOH concentration was performed over the range of 0.1 – 1.0 M. The increased in the intensity of CL signals was detected as the NaOH concentration increased up to 1 M (Figure 3.3b). Background CL at this concentration

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was very high, therefore NaOH of 0.75 M was selected as an optimum to be employed in future experiments. The oxidized product of lucigenin, i.e. N-methylacridone, has the property to adsorb on the inner surface of glass made spiral flow cell and therefore reduce the transmission of CL radiations. N-methylacridone has higher solubility in micellar medium than pure distilled water. For this reason, the effect of various nonionic, cationic and anionic surfactant was checked and the concentration of the selected one was optimized. First of all, the effect of 0.1% (w/v) solution each of Tween-20, Triton-X-100, Brij-35, CTAB and SDS for the quantitative analysis of Mn2+ was studied. For this purpose, each surfactant acting as carrier stream in the proposed manifold in the place of UHP water (R1). In comparison to UHP water, the CL signals intensity was higher in the case of Brij-35, CTAB, Triton-X-100 and Tween- 20, but among them, the highest intensity of CL signals was obtained when Tween-20 was used as carrier stream. This was the reason for which the Tween-20 concentration was optimized over the range of 0.001 – 1.0% (w/v). Figure 3.3c shows the increase in CL signal intensity with the increase in surfactant’s concentration up to 0.1% (w/v) and selected as an optimum. Beyond this concentration, the signals intensity decreased. N-methylacridone, the degraded product of lucigenin, is less soluble in water and therefore deposits on walls of reaction glass cell as mentioned before (Klopf, & Nieman, 1985). This deposition must be removed for enhanced intensity of CL signals. This is the first function which is performed by micellar medium as it increases the solubility of the N-methylacridone and as a result removes it from the inner walls of glass spiral flow cell. In addition, further research and investigation has concluded that quenching or sensitization of the lucigenin CL reactions in micellar medium can be ascribed to alteration of microenvironment pH and solubilization (Hinze, Reihl, Singh, & Baba, 1984; Kamidate, Kaneyasu, Segawa, & Watanabe, 1991) for electrostatic attraction.

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Figure 3.3 Optimization of chemical variables of FI-CL manifold for the quantitative analysis of Mn2+ and its fungicides: (a), Lucigenin; (b), NaOH; and (c), Tween-20. As the optimization proceeded, chemical and physical variables in optimized conditions were employed for further optimization. Selected optimized chemical and physical variables for Mn2+ and its fungicides were found as: lucigenin, 75 µM; NaOH, 0.75 and 0.5 M; Tween-20, 0.1 and 0.01% (w/v); flow rate, 1.6 and 2.1 mL min–1 per channel; sample loop volume, 180 µL and voltage of PMT, 1 kV ± 1 V, respectively. Each result was the average of measurements in triplicate.

Examination of the key physical parameters (flow rate, sample loop volume and PMT voltage) on the quantitative analysis of Mn2+ was performed. To enhance the sensitivity and sample throughput and reduce the reagent consumption, three stream’s flow rate effect was inspected simultaneously over the range of 0.6 – 3.2 mL min–1. The highest CL signal’s intensity with reproducible peak heights and steady baseline was achieved at 1.6 mL min–1 flow rate per channel and, therefore, was selected as an optimum flow rate for all streams and used in subsequent experiments. Similarly, to enhance the sensitivity and sample consumption economy, sample loop volume was examined over the range of 30 – 300 µL. Injection loop volume of 180 µL was found the most suitable in terms economy of sample consumption and kinetics of CL reaction i.e. speed of CL response. PMT voltage was examined over

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the range of 800 – 1050 V in terms of S/N and the CL intensity increased in a curvy linear fashion, but voltage of 1 kV ± 1 V due to high S/N was selected for subsequent experiments. Figure 3.3 and Table 3.1 show the optimization studies of chemical variables (concentration of lucigenin (range studied, 1.0 – 100 µM; optimum, 75 µM), NaOH (range studied, 0.01 – 1.0 M; optimum, 0.5 M ), Tween-20 (range studied, 0.001 – 0.1% (w/v); optimum, 0.01% (w/v)) and physical variables (flow rates (range studied, 0.6 – 3.2 mL min–1 channel–1; optimum, 2.1 mL min–1 channel–1), sample loop volume (range studied, 30 – 300 µL; optimum, 180 µL) PMT voltage (range studied, 800 – 1050 V; optimum, 1000 ± 1 V or 1 kV ± 1 V) were examined for the quantitative analysis of Mn containing fungicides (maneb and mancozeb, each of 0.5 ppm in UHP water). These optimized and selected parameters were employed in future experiments for the quantitative analysis of Mn2+ containing fungicides.

Table 3.1. Physical parameters optimization for the quantitative analysis of 0.25 ppm of Mn2+ and 0.5 ppm of Maneb. Optimized experimental parameter was used for the optimization of proceeded parameter i.e. sample loop volume, 180 µL; PMT voltage, 1000 ± 1V and flow rates for maneb and Mn2+ 2.1 and 1.6 mL min–1 channel–1, respectively. Each result was a mean of triplicate.

3.2.2 Analytical characteristics Table 3.2 shows various analytical characteristics concenrrned to the method developed for Mn2+ and its fungicides quantitative analysis in water samples of natural origin. These analytical figures of merit include LODs (based on three time of

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the S/N), injection throughputs, precisions, linear dynamic ranges, regression equations and coefficient of determinations for these analytes. LOD can be defined as the analyte concentration which gives CL response of three times of peak to peak base line noise. The LOD and injection throughput were found for Mn2+ as 1 × 10–4 ppm and 90, respectively, while for maneb and mancozeb, they were found as 1 × 10–3 ppm and 120, respectively.

Table 3.2. Analytical characteristics of the developed FI-CL method for the quantitative analysis of Mn2+ and its fungicides (maneb and mancozeb). LOD: S/N = 3.

3.2.3 Salinity effect on Mn2+ quantitative analysis Figure 3.4 shows the study of checking the applicability of the developed method for Mn2+ and its fungicides (mancozeb and maneb) quantitative analysis in marine water samples. For this purpose, the effect of salinity over the range of 0 – 35 g L–1 was checked on quantification of 0.01 ppm Mn2+. The salinity over the mentioned range was obtained by diluting the LNS water with 0.1% (w/v) Tween-20 solution in UHP water. The salinity was checked by injecting the solution of LNS both in the presence and absence (blank) of the Mn2+. LNS blank solution (in Mn2+ absence) both enhanced and inhibited the CL emission depending on its concentration. The highest CL enhancement as a blank signal was appeared at 0.6 g L–1 of salinity which decreased to minimum up to 17.5 g L–1 and beyond this

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concentration inhibition of the CL signal started and reached to maximum (– 4 mV) at about 35 g L–1. Similarly, the effect of salinity was checked in the presence of 0.01 ppm of Mn2+ and a successive decrease and quenching in its CL signals was observed as the concentration of salinity was increased from 0.6 to 35 g L–1 by comparing them with Mn2+ (0.01 ppm) CL signals in UHP water containing 0.1% (w/v) of Tween-20. This quenching of CL signal may be ascribed to the interfering effect of Cl– at higher concentrations. A calibration graph was obtained by injecting Mn2+ standards over the range of 0.01 – 1.5 ppm in LNS water, when LNS water was propelled as carrier stream instead of Tween-20 (0.1% w/v) in UHP water. In this experiment, the system was found very noisy because of the poor reproducibility and a noisy baseline was observed in comparison to the system when the carrier stream was 0.1 % (w/v) Tween-20 in UHP water. Because of this experiment, a regression or calibration equation was obtained as y = 151.45x + 21.736 (y = CL intensity in mV and x = Mn2+ concentration in ppm) and RSDs over range of 1.4 – 3.2% (n = 7) were obtained for studied range. A conclusion can be drawn from the results of these experiments that the method is unsuitable for Mn2+ and its fungicides quantitative analysis in the samples of marine water because of salinity interference at higher concentrations, however, it is suitable for their analyses in fresh water samples.

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Figure 3.4 Salinity effect over the range from 0 to 35 g L–1 in Mn2+ absence (blank) and in the Mn2+ presence of 0.01 ppm. The solid line in the figure shows Mn2+ CL response in the absence of salinity. Uncertainties were 3SD (n = 3).

3.2.4 Interference study Interfering effects of various cations especially metal ions were checked on the blank and on the quantitative analysis of Mn2+ at concentration levels present in fresh waters. For this purpose, each metal cation at concentration level of 0.01, 0.1 and 1 ppm were injected without and with 0.01 ppm Mn2+ standard was injected into proposed FI-CL manifold. Table 3.3 shows the list of metal ions which were checked as interferences and includes Co2+, Cd2+, Cr3+, Cu2+, Fe3+, Fe2+, Ni2+, Pb2+, V3+ and Zn2+. Metal cations such as Zn2+, Pb2+, Ni2+, Cr3+ and Cd2+ did not show any interferent effect on blank and on Mn2+ quantitative analysis. The metal cations such as Cu2+ and Co2+ at concentration level of 0.01 ppm did not show any significant effect, but at 0.1 and 1.0 ppm they suppressed the Mn2+ CL signal. In contrary, 0.1 and 1.0 ppm each of V3+, Fe2+ and Fe3+ enhanced the CL signals in blank and on the Mn2+ determination. Such behaviors of these metals on the CL reactions of lucigenin in the presence of dissolved oxygen may be attributed to their reducing and catalytic properties (Montano, & Ingle Jr., 1979; Veazey, & Nieman, 1979). The CL signals of iron was suppressed by Mn2+ at 0.1 and 1.0 ppm and the same effect has been

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observed and reported by Seitz and Hercules (1972) previously. This observation has been attributed by them to the oxidation of Fe2+ into Fe3+.

Table 3.3 Metal cations interfering effect on blank and on Mn2+ quantitive analysis (n = 3) CL Response (mV) in Concentration CL response (mV) Metal ions the presence of (ppm) in Mn2+ absence Mn2+ (0.01 ppm) Blank (Tween-20, 0 0.20.1 200.5 0.1% in UHP) Co2+ 0.01 0.20.1 201.0 0.1 1.00.2 160.4 1.0 1.50.3 3.00.1 Cu2+ 0.01 0.20.1 201.5 0.1 0.10.1 140.3 1.0 0 4.00.5 Fe3+ 0.01 0.20.1 201.0 0.1 3.00.4 241.2 1.0 382.2 562.4 Fe2+ 0.01 221.5 271.2 0.1 1363.2 803.5 1.0 7755.1 6406.2 V3+ 0.01 3.00.5 230.5 0.1 300.8 541.2 1.0 19706.5 20008.1

In drinking water, the Co2+ average concentration has been found as 20 ppb, but the concentration upto 107 ppb has also been reported (ATSDR, 1992). Its concentration in ground and surface water of pristine area is less than 1.0 ppb, while in populated area ranges over 1 – 10 ppb (Kim, Gibb, Howe, & Sheffer, 2006). The typical concentration of Co2+ in sea water is 0.02 nM and ranges less than 0.01 – 0.1 nM (Ellwood, & van den Berg, 2001), and addition of 20 µM of dimethylglyoxime to the CL reagent can effectively mask it (Ussher, et al., 2009). The concentrations of

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Cu2+ in river and sea waters have been reported as 5.8 ± 0.2 and 4.5 ± 0.1 ppb, respectively (Goudarzi, 2007) and its practical LOD has been established as 50 ppb by US EPA (USEPA., 1991). The total vanadium typical concentration in sea water ranges as 34 – 45 nM (Wang, & Sanudo-Wilhelmy, 2008), while in drinking water ranges over approximately 0.2 – 100 ppb (Vouk, et al., 1979) and the typical concentration ranges over 1 – 6 ppb in it, which depends on various parameters such as geographical location etc. (Davies, & Bennett, 1983). In oceans, Fe is present in very low concentration over the range of 0.05 – 2 nM (de Baar, & de Jong, 2001), in river water, 0.7 ppm has been reported, Fe2+ is present usually in anaerobic ground water over the range of 0.5 – 10 ppm, while 50 ppm has also been reported (National Research Council, 1979) and EPA has established Fe MCL level of 0.3 ppm and considered it safe in drinking water (WHO, 2003). The concentration ranges of dissolved Mn in sea, fresh and river waters are 0.08 – 10 nM (Bruland, 1983), 1 – 200 ppb (Barceloux, 1999) and <11 – >51 ppb (ATSDR, 2000), respectively. Immobilized 8-HQ makes complexes with different metals with variable selectivity depending on pH from 0 to 7 as reported previously (Sohrin, et al., 1998). Ussher et al. (2009) have effectively removed iron and vanadium by passing the sample at pH 3.1 and 3.7, respectively, through an immobilized 8-HQ column. In the proposed study, the interfering effect of iron was removed in-line by incorporating an immobilized 8-HQ column having dimensions of 10 × 2.4 mm i.d. in the carrier stream of the developed FI-CL manifold (Figure 3.2) after injection valve. The effect of HCl concentration in the carrier stream was investigated over the range of 6.0 – 80 × 10–5 M for the removal of Fe2+ from water samples for the quantitative analysis of Mn2+ and its fungicides as shown in Figure 3.4 a & b. At HCl concentration of 1.5 × 10–4 M, approximately 88% retention of Fe2+ in 8-HQ column and more than 86% of Mn2+ elution was obtained efficiently. These demonstrations show that Fe2+ species make complex with 8-HQ by passing its solution through immobilized 8-HQ column, incorporated in carrier stream when carrier stream’s pH is adjusted to 3.82. Thus Fe2+ is removed from sample in the carrier stream before the confluence point and stopped from reaction with alkaline lucigenin. The employment of 8-HQ resin for the analyses of metals in FI manifolds is coupled with many merits including large binding capacity, high selectivity, pre-concentration of trace metals, tolerance for regular exposure to bases and acids of molar concentrations, no swelling and good stability.

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The main advantage of this resin is that it can be used for the analysis of metals at trace level in water samples where complex mixture of elements exists. The column of 8-HQ has a capacity of 10 µM of resin for transition metals (Landing, Haraldsson, & Paxeus, 1986). Interfering effect of various cations, anion and organic compounds on the blank i.e. in maneb and mancozeb absence and on the quantitative analyses of maneb and mancozeb (0.05 ppm each) in water samples was investigated. The environmentally relevant concentrations of these possible interfering species were checked as shown in Figure 3.6. There was no effect of Mg2+ (30 ppm), Ca2+ (100 3+ 3+ 2+ 2+ 2+ – ppm), As (7.5 ppm) (Cr and Cd (0.5 ppm), Pb (0.5 ppm), Zn (5.5 ppm), NO2 – – – 3– 2– (0.5 ppm), NO3 (10 ppm), Cl (250 ppm), HCO3 (10 ppm), PO4 (2 ppm), SO4 (250 ppm), humic acid and phenol (2 ppm each) in the absebce and presence of Mn2+ containing fungicides (mancozeb and maneb). Fe2+, Mn2+ and V3+ (0.005 ppm each), Fe3+ (0.5 ppm) and ascorbic acid (0.1 ppm) acted as catalysts and reducing agents for the CL reaction of lucigenin in the presence of dissolved O2 and, therefore, enhanced the CL both in the blank and in the presence of maneb and mancozeb. In contrary, Cu2+ and Co2+ (0.1 ppm) suppressed both blank and maneb and mancozeb CL signals.

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Figure 3.5 HCl pH (eluent) effect on the intensity of CL. (a) Removal of 0.05 ppm Mn2+ and 0.1 ppm Fe2+; and (b) 0.05 ppm maneb and 0.01 ppm Fe2+ employing an in line 8-HQ column over 2.5 – 5.0 pH range.

Figure 3.6 Cations effect (Mg2+, 30 ppm; Ca2+, 100 ppm; Co2+, 0.1 ppm; Zn2+, 5.5 ppm; Pb2+, 0.5 ppm; Cu2+, 1.0 ppm; Mn2+, 0.005 ppm; Cd2+, 0.05 ppm; Cr3+, 0.1 ppm; Fe2+, 0.005 ppm; Fe3+, 0.5 ppm; V3+, 0.005 ppm and As3+, 7.5 ppm) on the blank signal and on the quantitative analysis of 0.05 ppm each of maneb and mancozeb. Maneb and mancozeb CL signals in the absence of any interfering species are shown by solid lines at the left most of the graph.

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3.2.5 Mn2+ analysis in the reference materials and water samples For the evaluation of the proposed method’s accuracy, river water samples for trace metals (SLRS-4 and SLRS-5 (National Research Council of Canalda)) were analyzed. The pH of these CRMs was adjusted to 3.82 ± 0.1 before injection to the proposed manifold with NaOH (0.1 M). These samples were then analyzed by the FI manifold containing an immobilized 8-HQ column, incorporated after the injection valve in the carrier stream (0.1% w/v Tween-20 containing HCl (1.5 × 10–4 M)). The labelled certified values for Mn2+ on the CRMs were 3.37 ± 0.18 and 4.33 ± 0.18 ppb and after analysis by proposed FI-CL method, the values obtained as 3.67 ± 0.10 and 4.65 ± 0.15 ppb, respectively. These results show that there is no substantial difference between the results of the proposed method and the labelled certified values on these CRMs. Water samples (tap, irrigation, lake and rain water samples) were collected from Quetta Valley in 10% (v/v) hydrochloric acid washed bottels made of HDPE (high density polyethylene) for the quantitative analyses of maneb and mancozeb. For the removal of suspended solids after collection, water samples were filtered through filter papers (cellulose acetate membrane, 47 mm diameter, pore size 0.45 µm, Whatman, Maidstone, UK). The filtered water samples were stored at 4 oC in dark until analyses. For recovery calculations, various standards (0.05, 0.1 and 0.15 ppm) each of maneb and mancozeb were spiked to the above-mentioned water samples and extracted by SPE reported by Waseem et al. (2009). After elution of the pesticides from SPE cartridges with absolute ethanol and drying the eluate under N2 stream, the residue was re-dissolved in suitable volume of 0.01% (w/v) of Tween-20 and after sonication for two minutes, analysed by the proposed FI-CL manifold without using 8-HQ column. Table 3.4 shows the results for recovery over the range of 92 ± 5 – 104 ± 3 and 91 ± 2 – 104 ± 3 (n = 3) for maneb and mancozeb, respectively.

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Table 3.4 Maneb and mancozeb recovery test results from natural water samples (n = 3).

3.2.6. Fungicides, herbicides and insecticides detection The influence of different fungicides, herbicides and insecticides was investigated by injecting their standard solutions (2 ppm each) into the proposed FI- manifold under optimized conditions. Their different physicochemical properties, chemical formulas, structures and names, and the intensity of generated blank CL signals are shown in Table 3.5.

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Table 3.5 Blank CL intensity (average of triplicate) on the proposed FI-CL manifold of thirty-two (32) pesticides (2 ppm each) including different fungicides (serial number: 1 to 11), herbicides (serial number: 12 to 19) and insecticides (serial number: 20 to 32) with their trivial names, chemical formulas and physicochemical properties (Tomlin, 2000).

Vapor Water Half life Trivial name and pressure Kow CL intensity S. No. solubility in soil Chemical formula (25oC) log P on blank mg/L (20oC) (Days) (mPa) (mV)

1. Maneb (C4H6MnN2S4) 0.014 –0.45 178 1.0 1252.0 2. Mancozeb 0.013 1.33 6.2 0.1 1402.5 [(C4H6MnN2S4)x(Zn)y]

3. Benomyl (C14H18N4O3) 0.005 1.4 2 67 0.60.1

4. Thiram (C6H12N2S4) 2.3 1.73 16.5 15.2 0.40.2

5. Dodemorph (C18H35NO) 0.48 4.6 100 41 0.50.1

6. Fenarimol (C17H12Cl2N2O) 0.065 3.69 13.7 250 0.80.2 –4 7. Fuberidazol (C11H8N2O) 9.0×10 2.71 71 6 3.50.4 8. Carbendazim (C9H9N3O2) 0.09 1.48 8 40 0.80.2

9. Tridemorph (C19H39NO) 12 4.2 1.1 24 1.00.1 -4 10. Thiabendazole (C10H7N3S) 5.30×10 2.39 30 500 2.50.3 -7 11. Nabam (C4H6N2Na2S4) 1.26×10 –4.24 200000 – 0.50.2 12. Paraquat (C12H14N2) 0.01 –4.5 620000 3000 1.50.4

13. Propanil (C9H9Cl2NO) 0.0193 2.29 95 0.4 1.00.2 –4 14. Asulam (C8H10N2O4S) 5.0×10 0.15 962000 3.2 0.50.1

15. Dinoseb (C10H12N2O5) 6.7 2.29 52 18 0.80.2 16. Atrazine (C8H14ClN5) 0.039 2.7 35 75 1.50.3

17. Simetryn (C8H15N5S) – 2.8 450 60 2.00.2

18. Molinate (C9H17NOS) 500 2.86 1100 28 2.50.4 19. Thiobencarb 2.39 4.23 16.7 21 2.00.2 (C12H16ClNOS)

20. Resmethrin (C22H26O3) 0.0015 5.43 0.01 30 0.80.1

21. Terbufos (C9H21O2PS3) 34.6 4.51 4.5 8 0.70.1

22. Amitraz (C19H23N3) 0.051 5.5 0.1 0.2 - 23. Aminocarb (C11H16N2O2) 2.3 1.90 915 6 2.50.3

24. Aldicarb (C7H14N2O2S) 3.87 1.15 4930 10 0.70.2 25. Aldrin (C12H8Cl6) 3 6.5 0.027 28 0.50.1

26. Diazinon (C12H21N2O3PS) 11.97 3.69 60 9.1 -ve 27. Permethrin (C21H20Cl2O3) 0.002 6.1 0.2 13 0.50.1 28. Malathion (C10H19O6PS2) 3.1 2.75 148 0.17 1.00.2

29. Bendiocarb (C11H13NO4) 4.6 1.7 280 3.5 1.00.2

30. Phoxim (C12H15N2O3PS) 2.1 3.38 1.5 6 0.80.1

31. Carbofuran (C12H15NO3) 0.08 1.8 322 29 2.00.5 32. Carbophenothion 1.07 4.75 0.34 100 5.00.3 (C11H16ClO2PS3)

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3.2.7 Spectrophotometric studies For spectrophotometric studies, UV-spectra of Mn2+, maneb and mancozeb were taken in aqueous and alkaline media using a UV-visible spectrophotometer (Model 1601, Shimadzu Corporation, Japan). A 10-ppm standard solution of Mn2+ shows maximum absorbance at wavelength of 279.5 nm, which matches with the wavelength reported previously for Mn2+ (Araujo, Dias, Macedo, dos Santos, & Ferreira, 2007). 5 ppm standard solution each of maneb and mancozeb in aqueous medium, absorbed UV radiation with wavelength maxima of 241 and 269 nm, respectively, but the single continuum spectrum of the same standards in alkaline medium of 5 M NaOH changed into dual peaks having wavelength maxima of 262 and 283 nm. This transformation of the single continuum spectrum of Mn2+ containing fungicides into dual peaks shows the dissociation of the fungicides into Mn2+ and nabam, because the wavelength maxima shown by the fungicides in alkaline medium resemble to the wavelength maxima of Mn2+ and nabam. (Gustafsson, & Thompson, 1981; Hayama, & Takada, 2008; Debbarh, Titier, Deridet, & Moore, 2004; Bohrer, Cicero do Nascimento, & Gomes, 1999). The generated Mn2+ reacts with dissolved oxygen in alkaline medium and generates superoxide radicles which further oxidize lucigenin and thus generate CL emission.

3.3 CONCLUSIONS The developed FI-CL method for the quantitative analyses of Mn2+ and its fungicides (mancozeb and maneb) in water samples of natural origin. The simplicity, rapidity and sensitivity of the method was good enough for analytical purposes. This method was applied for Mn2+ quantitative analysis in SLRS-5 and SLRS-4 river water CRMs and mancozeb and maneb in tap, river, rain and lake water samples. The LODs for Mn2+ and its fungicides were obtained as 0.1 and 1.0 ppm, with 90 and 120 injection throughputs respectively. Mn2+ played a role of catalyst for lucigenin oxidation in the presence of dissolved O2 in alkaline medium and the CL emission intensity was greatly enhanced by Tween-20 a non-ionic surfactant. The interfering effect of Fe2+, Co2+ and Cu2+ present in fresh water samples was selectively removed by employing an in-line immobilized 8-HQ column. These metals were retained by the column and Mn2+ was eluted by carrier stream constituted from HCl (1.5 × 10–4 M) containing 0.1% (w/v) Tween-20, when the sample was passed through the column. Recoveries were obtained over the range of 92 ± 5 – 104 ± 3 and 91 ± 2 –

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104 ± 3% (n = 3) for maneb and mancozeb, respectively. Comparison among the analytical characteristics, for example, analytical figures of merit and other analytical attributes obtained employing the proposed and developed approach for the quantitative analysis of maneb and mancozeb with previously established methods is shown in Table 3.6 (Waseem, et al., 2009; Kubo, Tsuda, Yoshimura, Homma, & Nakazawa, 2003; Nakazawa, et al., 2004; Hayama, & Takada, 2008; Ozhan, & Alpertunga, 2008; Debbarh, et al., 2004; Garcinuno, Hernando, & Camara, 2004; Malik, & Faubel, 2000; Bohrer, Cicero do Nascimento, & Gomes, 1999; Kaur, Malik, & Singh, 2011).

Table 3.6. FI-CL developed method assessment by comparing with other methods (batch or flow based) in terms of analytical characteristics for the quantitative analysis of mancozeb and maneb.

Linear Sample RSD Technique Analyte range LOD R2 Ref. matrix (%) (ppm) (ppm) FIA-CL Natural waters Maneb 0.9989 1.0–3.4 This work and 0.01–3.0 0.001 0.9992 Mancozeb FIA-CL Natural waters Maneb 0.01–4.0 0.01 0.9987 1.4–1.8 (Waseem, et al., 2009) FIA-CL Standard Mancozeb 0.01–10 0.0001 0.9999 1–2.5 (Kubo, et solutions al., 2003) RP-LC-CL Vegetables Mancozeb 0.01–10 0.0000899 0.9999 1–2.5 (Nakazawa, et al., 2004) LC-MS/MS Fruits and Mancozeb, 0.001–0.2 0.00012 NR 4.2 (Hayama, vegetables maneb and & Takada, zineb 2008) LC-DAD Fruit juices Maneb 1–100 0.1 0.9967 0.7–3.8 (Ozhan, & Alpertunga, 2008) LC-UV Human Mancozeb 0.25–100 0.1 0.9997 NR (Debbarh, plasma and et al., 2004) urine LC-DAD-UV Tomatoes Maneb 0.1–5.0 0.45 0.9993 4.9 (Garcinuno, (LOQ) et al., 2004) CE–UV Commercial Maneb 0.56–161.8 0.57 NR 3.24 (Malik, & samples Faubel, 2000) FIA- Vegetables Mancozeb 0.1–12.6 0.01 0.9999 0.06 (Bohrer, et Spectrophotometry al., 1999)

Spectrophotometry Wheat grains Maneb 0.4–8.0 0.0011 0.9994 NR (Kaur, et and soft al., 2011) drinks NR = Not given

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CHAPTER–IV DETERMINATION OF THIRAM AND AMINOCARB PESTICIDES IN NATURAL WATER SAMPLES USING FLOW INJECTION WITH TRIS(2,2/-BIPYRIDYL) RUTHENIUM(II)– DIPERIODATOARGENTATE(III) CHEMILUMINESCENCE DETECTION

4 INTRODUCTION Thiram also known as tetramethylthiuram disulfide, which belongs to the class of fungicides namely dithiocarbamate. It is commonly used in agriculture as a fungicide for the safety of seeds and crops of different vegetables. In rubber industry, it plays a role of vulcanizing agent to vulcanize the rubber. In environment, thiram comes as a product when the two most widely used fungicides namely ziram and ferbam are oxidized (Lowen, 1961), and for several months, thiram can exist and persist in soil (Edwards, 1981). Dithiocarbamate fungicides when dissociate produces

carbon disulfide (CS2), therefore, the maximum residue limit for these fungicides has –1 been set as 2 – 7 mg kg of CS2 by the European Union. Aminocarb (4- (dimethylamino)-3-methylphenyl N-methylcarbamate) is a phenylsubstituted methylcarbamate pesticide. This pesticide is commonly employed to control insects from agricultural products including fruits, stored grain and vegetables (Sundaram, Boyonoski, Wing, & Cadogan, 1987; Ni, Xiao, & Kokot, 2009). Figure 4.1 reports the chemical structures of thiram and aminocarb pesticides. The well-established analytical technique termed as CL has many advantages such as minimal instrumentation, no need of external light source, simple optical system and high selectivity and sensitivity (Campana, & Baeyens, 2001; Su, et al., 2007). It has become a valuable and suitable analytical tool in the liquid phase for the pesticides quantitative analysis when coupled with FI manifolds or HPLC systems (Gracia, et al., 2005).

Thiram (C6H12N2S4; Mw. 240.43)

Aminocarb (C11H16N2O2, Mw 208.257) Figure 4.1 Chemical structures of thiram and aminocarb.

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DPA, a Ag3+ complex, in alkaline medium shows a very strong oxidizing agent activity having a reduction potential of 1.74 V (Thabaj, Chimatadar, & Nandibewoor, 2008). This oxidizing agent can oxidize various organic compounds and the studies of the kinetics of their oxidation by DPA have been extensively performed (Thabaj et al., 2008; Seregar, Veeresh, & Nandibewoor, 2007; Veeresh, & Nandibewoor, 2008) Yang and Zhang (2010) have described a CL method in flow mode for the quantitative analysis of human serum uric acid and the CL reaction was based on uric acid reaction with alkaline DPA; it generates a strong CL signal with a LOD (3σ) of 0.12 µM. Yang, et al., (2010a) have established an FI–CL method for amikacin sulfate in human serum. This compound has enhanced the light emission of DPA and luminol CL reaction in alkaline conditions with a LOD (3σ) of 19 nM. Zhao, et al., (2013) have described another Flow based CL approach for gemifloxacin to be determined in biological fluids and pharmaceutical formulation based on a gemifloxacin–DPA CL reaction in a sulfuric acid medium sensitized by CTAB with a LOD of (3σ) of 0.73 ppb. FIA–CL methods are few in numbers, which have been established for quantitative analysis of thiram, but no FI–CL method has been still published for aminocarb quantification in natural water samples. Waseem, et al., (2010) have established an FI–CL approach for the analysis of thiram qunatitatively in water samples of natural origin based on a cerium(IV) sulfate–quinine CL system in aqueous sulfuric acid with a LOD (S/N = 3) and sample throughput of 7.5 ppb and 120 h–1, respectively. The same group (Waseem, et al., 2009) have also established another FI–CL method for the quatification of dithiocarbamate fungicides (maneb, nabam, and thiram) in water samples of natural origin based on their alkaline photo fragmentation via UV light; the produced photoproducts oxidize luminol and generate CL emission. The LOD (S/N = 3) were 5.0, 8.0 and 10 ppb for thiram, nabam and maneb, respectively. For this method, the sample throughput has been achieved as 100 h–1. Girotti et al. (2008) described a CL–enzyme-linked immunosorbent assay (ELISA) method for thiram in honeybees; they employed luminol, HRP as a labeling enzyme and polyclonal antibodies for thiram quantitative analysis. LOD of this method has been obtained as 9.0 ppb.

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The establishment of a sensitive, fast and simple FI–CL method for thiram and aminocarb quantitative analysis in water samples is the aim of the present study. The 2+ method is based on Ru(bipy)3 oxidation in the presence of DPA to generate 3+ 3+ 2+* Ru(bipy)3 . Thiram and/or aminocarb reduced the Ru(bipy)3 to Ru(bipy)3 , which underwent relaxation generating CL. Triton X-100 could be used to determine thiram in the presence of aminocarb.

4.1 EXPERIMENTAL

4.1.1 Reagents and solutions

All plastic and glassware were cleaned in a 2% (v/v) detergent (nutrient free, Neutrocon, Decon Laboratories, UK) and after keeping them in this detergent overnight, they were thoroughly rinsed with UHP water of conductivity of 0.067 µS cm–1 followd by storing and soaking in 10% v/v HCl for about 24 hours and again rinsed with UHP water several times to ensure complete removal of HCl. The cleaned wares were then stored in zip locked plastic bag to avoid any contamination. UHP water was used for the preparation of aqueous solutions when required. 2+ / A Ru(bipy)3 stock solution (0.01 M) was arranged when 0.374 g of tris(2,2 – bipyridyl) ruthenium(II) chloride hexahydrate (Fluka, UK) was dissolved in 50 mL of o an aqueous solution of H2SO4 (0.01 M), sonicated for 10 min and stored at 4 C. A 2+ –6 working standard solution of Ru(bipy)3 (50 × 10 M) was arranged from the stock solution by diluting its 1.25 mL up to 250 mL with H2SO4 (0.025 M) aqueous solution. The stock solution of potassium hydroxide (KOH: 0.5 M) was arranged when 2.8 g of KOH was dissolved in 100 mL water and stored at 4 oC until further usage. The working standard solutions of potassium hydroxide were arranged when the required aliquots of the KOH stock solution were diluted with UHP water. The stock solutions (100 ppm) of thiram, aminocarb, terbufose, thiabendazole, malathion, diazinone, propanil, antu, bendiocarb, aldrin, tridemorph, trimethoprim, maneb, nabam, carbofuran, fenarimol, simetryn, permethrin and dinoseb were arranged when 10 mg of each pesticide was dissolved in 100 mL of absolute ethanol, sonicated for 10 min and stored in brown colored bottles at 4 oC. Series of working standards of these pesticides were arranged by serial dilution of their required aliquots with aqueous ethanol solution (0.5% v/v). The stock solutions of various cations (Na+, + 2+ 2+ 2+ 2+ 2+ 3+ 2+ 2+ – – – K , Ca , Mg , Zn , Cu , Fe , Fe , Co and Mn ) and anions (NO3 , HCO3 , Cl ,

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2– 3– SO4 , and PO4 ) (250 ppm each) were arranged when required quantities of their respective salts were dissolved in water and working standard solutions were arranged by diluting the required aliquots from their stock solutions with ethanol (0.5% v/v). Analytical reagent grade surfactants were obtained from the sources described in the previous chapter. The stock solutions (1% w/v) of Brij-35, Triton X-100, Tween-20, CTAB and SDS were prepared when 100 mg of each surfactant was dissolved in 10 mL of UHP water in brown glass bottels at room temperature. Working standards of each surfactant were prepared by dilution in ethanol (0.5% v/v). Previously reported procedure was employed for the synthesis of DPA (Balikungeri, et al., 1977) as described in chapter-2. The UV-visible spectrum of DPA exhibited two absorption peaks at 253 and 362 nm, identical to those reported previously (Yang, & Zhang, 2010; Balikungeri, et al., 1977). The DPA solutions were freshly prepared when required and for this purpose the required and dried powder of the synthesized DPA was dissolved in a 0.05 M KOH solution before use; the concentration was then determined by the absorbency at 362 nm (є = 1.26 × 104 mol–1 L cm–1) (Balikungeri, & Pelletier, 1978) using a double–beam UV-Vis spectrophotometer (Model UV-1700, Shimadzu Corporation, Japan).

4.1.2 FI manifold and procedure Figure 4.2 shows a simple three channel proposed FI-CL manifold employed during the quantitative analysis of thiram and aminocarb. Each stream at a 3.2 mL min–1 flow rate was propelled by employing a four-channel peristaltic pump. Tubings made up of polytetrafluoroethylene (0.8 mm) was employed to connect different components of FIA manifold. Standard solutions of thiram and / or aminocarb (60

2+ L) were injected into an ethanol (0.5% v/v) carrier stream, merged with Ru(bipy)3 –6 –6 (50 × 10 M) in an aqueous H2SO4 solution (0.025 M) and oxidant DPA (60 × 10 M) in an aqueous KOH solution (0.05 M) stream at a T-piece. The merged stream was then allowed to pass and react in a spiral flow cell made of glass (18 mm dia., 1.5 mm i.d.) fitted and positioned infront of the window of the end window PMT. The assembly of PMT and glass coil and T-piece were encased in a light tight aluminum housing. PMT was provided a voltage of 800 V via a power supply of 2 kV. A flatbed chart recorder was employed for the recording of chart recorder output signals.

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Figure 4.2 FIA–CL manifold for the quantitative analysis of thiram and aminocarb: 2+ –6 R1, ethanol solution (0.5% v/v); R2, Ru(bipy)3 solution (50 × 10 M) in H2SO4 (0.025 M); R3, DPA solution (60 × 10–6 M) in KOH (0.05 M).

4.1.3 Samples collection and SPE procedure Thiram and aminocarb were determined in spiked natural water samples (tap, irrigation and lake waters) using proposed FI-CL manifold. For this purpose, the collection of these water samples from Quetta valley’s various locations were achieved in HCl (10% v/v) washed high density polyethylene made bottels. For the removal of suspended particles from water samples, first they were filtered through a cellulose acetate made filter membrane having a pore size of 0.45 µm (Whatman, Maidstone, UK) and then stored at 4 oC in dark until further analysis. A series of thiram and aminoccarb standard solutions in the range of 0.075 – 0.25 ppm were spiked in these water samples and extracted with a solid phase extraction (SPE) technique employing disposable Sep-Pak C18 cartridges (Water Associates, USA). The conditioning of the cartridges was performed by percolating 5.0 mL of deionized water and 5.0 mL of absolute ethanol and dried by passing air for about 2 minutes (Maggio, Damiani, & Olivieri, 2011). At a flow rate of 10 mL min–1, the spiked water samples were passed through the preconditioned SPE cartridge under vacuum followed by washing with an aliquot of 0.5 mL deionized water and dried by passing air through it for 10 minutes. The retained thiram and aminocarb pesticides from SPE cartridge was eluted with 5 mL absolute ethanol and dried under a stream of nitrogen. Re-dissolution of the dried thiram and aminocarb pesticides residue was

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accomplished with 0.5% (v/v) ethanol aqueous solution followed by injection for analysis into the optimized proposed FI-CL manifold.

4.2 RESULTS AND DISCUSSION 4.2.1 Kinetics curve of the CL reaction A batch method was used to evaluate the kinetic characteristics of the proposed CL reaction. The response curve between the CL intensity in mV on the ordinate and time in seconds on the abscissa was obtained as shown in Figure 4.3 of 2+ the Ru(bipy)3 – DPA CL reaction enhanced by thiram when all the experimental parameters were kept constant. The result demonstrates that the CL intensity increased rapidly at ca. 3 s after the thiram solution was injected and reached a maximum in ca. 6 s. After 18 s of injection, the intensity of CL emission was becoming weaker and reached almost to the baseline. These kinetic studies showed the reaction is fast enough and can be used for the sensitive quantitative analysis of thiram and aminocarb.

2+ 2+ Figure 4.3 CL kinetic curve of Ru(bipy)3 – DPA catalyzed by thiram: Ru(bipy)3 –6 –6 solution (50 × 10 M) in H2SO4, (0.025 M); DPA solution (60 × 10 M) in KOH (0.05 M); thiram standard solution (1.5 ppm) in ethanol (0.5% v/v).

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4.2.2 Chemical and physical parameters optimization studies To find the manifold’s optimums parameters for the quantitative analysis of thiram and aminocarb pesticides, the optimization studies of the important chemical and physical parameters were performed on the analytical CL signal on the basis of univariate approach. The optimization of chemical variables includes checking the 2+ effect of concentrations of H2SO4, Ru(bipy)3 , KOH, DPA and ethanol, and physical variables include checking the effect of three streams flow rates, volume of sample loop volume, length of the reaction coil and voltage of PMT for achieving the maximum CL emission. All studies were performed with thiram and aminocarb (0.1 ppm) standard solutions, 60 L of sample loop volume and 1000 V of PMT voltage. Each result was the average of triplicate measurements. The optimization studies of chemical variables have been shown in Figure 4.4. 2+ Acidic conditions are the prerequisite for Ru(bipy)3 efficient CL emission. For this reason, the effect of different mineral acids such as HCl, HNO3, H3PO4 and H2SO4 on 2+ the CL intensity was examined. The working standard solution of Ru(bipy)3 was prepared in the same concentration of each acid and propelled as a CL reagent stream.

H2SO4 was selected to be used in further experiments because the maximum CL signals were obtained when the CL reagent was dissolved in it. The effect of different concentrations of H2SO4 over the range of 0.001 – 0.1 M was studied. Increase in

H2SO4 concentration resulted in the increase of CL emission intensity and reached to the maximum value at 0.025 M (Figure 4.4a). Therefore, a H2SO4 optimum concentration of 0.025 M was designated to be employed in subsequent experimental studies. 2+ The Ru(bipy)3 concentration effect was inspected over the range of 1.0 – 100 –6 × 10 M using the optimized H2SO4 conditions (0.025 M). The increase in the 2+ –6 Ru(bipy)3 CL reagent concentration resulted in a steep increase up to 50 × 10 M (Figure 4.4b), beyond this concentration the background CL intensity increased, 2+ causing a decrease in the signal CL intensity of analytes. Therefore, a Ru(bipy)3 concentration of 50 × 10–6 M was opted for selection to be employed in future experimental studies. In the proposed CL reaction, DPA acts as an oxidant, the molar strength of which impacts the CL sensitivity, as well as the linearity of the technique. DPA

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concentration effect was examined on the intensity of CL signal for the analytes over the range of 7.0 – 85 × 10–6 M. Increase in the intensities of CL signals were observed with the increase DPA concentration up to 60 × 10–6 M (Figure 4.4c), above which the CL intensity declined. Therefore, a DPA concentration of 60 × 10–6 M was selected and employed in subsequent experimental studies. Yang and Zhang (2010) reported that the DPA complex at low concentration is not stable in UHP water, while Blaikungeri et al. (1977) reported that this reagent is insoluble in concentrated KOH. Keeping in view both properties for DPA, the effect of the KOH concentration over the range of 0.001 – 0.1 M was examined. The optimal KOH concentration was 0.05 M (Figure 4.4d) and therefore, a KOH concentration of 0.05 M was selected and used 2+ subsequently. The pH value of the waste solution [Ru(bipy)3 in H2SO4 (0.025 M) and DPA in KOH (0.05 M)] was 2.71, using a pH meter (3305, Jenway Ltd. Felsted, Dunmow, Essex, UK).

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Figure 4.4: Effect of chemical variables on the quantitative analysis of thiram and aminocarb: (a), effect of H2SO4 concentration of CL reagent stream; (b), effect of 2+ Ru(bipy)3 concentration prepared in 0.025 M H2SO4; (c), effect of DPA concentration stream and (d) effect of KOH concentration of DPA stream. Each optimized variable was employed in the subsequent experimemt. Physical variable employed during these optimization studies were as: flow rate, 2.8 mL min–1 channel– 1; sample loop volume, 60 µL and PMT voltage 1000 V.

Thiram and aminocarb stock solutions were prepared in absolute ethanol. It has been conmmonly observed that the intensity of CL emission is influenced by organic solvents; in this manner, the impact of the ethanol concentration on the CL signal’s intensity was contemplated over the range of 0.01 – 5% (v/v). The maximum CL signal intensities were observed at 0.5% of the ethanol concentration and a further increase in the ethanol concentration caused high background CL noise. Therefore, an ethanol concentration of 0.5% (v/v) was carefully chosen and used as a sample carrier stream. The effect of various surfactants, including SDS, Brij-35, CTAB, Tween-20 and Triton X–100, in the range of 1 – 5 × 10–4% (w/v) on the blank (in thiram and / or aminocarb absence) and on thiram and aminocarb quantitative analysis was examined. Among these surfactants, CTAB was found to inhibit the CL of both pesticides, while

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the inhibition of the aminocarb CL emission in the presence of Triton X–100 was obtained without effecting the thiram CL signal. The inhibiting effect of Triton X–100 on the quantitative analysis of aminocarb was investigated, as shown in Figure 4.5. The CL signal of aminocarb was almost quenched by Triton X–100, possibly due to a strong interaction between Triton X–100 and aminocarb. The CL signal from thiram remained almost unchanged in the presence of Triton X–100 at 3 × 10–4% (w/v), while aminocarb CL was quenched at >90%, which means that Triton X–100 could be used while determining thiram in the presence of aminocarb. In the presence of surfactants or micellar media, the CL reactions could be quenched or enhanced possibly due to the enhanced solubility or pH alteration of the microenvironment (Zhang, & Chen, 2000; Chong et al., 2013) for electrostatic attraction.

Figure 4.5 Effect of the Triton X–100 concentration on the CL intensity of thiram (0.01 ppm, triangle) and aminocarb (0.2 ppm, circle) standard solutions. Experimental

–1 2+ conditions: sample volume, 60 L; flow rate, 3.2 mL min ; Ru(bipy)3 solution (50 –6 × 10 M) in H2SO4, (0.025 M); PMT voltage, 800 V

To achieve high sensitivity, sample throughput and economy of reagent consumption, various physical parameters such as flow rate, sample loop volume and PMT’s voltage were optimized. The simultaneous effect of flow rate of three streams was examined from 0.5 to 5 mL min–1 on the quantitative analysis of thiram and aminocarb. A linear positive correlation was observed between the increase in flow

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rate and CL signal intensity of thiram and aminocarb upto 3.2 mL min–1 and an infinitesimal decline in CL signal intensity was achieved when flow rate was furhter increased. Therefore, flow rate of 3.2 mL min–1 was employed subsequently. Sensitivity and dynamic linear range both are highly dependent on the sample volume and its effect was investigated from 60 – 360 µL. For the speed of response and economy of sample consumption, 60 µL was employed subsequently. The effect of PMT voltage from 700 – 1000 V was examined to achieve the best signal to noise ratio and an optimum CL intensity was observed at 800 V which was used for further studies. 4.2.3 Analytical characteristics Under the optimum chemical and physical parameters, a series of thiram standard solutions were injected into the proposed FI-CL manifold and a linear calibration curves over the ranges of 1.0 – 1000 (R2 = 0.9998 (n = 7)) and 1.0 – 10,000 (R2 = 0.9994 (n = 11)) ppb were obtained for thiram and aminocarb as shown in Figure 4.6 A and B respectively. The limit of detections based on the concentrations giving mean responses equal to three times of peak to peak base line noise (S/N = 3), were 0.1 ppb each for thiram and aminocarb with the injection throughputs of 200 h–1. All the analytical charateristics for the qnatitative analysis of thiram and aminocarb such as dynamic linear ranges, coefficient of determinations, relative standard deviations, LODs, injection throughputs and blank CL intensities have been shown in Table 4.1.

Table 4.1 Analytical characterisitics of thiram and aminocarb pesticides. The detection limit is based on S/N = 3.

Parameters Thiram Aminocarb Linear range (ppb) 1.0–1000 1.0–10000 LOD (ppb) 0.1 0.1 Calibration equation (I = intensity (mV), I = 2.533C + 3.5822 I = 0.5986C + 14.992 C = concentration (ppb) Blank signal (mV) 0.7 0.5 R2 0.9998 (n = 7) 0.9994 (n = 11) RSD range, % (n = 4) 1.3–2.6 1.0–2.3 Injection throughput (h–1) 150 150

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Figure 4.6 Calibration curves for (A) thiram and (B) aminocarb under optimum conditions.

4.2.4 Interference study Under the optimized chemical and physical experimental conditions, the effect of several pesticides and some common cations and anions at the concentration levels relevant to environmental water samples was investigated in the absence of thiram and aminocarb (on the blank) and on the quantitative analysis of thiram (0.025 ppm)

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and aminocarb (0.05 ppm). Propanil, bendiocarb, aldrin, tridemorph, carbofuran, fenarimol, simetryn, permethrin and dinoseb pesticides (40–fold) did not show any significant interferent effect on the quantitative analysis of aminocarb and thiram. However, nabam (0.5-fold) had a slight enhancing effect on both the CL signal blank and on the thiram and aminocarb response. The interfering effect of different anions and cations was examined in thiram and aminocab absence (on the blank) and on the quantitative analysis of thiram and aminocarb. The foreign chemical species tolerance limit was selected as that concentration which caused <±5% relative error in CL signal of blank or on the quantitative analysis of thiram and aminocarb. Different cations and anions such as + + 2+ 2+ 3– 2– Na (100 ppm), K (40 ppm), Ca (75 ppm), Mg (50 ppm), PO4 (1.0 ppm), SO4 – – – (250 ppm), Cl (100 ppm), NO3 (10 ppm) and HCO3 (25 ppm) did not show any interferent effect on the blank signals and on the quantitative analysis of thiram and aminocarb. Fe2+, Mn2+ and Co2+ (at 1.0 ppm each) had an enhancing effect on both the blank CL signal and on the quantitative analysis of thiram and aminocarb. The effect 2+ 2+ 2+ of cations e.g., Fe , Co and Mn can be masked online with Na2–EDTA, and could be easily removed from natural water samples during SPE extraction.

4.2.5 Validation of the proposed method The evaluation of the validity of the proposed manifold was performed by the quantitative analysis of thiram and amincarb in water samples of natural origin. All the water samples were spiked with 0.075, 0.15 and 0.25 ppm of thiram and aminocarb within the linear dynamic range of the methods, respectively. After using the SPE procedure (described above), the processed samples were analysed quantitatively by injecting them into the developed FI-CL method and the Table 4.2 presents the obtained results. These obtained results were then matched with the reported FI-CL (Waseem et al., 2009) and HPLC (Sundaram, 1997) methods with % recovery in the range of 95  3 – 102  2% for thiram and 95  5 – 105  3% for aminocarb, respectively. They were in good agreement with reported methods.

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Table 4.2 Thiram and aminocarb recovery results from spiked natural water samples (n = 3).

Proposed FIA-CL method Reported methods Spiked Recovery, % Recovery, % Sample (ppm) Thiram Aminocarb Thiram Aminocarb (Waseem et (Sundaram, al., 2009). 1997) Tap water 0.075 1032 1043 972 984 0.15 984 1024 1014 1002 0.25 1011 933 962 955 Irrigation 0.075 99.53 985 983 962 water 0.15 96.52 1012 1002 984 0.25 1051 95.84 953 975 Lake water 0.075 954 1023 985 1053 0.15 982 966 1022 1005 0.25 1062 1034 954 972

4.2.6 CL reaction mechanism 2+ Since the emergence of Ru(bipy)3 as a CL reagent (Hercules, & Lytle,

1966), it has found many applications for pharmaceuticals and pesticides. The methodology relies upon the fundamental sequence of reaction presented below:

Chemical oxidation can be achieved by using concentrated HNO3, PbO2, Ce(IV) 2+ and Cl2 (Iranifam, 2013). Ru(bipy)3 with Ce(IV) as an oxidizing agent has been applied for the quantitative analysis of antibiotics of the fluoroquinolones family (Murillo, Molina, de la Pena, Meras, & Giron, 2007). Pharmaceuticals/pesticides containing nitrogen in their chemical structure (heterocyclic compounds/amines) have been used as reductants to produce orange emission, and hence detected by the Ru2+ complex (Gerardi, Barnett, & Lewis, 1999; Gorman, et al., 2006).

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In recent years, the stabilized complexes of transition metals including Cu3+, 4+ 3+ – Ni and Ag with polydentate ligands such as periodate (IO4 ) have been commonly used in different CL reactions as oxidizing agents as discussed in Chapter 1. These complexes have been employed both in the presence or in absence of any CL reagent and the CL light emission has been brought about during redox CL reaction between these oxidant and CL reagent or analyte acting as reducing agent. DPA is a powerful oxidizing agent that has been employed previously for the oxidation of different inorganic and organic chemical species (Thabaj et al., 2008; Rao, Sethuram, & Navaneeth Rao, 1985; Jaiswal, & Yadava, 1970). DPA played a strong oxidizing role with luminol, and some drugs were capable of enhancing the CL reaction (Yang et al., 2010). In the present study, the maximum CL in the presence and absence of thiram and aminocarb containing nitrogen appeared at a 610 nm and a much higher CL peak was observed in the presence of thiram and aminocarb, indicating that Ru2+ is the 3+ luminophore. DPA is used as an oxidizing agent for generating Ru(bipy)3 , thiram and aminocarb as reductant enhancing CL emission.

4.3 CONCLUSIONS The proposed FI–CL method for the quantitative analysis of thiram and aminocarb in water samples of natural origin is simple; has low LODs [(S/N = 3) 0.1 ppb and high injection throughputs (150 h–1)]. The achieved results by employing the proposed FI-CL manifolds were close to that of the FI–CL and HPLC methods with % recovery in the range of 95  3 – 102  2% for thiram and 95  5 – 105  3% for aminocarb, respectively. Thiram could be selectively estimated in the presence of aminocarb employing Triton X–100. The low LODs, good precision (RSD 3.0%), high injection throughput, low reagents concentration and easy manifold to assemble and operate, all compare favorably with previously reported flow-based methods. In addition, DPA can be readily synthesized and this chemical reagent showed high stability in alkaline solution.

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CHAPTER–V FLOW INJECTION DETERMINATION OF THIABENDAZOLE FUNGICIDE IN WATER SAMPLES USING DIPERIODATOCUPRATE(III) – SULFURIC ACID – CHEMILUMINESCENCE SYSTEM

5 INTRODUCTION Benzimidazole fungicides are extensively employed in agriculture for pre- and post-harvest treatment for the control of a wide range of pathogens. There are two classes of these fungicides, they may be either over the fields of crops or applied directly to the soil. The clear majority of these compounds persist in the environment for a long time (Danaher, De Ruyck, Crooks, Dowling, & O’Keeffe, 2007). Thiabendazole [2-(4-thiazolyl)-1H-benzimidazole, TBZ] fungicide is used to control a variety of vegetable and fruit fungal diseases, such as stains, blight, rot and mold (USEPA, 2002). It is also used as a food preservative (Rosenblum, 1977) to preserve freshness in fruits and vegetables (Zhang, & Quantick, 1998), as a pre- or post-harvest fungicide (Artes, Rodriguez, Martinez, & Marin, 1995) and in industry as mildew proof agent. Human well being hazard and health risk evaluation and assessment did by European Water Framework Directive has set a MCL (maximum concentration level) in natural water samples of 0.1 ppb for most of the benzimidazole pesticides and of 0.5 ppb for total pesticides (Directive (2006)/11/EC of the European Parliament, 2006). The chemical structure of TBZ is given in Figure 5.1.

Figure 5.1 Chemical structure of TBZ fungicide (C10H7N3S, Mw. 201.25).

Numerous analytical approaches have been reported for the quantitative analysis of TBZ in a variety of matrices, such as animal bodies, natural waters, pesticide residues, pharmaceutical preparations, plasma, fruits, saliva, serum, soil, urine, and vegetables. These approaches are based on spectrophotometry (Tang, Wang, Liang, Jia, & Chen, 2005; Onur, & Tekin, 1994; El-Bardicy, Mohamed, & Tawakkol, 1990), HPLC and mass spectrometry (MS) (Han, Tang, Tian, & Row, 2013; Moral, Sicilia, & Rubio, 2009; Wu, et al., 2009; Boeris, Arancibia, & Olivieri, 2014; Yoshioka, Akiyama, Matsuoka, & Mitsuhashi, 2010), gas chromatography (GC) with thermionic specific detection (Navickiene, & Ribeiro, 2002) and GC/MS (Silva, Aquino, Dorea, & Navickiene, 2008; Filho, Santos, & Pereira, 2010; Mercer, & Hurlbut, 2004), CE with electrospray-MS (Rodrıguez, Pico, Font, & Manes, 2002),

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micellar electrokinetic chromatography (Rodrıguez, Pico, Font, & Manes, 2001), thin layer chromatography (Wang, & Huang, 2000), polarography (Lopez, Muniz, Garcia, Miranda, & Fojon, 1998), ELISA (Bushway, Larkin, & Perkins, 1997), fluorescence (Li et al., 2015), sequential injection analysis with fluorescence (Martínez, Cordova, Medina, & Ortega-Barrales, 2012), FIA-fluorescence (Garcia, & Aaron, 1997; Piccirilli, & Escandar, 2007) and room temperature phosphorescence (Piccirilli, & Escandar, 2009). Recently, new oxidizing agents have been introduced to be employed in the CL reactions and they have acquired analytical importance and considerable attention of analytical researchers. For instance, transition metals such as Ag3+, Cu3+ and Ni4+ have been used as oxidizing agents in various CL reaction systems. These transition metals in higher oxidation states have been stabilized by polydentate ligands such as – IO4 . By and large, the emission of CL radiation is the result of a redox reaction between these oxidizing agents and reductants. The oxidizing agents are these tranisition metals in uncommon oxidation states while the reductants are the luminophore, which may be either a CL reagent, for example luminol or an analyte. The complexes of Cu have got the most important position in redox chemistry because they are easily available and important for biological chemistry (Kitajima, & Moro-oka, 1994; Karlin, Kaderli, & Zuberbühler, 1997; Pierre, 2000; Solomon, Chen, Metz, Lee, & Palmer, 2001; Halcrow, 2001). A limited number of FI-CL approaches have been reported based on the use of Cu(III) complex in aqueous phosphoric acid and sulfuric acid solutions for the quantitative analysis of ofloxacin, levofloxacin and enrofloxacin in urine, pharmaceutical preparations and veterinary preparations with LODs (S/N=3) of 8.7, 9.2 and 15.4 ppb respectively (Shi, Chen, & Sun, 2011; Sun, et al., 2011). Several other methods have also been reported based on luminol-DPC-CL reaction in an alkaline medium for the quantitative analysis of chlortetracycline, cholesterol, ergometrine maleate, lincomycin, cefazoline, N-(4-aminobutyl)-N-ethylisoluminol and mitoxantrone (Li, Zhang, & Liu, 2001; Hu, & Zhang, 2008; Hu, et al., 2010; Hu, et al., 2011; Sun, et al., 2013; Fu, Li, Xie, Li, & Li, 2010; Yao, et al., 2014) in pharmaceutical and biological fluids. Table 5.1 compares the analytical characteristics of the various analytical methods for the quantitative analysis of TBZ.

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Table 5.1 Comparison of analytical characteristics of various analytical approaches for the quantitative analysis of TBZ.

Linear range LOD Sample Technique Matrix Ref. (ppb) (ppb) (h–1)

Spectrophotometry Fruits 178.3–2918 54.54 NR (Tang et al., 2005)

Spectrophotometry Tablets 0.2–1.2 0.2 NR (Onur & Tekin, 1994)

HPLC–UV Tomatoes 5–200 0.24 03 (Han et al., 2013)

HPLC–FL River and underground 2.5–3000 4×10–3 03 (Moral et al., 2009) water

HPLC–FL Water and soil 5–1000 0.5–1.6 NR (Wu et al., 2009)

HPLC–DAD Juice, fruit and vegetable 0–99.2 0.90 ˃6 (Boeris et al., 2014)

(Navickiene & Ribeiro, GC–TSD Lemons: peel and pulp 200–10000 200 NR 2002)

CE–ESI–MS Fruits and vegetables 1000–10000 10 NR (Rodrıguez et al., 2002)

FL–spectroscopy Apple juice 5–50 2.2 NR (Li et al., 2015)

SIA–FL Mushrooms 1600–40000 500 NR (Martínez et al., 2012)

FIA–FL Water 2–242 0.7 60 (Garcia & Aaron, 1997)

(Piccirilli & Escandar, FIA–FL Water 8–120 2.8 14 2007)

(Piccirilli & Escandar, FIA–RTP Natural waters 12.9–110 4.5 14 2009)

FIA–CL Natural waters 1–2000 0.3 160 This method

NR = not reported; GC–TSD = gas chromatography–thermionic specific detection; CE = capillary electrophoresis, ESI–MS = electrospray ionization–mass spectrometry; SIA = sequential injection analysis; FIA–RTP = flow injection analysis–room temperature phosphorescence.

To our knowledge, there has been no work reported using the oxidation of DPC for TBZ by the CL method in an acidic medium. In this manuscript, we report a novel FI–CL technique for the quantitative analysis of TBZ, based on its enhancement 5− effect on [Cu(HIO6)2] –sulfuric acid–CL system. The method is simple, sensitive, high injection throughput with a LOD (S/N = 3) and RSD (n = 4) of 0.3 ppb and 1.1 – 2.9%, respectively in the studied concentration range.

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5.1 EXPERIMENTAL 5.1.1 Reagents and solutions Analytical grade chemicals and reagents were employed during all the experiments, unless stated otherwise. Stocks and working standard solutions were prepared in deionized UHP water. The plastic and glass wares used throughout the experimental studies were stored and cleaned in 10% HCl and after one week, they were thoroughly rinsed with deionized UHP water. These wares were then saved from any contamination by storing them in ziplocked plastic bags. The KOH stock solution (1 M) was arranged when 5.61 g of it was dissolved in deionized UHP water (100 mL) and the arrangement of working standard solutions were achieved when required aliquots of the stock were diluted with deionized UHP water.

The stock solutions (1 M) of H2SO4, H3PO4, HCl, HNO3 and CH3COOH were arranged by diluting the required volumes from commercial stock solutions in UHP water and working standard solutions (50 mM) were arranged by diluting with UHP water. The stock solutions (100 ppm) of thiabendazole, carbophenothion, carbofuran, nabam, antu, permethrin, propanil, fubridazol, carbendazim, thiabencarb, aminocarb, aldicarb, asulam, fenarimol, benomyl, resmethrin, dodemorph, amitraz, paraquat, digoxim, ribavirin, aldrin, thiram, terbufos, diazinon, mancozeb, trimethoprim, atrazin, simetryn, tridemorph, gabapentin, phoxim, bendiocarb, molinate, maneb, malathion, and dianoseb (Dr. Ehrenstorfer, Augsburg, Germany) were arranged when the required quantities of each pesticide were dissolved in absolute ethanol and stored at –4 oC in dark brown bottles. The series of working standard solutions of these pesticides were arranged day-to-day by serial dilution of these stock solutions with ethanol (0.1%, v/v). The stock solutions (1000 ppm) of various organic compounds (ascorbic acid, phenol and humic acid), cation (Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Fe3+, Co2+ and 2+ – – – 2– 3– Mn ) and anion (NO3 , HCO3 , Cl , SO4 , and PO4 ) were arranged in UHP water; further dilutions were achieved with 0.1% ethanol for arranging series of working standard solutions for investigation during interference studies.

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DPC was prepared according to procedures reported previously (Jaiswal, &

Yadava, 1973; Jose, & Tuwar, 2007). Copper(II)sulfate·5H2O (1.56 g), sodium metaperiodate (2.67 g), potassium persulfate (1 g) and KOH (8 g) were dissolved in 100 mL UHP water. The mixture of solutions was kept at hot plate and heated to boiling for about 20 minutes and continuously stirred. After this time and boiling the mixture turned into intense red color. For the completion of the reaction, the mixture was boiled with stirring for another 20 minutes. The mixture was filtered through a sintered crucible after cooling at room temperature. The filterate was again filtered after cooling on an ice bath. To attain room temperature, the dark red color filterate left for some time standing at room temperature. For the complex isolation and crystallization, the filterate solution was added by sodium nitrite solution (50% w/v). When the crystallization reached to completion the filterate turned colorless. The supernatant was separated from the crystals by means of filteration and crystals left behind were washed with UHP water three time (3 × 10 mL) until the drops of brown color were formed under the cruicible. The complex’s alkaline solution was stable for at least one month when kept in dark at room temperature. For the characterization of the complex, UV-Visible spectrum of DPC exhibited maximum absorbance at 263 and 415 nm. Fresh solutions of DPC were arranged when appropriate amounts of the synthesized compound were dissolved in a 5 mM KOH solution; the concentration was then determined by measuring the absorbance at 415 nm before use (molar absorbtivity є = 6230 mol–1 L cm–1) using a double–beam UV-Vis spectrophotometer (Shimadzu, Model UV-1700, Japan).

5.1.2 FI manifold and procedure Figure 5.2 shows the FI–CL manifold used for TBZ quantitative analysis. A peristaltic pump was employed for the propulsion of reagents and carrier streams at 2.8 mL min–1 flow rate. Standards and samples of TBZ (180 µL), were injected into 0.1% (v/v) ethanol carrier stream using a six-way rotary injection valve and allowed to mix with 50 mM sulfuric acid stream. This mixed stream of 0.1% ethanol and 50 mM sulfuric acid was then merged with an aqueous alkaline DPC reagent (0.25 mM in potassium hydroxide, 5.0 mM) stream at a T-piece. These mixed streams travelled 2.0 cm before they were allowed to pass through glass spiral flow through cell with dimension of 18mm dia and 1.5 mm i.d. for CL reaction. This flow through cell was positione in a straight line to the window of an end window PMT. The whole assembly including T-

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piece, glass coil and PMT were saved from light by enclosing it in a light tight aluminum casing. A power supply of 2-kV was employed to provide voltage of 1250 V to the PMT. Flatbed strip chart recorder was employed for the recording of PMT output in the form of CL signal peaks. PTFE Tygon tubes of 1.02 mm, id., were used for the whole manifold connections.

Figure 5.2 FI–CL manifold for the quantitative analysis of TBZ.

5.2 RESULTS AND DISCUSSION 5.2.1 DPC-sulfuric acid CL system

DPC produced weak CL directly with different acids e.g., H2SO4, H3PO4, HCl,

HNO3 and CH3COOH (50 mM). The maximum CL intensity and better reproducibility were observed with sulfuric acid when compared with other acids. When TBZ was added to the DPC–sulfuric acid system, the CL signal was enhanced significantly. The pH value of the waste solution (DPC 0.25 mM in 5 mM KOH– sulfuric acid 50 mM–TBZ 100 ppb in 0.1% ethanol) system was 2.5, using a pH meter (3305, Jenway Ltd. Essex, UK). Further, some compounds and ions, e.g., rhodamine B, fluorescein, quinine sulfate, pyrogallol and sodium sulfite (0.1 mM each) when added in DPC–sulfuric acid–TBZ system, resulted in a decrease in the analyte CL response and in an increase of background CL noise. This is contrary to what had been being reported previously (Sun, et al., 2011), where sodium sulfite enhanced the CL intensity in Cu3+ complex- sulfuric acid system. Therefore, idea of using these compounds was abandoned for

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further study. In the absence of sulfuric acid, the DPC–KOH–TBZ system gave no CL signal under optimized conditions.

5.2.2 Kinetic profile and CL spectrum of DPC–sulfuric acid–TBZ For the assessment of kinetic behavior of the CL reaction, the optimized FI- CL manifold was employed for the recording of the response curve under the optimized conditions. The curve was obtained between the CL intensity on the ordinate and time along the abscissa and the curve has not been shown here. The CL emission intensity reached to its peak maximum in 8 seconds after the mixing of reagents and after almost 14 seconds it reached to the baseline. These results showed that the proposed FI-CL reaction is fast and appropriate for quantitative analysis of TBZ in water samples when flow rate is controlled.

5.2.3 Optimization of key chemical and physical variables Various chemical and physical parameters were investigated to find the optimized conditions for the quantitative analysis of TBZ. The parameters optimized were DPC, potassium hydroxide, sulfuric acid and ethanol concentrations, flow rates of all channels, sample injection volume and PMT voltage. Figure 5.3 and Table 5.2 present the results of optimization experiments. All these studies were performed with 100 ppb TBZ and all measurements were performed in triplicate. In the CL system, DPC was used as an oxidant. DPC concentration not only changed sensitivity but also the linear range for the assay. This influence was investigated over the range of 0.02 – 0.6 mM on the CL intensity. The results showed that the CL intensity increased with an increase in the DPC concentration up to 0.25 mM, above which the CL intensity was leveled off (Figure 5.3a). Therefore, a DPC concentration of 0.25 mM was selected and employed for subsequent experiments. The effect of KOH concentration on the CL reaction was explored over the range of 1 – 20 mM. Optimum and reproducible CL signals were observed at KOH concentration of 5 mM (Figure 5.3b), and therefore, a KOH concentration of 5 mM was selected and used in further experiments. The intensity of CL emission was significantly influenced by sulfuric acid concentration. The effect of the sulfuric acid concentration on the CL emission intensity was further explored over the range of 1.0 – 100 mM. The CL intensity increased as the concentration of sulfuric acid was increased up to 50 mM; above this

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concentration, the CL intensity decreased (Figure 5.3c). Therefore, 50 mM sulfuric acid was selected and employed in succeeding experiments. The solubility of TBZ is comparatively higher in ethanol (2.1 g L–1) than water (0.05 g L–1) at 20 oC (http://pmep.cce.cornell.edu/profiles/extoxnet/pyrethrins- ziram/thiabendazole). Therefore, due to its high solubility in ethanol, the stock solution of TBZ was prepared in ethanol. It is also known that the CL behavior is generally influenced by organic solvent; therefore, the effect of ethanol concentration was examined over the range 0 – 1.0% (v/v) based on signal to noise ratio. A small decrease in signal to noise ratio was observed from 0.01 – 0.1% ethanol, above which a considerable decrease in signal to noise ratio was examined. Therefore, an ethanol concentration of 0.1% (v/v) was selected and used as a sample carrier stream.

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Figure 5.3 Effect of concentraton of chemical variables on the variation of CL signal intensity (a) effect of diperiodatocuprate(III) concentration (b) effect of potassium hydroxide concentration (c) effect of sulfuric acid concentration. Each optimized variable was used during the optimization of next optimizing parameter. Physical parameters employed during these studies were 2.8 mL min–1 channel–1 flow rate, 180 µL of sample loop volume and 1250 ± 1.0 V of PMT voltage.

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The effect of physical parameters including flow rate of steams, volume of sample loop and voltage of PMT, in terms of speed, economy of reagents and samples consumption and sensitivity, were checked and investigated as shown in Table 5.2. The flow rates for each of three channels were explored over the range of 0.6 – 4.0 mL min–1. The CL intensity reached the maximum at a flow rate of 2.8 mL min–1, therefore, this flow rate was used by considering the sensitivity, reagents consumption and reproducibility. Similarly, the sample loop volume was examined over the range of 60 – 300 µL. The CL intensity of TBZ gradually increased with increasing sample injection volume. For the selection of optimum sample loop volume two parameters, namely, measurements sensitivity and analysis time were also kept in consideration. As a consequence, sample loop volume of 180 µL was opted as an optimum volume and employed throughout the further experimental studies. The effect of PMT voltage was also inspected from 900 to 1300 V and intensity of CL signals was increased in a curvi linear fashion with the increase in PMT voltage, however, 1250 V was employed for exclusively further experiments which gave steady baseline and reproducible CL signals.

Table 5.2 Physical parameters effect on the quantitative analysis of TBZ (100 ppb) using FI–CL manifold. Each optimized variable was used during the optimization of next optimizing parameter. Physical parameters employed during these studies were 2.8 mL min–1 channel–1 flow rate, 180 µL sample loop volume and 1250 ± 1.0 V of PMT voltage.

Parameters Range studied Optimized values

Flow rate (mL min–1 channel–1) 0.6–4.0 2.8

Sample loop volume (µL) 60–300 180

Voltage of PMT (V) 900–1300 1250

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5.2.4 Analytical figures of merit Under the carefully chosen physical and chemical parameters, a series of TBZ standards were injected into the proposed FI-CL manifold and a linear calibration graph of CL intensity . TBZ concentration over the range of 1 – 2000 ppb (r2 = 0.9999; n = 8) was obtained as shown in Figure 5.4, described by the equation y = (3.33 ± 0.1)x + (1.2 ± 0.2), where y = CL intensity and x = concentration in ppb. The injection throughput was 160 h–1 and RSD (n = 4) 1.1–2.9% over the range studied. The LOD (S/N = 3) (i.e. the concentration giving a mean response that was three times the peak-to-peak baseline noise) was 0.3 ppb without applying dispersive liquid–liquid micro-extraction (DLLME) method.

Figure 5.4 Calibration curve for TBZ under optimum conditions.

5.2.5 Interference study The effect of major freshwater ions at environmentally relevant concentration, and some organic compounds on the blank (in the absence of TBZ) and on the quantitative analysis of TBZ (50 ppb) was studies without applying DLLME method. The acceptable and tolerable concentration of extraneous chemical species were taken as a relative error not greater than 5%. Ca2+ 100000 ppb; Mg2+ 30000 ppb; Zn2+ 5500 ppb; Fe2+ 300 ppb; Cr3+, Cd2+, V4+, Fe3+, Co2+ and Mn2+ 500 ppb; Pb2+ and Cu3+

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+ + − 2− − 1,000 ppb; K 30000 ppb; NH4 25000 ppb; Cl and SO4 250000 ppb; HCO3 − − 3− 100000 ppb; NO3 20000 ppb; NO2 and PO4 1000 ppb; ascorbic acid, phenol and humic acid 1000 ppb had no significant effect on the blank CL signal and on the quantitative analysis of TBZ (50 ppb).

5.2.6 Detection of insecticides, herbicides and fungicides Under the carefully chosen and selected chemical and physical parameters discussed in the preceding paragraphs, the CL responses for different insecticides, herbicides and fungicides were also checked in the absence of TBZ (on the blank) and on the quantitative analysis of TBZ (50 ppb) without applying DLLME method. These include carbophenothion, carbofuran, and nabam 250 ppb; antu, permethrin, propanil, fubridazol, carbendazim, thiabencarb, aminocarb, aldicarb, asulam, fenarimol, benomyl, resmethrin, dodemorph, amitraz, paraquat, digoxim, ribavirin, aldrin, thiram, terbufos, diazinon, mancozeb, trimethoprim, atrazin, simetryn, tridemorph, gabapentin, phoxim, bendiocarb, molinate, maneb, malathion, and dianoseb 2000 ppb. No significant CL responses of these pesticides were observed on the blank as well as on the quantitative analysis of TBZ.

5.2.7 Application to water samples The applicability of the method was assesed by analyzing TBZ in tap water, rain water and irrigation water samples, collected from Quetta valley’s different locations in acid washed (hydrochloric acid 10%) bottles made up of high-density polyethylene. These samples were then filtered for the removal of suspended solids through cellulose acetate made filter paper having dimensions of 0.45 mm pore size and 47 mm diameter (Whatman, Maidstone, UK) and stored at 4 oC until further analysis. Recovery experiments were carried out with spiked water samples using 50 – 500 g L–1 spikes for TBZ. The obtained results are given in Table 5.3. The recoveries were in the range of 92 – 108%. For extraction of spiked TBZ, the DLLME procedure was adopted (Li, et al., 2015). In brief, 10 mL of water sample spiked with TBZ was taken in a screw-cap centrifuge tube (5 mL). Subsequently, ethanol (1.2 mL) acting as a disperser solvent which also contained chloroform (CHCl3) (0.155 mL) acting as an extraction solvent was injected into the sample solution rapidly. Almost for one min this mixture was shaken vigorously and as a result the mixture turned

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cloudy due to the dispersion of small droplets of CHCl3 throughout the water sample.

TBZ from the water sample was extracted into these small droplets of CHCl3. For the o separation of the CHCl3 from the sample, the tube was centrifuged for 2 min at 5 C and 3000 rpm. The CHCl3 was denser both than water and ethanol and therefore sedimented to the bottom of the tube. This sedimented CHCl3 was recovered and transferred with the help of a microsyringe into another tube and then dried with help of purging of N2 gas. The residue was dissolved in 10 mL of ethanol (0.1% v/v) and then analysed quantitatively by the help of developed FI-CL manifold.

Table 5.3 Recovery study of TBZ in spiked water samples (n = 3).

Recovery Sample Taken (ppb) Found (ppb) ± RDS (%)

Tap water 50 48 96±2.1

250 230 92±3.0

500 520 104±2.6

Rain water 50 47 94±3.5

250 260 104±2.8

500 530 106±2.5

Irrigation water 50 46 92±2.2

250 270 108±3.0

500 480 963.2

The effect of DLLME was also examined concerning the quantitative analysis of TBZ. Under optimum conditions, TBZ standards 50, 250 and 500 ppb prepared in tap water, were injected into the proposed FI–CL manifold and the CL intensity was found to be inhibited up to 35 ± 5% compared with standards of TBZ prepared in UHP water. This was probably due to cationic and anionic interactions of ions in tap water with TBZ. To avoid CL inhibition effect, the DLLME technique was adopted which almost recovered up to 98 ± 3% CL response for these standards.

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The proposed method was validated, for TBZ concentrations in three different mineral water samples (purchased from local market). Each sample was spiked using 50, 100 and 150 ppb TBZ and analyzed with proposed FI–CL method and reported fluorescence method (Li, et al., 2015) using a fluorescence spectrophotometer (KF- 1501, Shimadzu, Japan), equipped with a 150-W continuous xenon lamp and a 1-cm quartz cell holder. The excitation and emission slits of monochromators were both adjusted to 5 nm. The fluorescence intensity was measured at 345 nm with an excitation wavelength at 302 nm. The obtained results by these methods were not significantly different by applying Student t-test at 95% confidence level (tcalc. = 1.20; ttab. = 2.31).

5.2.8 Possible CL reaction mechanism To study the most propable CL reaction mechanism of TBZ with DPC in acidic conditions, the UV–Visible absorption spectra were recorded via a double beam UV–Vis spectrophotometer (Shimadzu, Model UV-1700, Japan). As shown in Figure 5.5, DPC (0.25 mM) in KOH solution (5.0 mM) has two distinct absorption peaks at about 263 and 415 nm as reported previously (curve a) (Jaiswal, & Yadava, 1973; Jose, & Tuwar, 2007), when aqueous sulfuric acid (50 mM) was added in DPC solution, the absorption peaks of DPC disappeared (curve b) and when TBZ (10000 ppb) was added to DPC and aqueous sulfuric acid mixture, a single absorption peak at about 301.4 nm (curve c) re-appeared.

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Figure 5.5 UV–Vis absorption spectra. (a) DPC (0.25 mM in KOH (5 mM)); (b) DPC + sulfuric acid (50 mM) and (c) DPC + sulfuric acid + TBZ (10000 ppb).

5− [Cu(HIO6)2] is a water-soluble complex of Cu(III) complexed with periodate 5− (Reddy, Sethuram, & Navaneeth, 1987) to be [Cu(HIO6)2] . Sun, et al. (2011) 5– reported that [Cu(HIO6)2] –H2SO4 system gave CL emission at 490 nm suggested the

–• –• production of O2 in the CL reaction, a part of O2 may recombine and generate energy rich dimer of oxygen molecule in electronically excited state represented as

(O2)2* which decomposed to O2 and emitting bright CL radiations at 491.6 nm. The 3– CL emission produced from [Cu(H2IO6)2] –sulfuric acid–TBZ system was suggested via the intermolecular energy transfer from (O2)2* to TBZ then, the excited TBZ de- excited to its ground state, producing CL emission at 345 nm (Garcia, & Aaron, 1997). According to the above discussion and experimental results, the possible CL reaction mechanism for the quantitative analysis of TBZ can be written as as follows:

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The CL intensity profile of DPC–sulfuric acid reaction in the TBZ absence and presence were also inspected as shown in Figure 5.6. The sample carrier, ethanol (0.1% v/v) was propelled via all three streams and the DPC solution (0.25 mM in KOH 5.0 mM) was injected in the sample carrier stream and no CL signal was observed (curve a). When a DPC solution was pumped in the third channel in place of the sample carrier an ethanol and sulfuric acid aqueous solution (50 mM) was injected in the sample carrier stream in place of the DPC solution, a weak CL signal appeared (curve b). Then, a sulfuric acid aqueous solution was pumped in the second channel in place of the sample carrier ethanol and when TBZ solution (25 ppb) was injected in the sample carrier stream, a strikingly enhanced CL signal was acquired (curve c). Therefore, it can be presumed that TBZ most likely assumed the part of an enhancer in the DPC– sulfuric acid CL response.

5.3 CONCLUSIONS A simple CL emission system was developed for the quantitative analysis of 5– TBZ in water samples based on [Cu(HIO6)2] –sulfuric acid–TBZ reaction system. The proposed FI–CL method is very simple, has low LOD (0.3 ppb) and high injection throughputs (160 h–1). Common inorganic ions and some organic compounds present in water samples, and a number of pesticides had no effect on the quantitative analysis of TBZ. The method was used for TBZ quantitative analysis in water samples of natural origin. It is better in terms of detection limit, reagent consumption and sample throughputs compared with other flow-based methods. A micro liquid-liquid extraction, namely DLLME, is principally based on the dispersion of small sized extraction solvent droplets inside the aqueous sample of pesticides (Rezaee, et al., 2006) and seems to be an environmentally friendly approach (Ojeda, & Rojas, 2011). Transition metals in higher oxidation states, for example, Cu3+, Ni4+ and

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Ag3+ have been used in various CL reaction framworks acting as oxidizing agents in acidic as well as in basic conditions.

Figure 5.6 The CL profile of DPC–sulfuric acid–TBZ system. (a) DPC: 0.25 mM in potassium hydroxide 5.0 mM; (b) DPC–sulfuric acid solution (50 mM) and (c) DPC–

H2SO4–TBZ: (25 ppb); sample volume: 180 µL.

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CHAPTER–VI FLOW INJECTION DETERMINATION OF CYROMAZINE IN NATURAL WATER SAMPLES USING

DIPERIODATOARGENTATE(III)-H2SO4- CHEMILUMINESCENCE SYSTEM

6 INTRODUCTION

Cyromazine (CYR), N-cyclopropyl-1,3,5-triazine-2,4,6-triamine (C6H10N6, RMM 166.19) belongs to the insect growth regulator class of pesticides and is widely used to inhibit insect growth and control flies from vegetables, field crops, fruits and cattle manure (Thomson, 1994). CYR is highly effective in pest control, and may be toxic to humans and the environment. Melamine, 1,3,5-triazine-2,4,6-triamine is a suspected carcinogen (Aldrich Chemicals Co., 1987; Cabras, Meloni, & Spanedda, 1990) and a potential degradation product or metabolite of CYR formed through de- alkylation reactions in plants, animals, and the environment (Goutailler, Valette, Guillard, Paısse, & Faure, 2001; Ingelfinger, 2008). CYR is water soluble and stable and can enter from soil to the food chain, causing potential hazards to organisms and human health (Pote, Daniel, Edwards, Mattice, & Wickliff, 1994). The allowable concentration of CYR is 10 and 100 µg kg–1 for milk products and edible poultry tissue, respectively (Codex Alimentarius Commission, 2012). The European Economic Community (EEC) sets a maximum admissible concentration (MAC) of 0.1 and 0.5 ppb for individual and total pesticides, respectively (EU, Council Directive 98/83/EC., 1998). The chemical structure of CYR is shown in Figure 6.1.

NH

N N

NH2 N NH2

Figure 6.1 Chemical structure of CYR (C6H10N6, RMM. 166.2)

Various analytical approaches have been established for the quantitative estimation of CYR and its metabolite melamine in biochemical and environmental samples. These include high performance liquid chromatography (Chen, Zhao, Miao, & Wu, 2015; Meng, et al., 2015; Wu, Ge, Liang, & Sun, 2014; Wang, et al., 2014; Zhang, et al., 2014; Hou, et al., 2013; Furusawa, 2012: Ge, Wu, Liang, & Sun, 2013; Sun, Qin, Ge, & Wang, 2011), GC (Zhu et al., 2009; Mansilha et al., 2010; Shang, Chen, Wang, Yang, & Zhang, 2011), voltammetry (Mercan, Inam, & Enein, 2011), IA (Liu, et al., 2010; Le, Yan, Xu, & Hao, 2013), vibrational spectrometric

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procedures (Armenta, Quintas, Garrigues, & de la Guardia, 2004), capillary electrophoresis (Wang, Qi, & Liu, 2014), fluorescence (Zhou, Yang, Liu, Wang, & Lu, 2010; Su, Chen, Sun, & Ai, 2012) and colorimetry (Bai, et al., 2016; Liu, et al., 2015). These methods are accurate and sensitive, but some of these instruments are not widely used in general laboratories due to large consumption of reagents, lengthy procedures, low sample throughputs and availability of expertise. The emission of light during a chemical reaction from an electronically excited intermediate or product is termed as CL (Campana, & Baeyens, 2001). This technique is attractive due to instrument simplicity, high sensitivity, wide analyte calibration range and small sample size. The inorganic complexes of some transition metals in uncommon highest oxidation states (Ag3+, Cu3+ and Ni4+) have been used as oxidants in various CL systems. The metals in their highest oxidation states have been stabilised by complexing with ligands mainly periodate and have found analytical applications in alkaline or acidic condition in environmental and pharmaceutical analyses (Asghar, et al., 2013; Asghar, Yaqoob, Munawar, & Nabi, 2016; Yang, Chen, Chang, Sun, & Zhang, 2014; Iranifam, 2013; Waseem, et al., 2013). The DPA in an alkaline medium has reduction potential of 1.74 V which shows its high oxidizing power (Sethuram, 2003). DPA-CL methods have been used for the quantitative analysis of fluoroquinolones in biological and pharmaceutical samples (Sun, et al., 2009; Sun, et al., 2010; Chen, & Sun, 2010). Wang et al., (2015) reported an inhibition CL method for CYR using FI technique based on luminol-DPA-CTAB system. The LOD (S/N = 3) was 6 µg kg–1 and the recovery of CYR spiked in bovine milk and milk powder samples ranged from 90.1 to 109% with RSD of 1.2 – 2.7%.

This paper reports DPA-H2SO4 reaction to devise a simple and sensitive FI- CL procedure to determine the amount of CYR based on its strong enhancing effect on the system. SPE procedure or incorporating an ion exchange resin column (IERC; OH-form) in the sample carrier stream could be employed to remove interference of chloride. The LOD (S/N = 3) were 0.029, 2.5 and 0.5 ppb with sampling throughput of 120, 80 and 120 h–1 with SPE technique and with and without IERC, respectively. The strategy was effectively connected to quantify the amount of CYR in water samples of natural origin.

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6.1 EXPERIMENTAL 6.1.1 Reagents and materials Glassware and bottles used during experiments were pre-cleaned in an acid bath (10% HCl) for a week, thoroughly rinsed with deionized water and stored. The analytical reagent grade materials (BDH chemicals Ltd., Poole and Fisher Scientific, Loughborough, UK) and deionized water were used for the preparation of stocks and working standard solutions. Stock solutions of KOH (1.0 M), organic compounds such as phenol, ascorbic acid, humic acid, melamine (Sigma-Aldrich, chemical Co. St. Louis, USA), cations such as Ca2+, Mg2+, Zn2+, Fe2+, Fe3+, Cr3+, Cd2+, V4+, Co2+, Mn2+, Pb2+, Cu2+, Na+, K+ + – 2– – – – 3– 5 and NH4 and anions such as Cl , SO4 , HCO3 , NO3 , NO2 and PO4 (5 × 10 ppb) from their respective salts were arranged in deionized water. Working standards were arranged in deionised water when required.

Stock solutions of H2SO4, H3PO4, HCl, HNO3 and CH3COOH (1.0 M) were organized by diluting the required volumes from their commercially available stocks in deionised water and working standard solutions (0.1 M) were set from these stocks (1.0 M) in deionised water. The DPA solution was organised in 0.05 M KOH by weighing an appropriate quantity of the synthesized compound and dissolved in 100 ml of deionized water. Its concentration was determined spectrophotometrically (Shimadzu, Model UV-1700, Japan) by monitoring the absorbance at 362 nm (molar absorptivity є = 1.26 × 104 M cm–1). Stock solutions of aldicarb, aldrin, aminocarb, asulam, atrazine, bendiocarb, carbofuran, CYR, diazinon, dinoseb, dodemorph, fubridazol, malathion, maneb, molinate, nabam, paraquat, propanil and simetryn (Dr. Ehrenstorfer, Augsburg, Germany) (1 × 105 ppb) were prepared in deionized water or absolute ethanol and refrigerated at –4 oC. The working standard solutions of each pesticide from their respective stocks were prepared in deionized water.

6.1.2 Preparation of DPA DPA complex was prepared and synthesized according to the previously reported procedure (Balikungeri, et al., 1977). The compound was dried and stored in brown bottle at room temperature for subsequent use. The UV-Vis spectrum of the

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compound has shown three bands at 216, 255 and 362 nm as reported previously (Yang, & Zhang, 2010).

6.1.3 Amberlite resin column Amberlite IRA-400 in Cl-form resin (particle size 0.30 – 1.18 mm, 14 – 52 mesh, standard grade, BDH Chemicals Ltd.) is a strongly basic anion exchange resin, cross linked copolymer of polystyrene and divinylbenzene. In order to introduce hydroxyl ions and obtain an effective removal of chloride ions, the ion exchanger was transformed into the OH-form by treating 20 g of resin with 100 mL of aqueous 0.5 M KOH solution. The mixture was left in contact for about 48 hours with intermittent shaking. After separation from supernatant, the resin was washed and rinsed several times with deionized water for complete removal of unbound ions and dried in an oven at 50 oC. The dried resin was then packed in an acid washed nylon tube (200 mm × 2.5 mm i.d.). Both ends of the tube were obstructed with clean cotton wool, incorporated in a carrier stream after the injection valve of FI manifold and washed with the stream of water for about 10 min. The incorporated-packed IERC was employed for removal of the possible interfering effect of chloride and other anions present in natural water samples.

6.1.4 Water samples collection and SPE procedure Natural water samples (irrigation, river and rain water) were collected in acid washed (10% HCl) high density polyethylene bottles and stored in dark at 4 oC until analysis. For removal of suspended solids, samples were filtered through a 0.45-mm cellulose acetate membrane filter (Whatman, Madistone, UK). Spiking was performed by adding CYR standards (75, 100, 125 and 150 ppb) to 10 mL of water sample. Three replicates of each concentration were prepared. Extraction and pre-concentration were obtained by SPE using disposable Sep-Pak C18 cartridges (Water Associates, USA) (Chou, Hwang, & Lee, 2003). These cartridges were preconditioned with 10 mL deionized water, 10 mL methanol and dried under vacuum. Spiking was performed by adding CYR standards (5, 10, 50 and 100 ppb) to 50 mL of water sample, passed through cartridge at a flow rate of 10 mL min–1, washed with 1 mL deionized water and dried with air for 10 min. The retained CYR was eluted with a mixture of concentrated (25%) NH4OH-methanol solution (1:3) (Sun, Wang, Liu,

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o Qiao, & Liang, 2009) and the eluent was evaporated at 45 C under N2 stream. The dried residue was redissolved in 3 mL deionized water and injected into the proposed FI-CL manifold for analysis. For calibration curve, a series of CYR standard solutions in the range 0.1 – 200 ppb prepared in 50 mL deionized water was extracted and pre- concentrated according to the procedure described above.

6.1.5 FI manifold and procedure The components of proposed FI-CL manifold (Figure 6.2) were connected using PTFE flow tubes (0.8 mm i.d.) and a transparent spiral flow cell (18 mm dia, 1.5 mm i.d.) was employed for CL measurements. Light emission was measured with a 9798B end window PMT operated at 1250 V connected with PM30D power supply. A 4-channel Ismatec peristaltic pump was used to propel the reagents and carrier at a flow rate of 2.0 mL min–1. The CYR standards and samples (180 μL) were introduced into the deionized H2O stream via a solenoid-activated rotary injection valve, and merged with the H2SO4 stream (0.1 M). This stream was then combined with DPA (3 × 10–4 M in 0.05 M KOH) stream in the flow cell. The CL signals were recorded on a Kipp & Zonen chart recorder. For the removal of chloride ions, an IERC (OH-form; 200 × 2.5 mm i.d.) was connected in the deionized water stream.

Figure 6.2 FI-CL manifold for the quantitative analysis of CYR. IERC: ion exchange resin column (Amberlite IRA-400 in OH-form; 200 × 2.5 mm, i.d.), HV = high voltage power supply.

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6.2 RESULTS AND DISCUSSION 6.2.1 Kinetic curve of the CL reaction A batch CL procedure was used to evaluate the kinetic characteristics of the proposed CL reaction based upon DPA and CYR under acidic conditions. In this study, a 2.0 mL volume capacity quartz cuvette was fixed in front of a PMT with a manual injection of (0.5 mL) DPA (3 × 10–4 M in 0.05 M KOH) with the help of a syringe followed by (1 mL) CYR standard solution (400 ppb in 0.1 M H2SO4). Figure 6.3 shows that the CL emission increased rapidly to the peak maximum in 1 s and declined to the baseline in 3 s. It has been shown that CYR strongly enhanced the CL response when reacted with DPA-H2SO4 system and is suitable for the assay of CYR.

Figure 6.3 Kinetic curve of CYR–DPA–H2SO4 system. Experimental conditions: 3 × –4 10 M DPA (0.5 mL) in 0.05 M KOH; 400 ppb CYR (1 mL) in 0.1 M H2SO4; 1000 V PMT voltage and 5 mm/s chart recorder speed.

6.2.2 Chemical and physical parameters optimization

The influence of experimental variables including DPA complex and H2SO4 concentrations, flow rates of three streams, sample loop volume, PMT voltage and IERC length on the CL intensity was investigated. A 50 ppb CYR standard solution was used throughout the optimisation studies.

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It has been reported that acid concentration is an important variable for the DPA complex CL reactions (Sethuram, 2003; Sun, et al., 2009). In this regard, the influence of different acids such as 0.1 M of H2SO4, H3PO4, HCl, HNO3 and

CH3COOH was examined. Among these, HCl and CH3COOH completely quenched the CL emission of CYR with DPA while H2SO4 was found suitable with better reproducibility and high CL intensity. Therefore, the effect of H2SO4 concentration in the range of 0 – 0.25 M was investigated. At 0.1 M H2SO4 concentration, maximum and reproducible CL signals were obtained and above this concentration, non- reproducible CL signals were observed. Thus, 0.1 M H2SO4 was chosen and selected as the ideal and optimum concentration to be employed in further experiments (waste pH 2.3 ± 0.1). The effect of DPA complex concentration in the range of 1 × 10–6 – 4 × 10–4 M was investigated. The best CL response was obtained at 3 × 10–4 M, and above this concentration, the CL response decreased. Therefore, 3 × 10–4 M DPA concentration was chosen as the optimum. The stability of 1 × 10–5 M DPA concentration prepared in 0.05 M KOH as well as in deionized water was also examined by measuring its absorbance (362 nm) versus time (24 h). At zero time, the DPA complex prepared in 0.05 M KOH, gave absorbance of 0.16 and after 24 h, its absorbance was decreased to 0.15 (6.3%). In deionized water, the initial absorbance was 0.1 and after 24 h, its absorbance decreased to 0.012 (88%). It has been reported (Yang, & Zhang, 2010) that the DPA complex is not stable in deionized water at low concentration. Therefore, based on these observations, a fresh solution of DPA complex was prepared in 0.05 M KOH when required. To achieve high sensitivity, linear calibration range and economy of reagents consumption, the influence of reagent streams flow rates, sample loop volume and PMT voltage was investigated for the quantitative analysis of CYR. The kinetic studies of the proposed CL reaction revealed that the CL reaction is very fast and thus, the flow rates for three reagent streams were checked simultaneously from 0.5 to 3 mL min–1. At 2.0 mL min–1 flow rate, reproducible CL peak heights and stable/steady baseline were observed; thus, this flow rate was selected as optimum for further studies. A sample loop volume of 180 µL was selected to minimize sample consumption and reduce response time (range studied 60 – 300 µL). A steady increase

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in the CL intensity was detected when increasing the PMT voltage in the range of 800 – 1300 V. Then, the PMT voltage of 1250 V was selected afterward.

6.2.3 Analytical figures of merit Under the optimized experimental conditions, the analytical characteristics including linear dynamic range, LOD, LOQ, precision and sample throughput for the proposed FI–CL manifold using the following three working conditions are presented in Table 6.1. CYR standards prepared in deionized water (a) after extraction and pre- concentration by SPE without using IERC, (b) incorporating an in-line IERC and (c) without using the IERC were injected into the deionized water stream of FI manifold for analysis.

Table 6.1 Analytical attributes of the proposed FI-CL technique for the quantitative

analysis of CYR utilizing DPA– H2SO4 CL framework.

Parameters SPE procedure a with IER column b without IER column c

Linear range (ppb) 0.1–200 10–1000 2–2500

LOD (ppb) 0.029 2.5 0.5

LOQ (ppb) 0.096 8.3 1.7

Correlation coefficient (R2) 0.9974 0.9980 0.9990

Precision (%, RSD, n = 3) 1.9–3.6 1.4–2.7 1.0–3.0

Regression equation y = 7.630x + 2.3372 y = 0.2947x + 14.06 y = 0.5478x + 25.47 (x = ppb; y = mV)

Sample throughput (h–1) 120 80 120 CYR standards (a) after extraction and pre-concentration by SPE without using IERC, (b) incorporating an in-line IERC and (c) without using the IERC.

The LOD and LOQ were calculated and determined as that concentrations of the analyte which give CL signals of three and ten times of signal to noise ratios, respectively. The LODs were 0.029, 2.5 and 0.5 ppb CYR with sample throughput of 120, 80 and 120 h–1 with SPE procedure and with and without using IERC in the FI manifold and their LOQs were 0.096, 8.3 and 1.7 ppb, respectively. Figure 6.4 shows

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chart recorder traces of CL signals and calibration curve in the inset for a series of CYR standard solutions (2.5 – 100 ppb) without using IERC in the FI manifold.

Figure 6.4 CL signals obtained for a series of CYR standard solutions injected in triplicate (inset shows calibration curve).

Analytical figures of merit for the quantitative analysis of CYR concerned to the proposed approach have been evaluated by comparing with the quantitative approaches developed previously as presented in Table 6.2. The proposed method has a lower LOD with comparison to the literature (Sun, et al., 2011; Mercan, et al., 2011; Bai, et al., 2016), while of the same order obtained with GC–MS method (Mansilha, et al., 2010). The method could be applied to analyse low concentration of CYR in water samples. The proposed CL system has satisfactory sensitivity, precision and linearity.

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Table 6.2 Evaluation of the analytical attributes of the proposed FI-CL method with other techniques for the quantitative analysis of CYR.

Technique Sample Linear range (ppb) LOD (ppb) Ref.

HPLC-DAD Environmental water 40–10,000 20 (Sun, et al., 2011) GC-MS Water 15–360 0.02880 (Mansilha, et al., 2010) Voltammetry Natural water 410–83,300 120 (Mercan, et al., 2011) Colorimetry River water 100–1000 100 (Bai, et al., 2016) FI-CL Natural water 0.1–200 (SPE) 0.029 This method 10–1000 (with IERC) 2.5 2–2500 (without IERC) 0.5

6.2.4 Interference studies The effect of several inorganic ions, present in fresh waters and some related organic compounds on 50 ppb CYR concentration was examined. The tolerable limit of each chemical species was assigned as the largest concentration producing an error  ±5% in the CYR CL signal. The possible interfering inorganic ions and organic compounds examined were 1 × 105 μg L–1 Ca2+, 30,000 ppb Mg2+, 5000 ppb Zn2+, 300 ppb Fe2+, 500 ppb Cr3+, Cd2+, V+4, Fe3+, Co2+ and Mn2+, 1000 ppb Pb2+ and Cu2+, 5 + + + 5 − 2− 1 × 10 ppb Na , 30,000 ppb K , 25,000 ppb NH4 , 2.5 × 10 ppb Cl and SO4 , 1 × 5 − − − 3− 10 ppb HCO3 , 20,000 ppb NO3 , 2000 ppb melamine and 1000 ppb NO2 , PO4 , ascorbic acid, phenol and humic acid. These foreign substances did not show any significant interfering activity on 50 ppb CYR concentration except chloride ions. During the interference study, it was observed that chloride at 2.5 × 105 ppb concentration completely quenched the CL emission of CYR. This quenching effect of chloride concentrations (1000 – 3 × 105 ppb) was investigated on the blank (without CYR) and on 500 ppb CYR concentration (Figure 6.5). When chloride standards (1000 – 10,000 ppb) were injected as blank, a high CL intensity was obtained at 1000 ppb then a gradual decrease in the CL response up to 10,000 ppb, above which negative CL signals were observed by decreasing the background CL intensity. When chloride standards 1000 – 10,000 ppb were injected in the presence of 500 ppb CYR standard, a high CL intensity was obtained at 1000 ppb (112% high

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CL intensity relative to 500 ppb CYR CL intensity). The CL response was decreased up to 10,000 ppb, above which quenching effect was observed on the CYR CL intensity. The quenching of background CL intensity and inhibition of CYR CL signal intensity could be ascribed to the formation of silver chloride white precipitates from the reaction of chloride anions with silver cations of DPA complex.

Figure 6.5 Effect of chloride in CYR absence (on the blank) and on the quantitative analysis of CYR 500 ppb. The solid line shows the response for CYR in Cl– ions absence.

To overcome chloride interference, an IERC (OH-form; 200 × 2.5 mm, i.d.) was incorporated in the sample carrier stream. Also, 20 replicate injections of 2.5 × 105 ppb chloride standard solutions did not show any harmful and deleterious effect on the CL response of CYR response for about 20 replicate injections of a 50 ppb CYR standard. The column was then regenerated by passing 0.5 M KOH solution at a flow rate of 0.5 mL min–1 for 10 min, followed by rinsing with deionized water until getting pH 6.0 of waste solution. The effect of IERC length (50 – 250 length × 2.5 mm, i.d.) was examined and a column length of 200 × 2.5 mm, i.d. provided low background CL noise and back pressure coupled with high reproducible CL signals

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and thus, a column length of 200 × 2.5 mm, i.d. was chosen for the removal of chloride interference. The effect of aldicarb, aldrin, aminocarb, asulam, atrazine, bendiocarb, carbofuran, diazinon, dinoseb, dodemorph, fubridazol, malathion, maneb, molinate, nabam, paraquat, propanil and simetryn pesticides (2000 ppb each) was also examined. No significant effect of these pesticides was observed on the DPA-H2SO4- CL system and CYR (50 ppb).

6.2.5 Applications to water samples Recovery experiments were carried out with spiked irrigation, river and rain water samples fortified with 75, 100, 125 and 150 ppb CYR standards incorporating an IERC (OH-form; 200 × 2.5 mm, i.d.) in the manifold. Recoveries were in the range of 91 – 105% for the proposed FI-CL method and 91 – 102% for the reported HPLC method (Sun, et al., 2011) (Table 6.3). The accuracy was verified by the Student’s t- test (paired) with value tcal. = 1.72, ttab. = 2.201 at a confidence level of 95%.

Table 6.3 Recovery study of CYR in spiked natural water samples, using an in-line IERC (OH-form; 200 × 2.5 mm, i.d.) in the proposed FI-CL manifold and its comparison with a reported HPLC method. FI-CL HPLC RSD RSD Spiked method Recovery method* Recovery Sample % % (ppb) Found % Found % (n=3) (n=3) (ppb) (ppb) Irrigation 75 70 93 2.5 69 92 3.0 water 100 96 96 2.0 92 92 4.0 125 123 98.4 3.8 122 97.6 5.0 150 153 102 3.0 145 97 6.0 River water 75 72 96 4.5 71 94.6 5.0 100 91 91 3.0 94 94 4.0 125 124 99 3.5 127 102 3.0 150 144 96 2.7 142 95 3.5 Rain water 75 72 96 3.1 68 91 4.0 100 92 92 2.0 94 94 7.0 125 123 98.4 3.5 121 96.8 5.0 150 157 105 2.0 142 94.6 6.0 Student t-test value: t = 1.72, t-distributed (95%) = 2.201 * Sun, et al., 2011

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Using the above described SPE procedure, recovery experiments were also performed spiking CYR standard solutions of 5, 10, 50 and 100 ppb in 50 mL irrigation, river and rain water samples. The residue was redissolved in 3 mL deionized water and injected in the proposed FI-CL system without using IERC. The % recovery ranged from 89 to 96.7 (Table 6.4). The pre-concentration factor for CYR standards in 50 mL deionised water with SPE procedure and redissolving in 3 mL deionised water was calculated as 16.7. Therefore, the method could easily be applied for the CYR quantitative analysis in water samples of natural origin at the minimum residue limit (0.1 ppb) for individual pesticides.

Table 6.4 Recovery study of CYR in spiked natural water samples using SPE technique by the proposed FI-CL manifold without using IERC. FI-CL method Spiked Recovery RSD % Sample Found (ppb) % (n=3) (ppb) Irrigation 5 4.6 92 2.8 water 10 9.0 90 1.6 50 45.6 91.2 3.0 100 96.7 96.7 2.2 River water 5 4.6 92 3.4 10 9.2 92 2.5 50 44.5 89 3.6 100 94.7 94.7 2.6 Rain water 5 4.8 96 2.0 10 9.4 94 1.8 50 45.3 90.6 2.5 100 96.4 96.4 2.7

6.2.6 CL reaction mechanism The DPA in aqueous solution converts into monoperiodatoargentate (MPA), which is an active form for complex formation and oxidation of other compounds

(Step 1) (Shen, et al., 2007). Under the aqueous H2SO4 medium, MPA dissociates and form orthoperiodic acid (H5IO6) and silver(III) cation (Step 2). The water molecule is

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oxidized with silver(III) cation liberating protons (H+) and superoxide-anion radicals –• (O2 ) (Step 3). The recombination of these superoxide-anion radicals forms energy rich dimer of oxygen molecule (step 4) (Sun, et al., 2010; Sun, Chen, et al., 2009). In acidic environment, the dimer on de-excitation emits light at 490 nm (step 5) (Lin, & Yamada, 1999). The CYR was found as a fluorophore in strong acidic solution (0.1 M

H2SO4) showing the λex./λem. at 213 and 462 nm respectively. The energy from the excited oxygen dimer could be transferred to CYR in acidic solution via intermolecular collision (Step 6) and the excited CYR molecule emits its energy in the form of light at 462 nm when comes to ground state (Step 7). The most possible CL reaction mechanism of DPA with CYR in acidic solution can be written as follows:

6.3 CONCLUSIONS A sensitive, selective and simple flow CL procedure has been reported for the quantitative analysis of CYR in natural water samples with the LODs (S/N = 3) of 0.029, 2.5 and 0.5 ppb with sampling throughput of 120, 80 and 120 h–1 employing SPE technique and with and without IERC respectively. Chloride was found to quench the CL signals which could be eliminated effectively by using IERC (OH- form) and SPE procedure. Different pesticides had no significant effect on the CL system. Superoxide anion radical generated between the reaction of DPA and H2SO4 is suggested to be involved in the CL emission.

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CHAPTER–VII POTASSIUM BROMATE-QUININE CHEMILUMINESCENCE DETECTION OF THIRAM IN WATER SAMPLES USING FLOW INJECTION ANALYSIS

7 INTRODUCTION

Thiram (N,N-dimethylcarbamodithioate, C6H12N2S4, RMM 240.42), a dithiocarbamate fungicide, is commonly used as a protectant of seeds and vegetable crops seeds from various fungal diseases and is also used as a vulcanizing agent in rubber industry (Lowen, 1961; Edwards, 1981). It persists in the environment for months and is introduced when two commonly used dithiocarbamate fungicide ferbam and ziram are oxidized (Lowen, 1961; Edwards, 1981). Different experiments concerned with thiram toxicology using rat as a test subject has shown several toxic effects. It may cause lesions of skin (Saunders, & Watkins, 2001), liver dysfunction (Edwards, Ferry, & Temple, 1990), effect nervous system (Han, et al., 2003) and cells (Cereser, Boget, Parvaz, & Revol, 2001). The residue limit of dithiocarbamates is generally expressed as carbon disulphide and the maximum residue limits for all dithiocarbamates have been assigned from 2 – 7 mg kg–1. A two-dimensional chemical structure of thiram is shown in Figure 7.1.

Figure 7.1 Chemical structure of thiram

Several analytical approaches have been established for the quantitative analysis of thiram, including HPLC (Aulakh, Malik, & Mahajan, 2005; Filipe, Vidal, Duarte, & Santos, 2008; Gupta, Rani, & Kumar, 2012), GC (Vryzas, Papadakis, & Mourkidou, 2002), MS (Peruga, Grimalt, Lopez, Sancho, & Hernandez, 2012), polarography and voltammetry (Sharma, Gupta, & Kashyap, 2011; Zhao, Zheng, Huang, & Yang, 2003), surface-enhanced Raman scattering spectroscopy (Zheng, et al., 2012), FI-Fourier transform infrared spectrometry (Cassella, et al., 2000) and spectrophotometry (Rastegarzadeh, & Abdali, 2013; Rastegarzadeh, Pourreza, & Larki, 2013).

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A well-established spectrometric technique termed as CL refers to the production of electromagnetic radiation during a chemical reaction (Campana, & Baeyens, 2001; Su, et al., 2007). CL possess many advantages such as simple and cheap instrumentation, require no external light source, limited light scattering due to simple optical components, low detection limit, high reproducibility and selectivity. Due to these advantages, CL is commonly employed as an analytical tool for the quantitative analysis of various fungicides when hyphenated to FI or HPLC techniques (Gracia, et al., 2005). Table 7.1 compares analytical characteristics of the proposed method and previously developed FI methods based on spectrophotometric and chemiluminescence detectors for the quantitative analysis of thiram (Asghar, Yaqoob & Nabi, 2014; Waseem, et al., 2010; Asghar et al., 2013; Waseem, et al., 2009) in natural water samples.

Table 7.1 Evaluation of analytical attributes of the proposed flow based approach with the previously reported flow based approaches for the quantitative analysis of thiram in water samples.

Linear range LOD Sample RSD Method Reaction and  R2 Ref. Abs/em (ppm) (ppm) (h–1) (%) rFI– 0.01–8.0 0.01 60 0.9999 0.8–1.6 (Asghar, et al., Fe(II)-K3[Fe(CN)6] Spec. complex, 790 nm 2014)

FI-CL Ce(III)-quinine-CL 0.0075–2.5 0.0075 120 0.9988 2.5 (Waseem, et al., 450 nm 2010) FI-CL 0.001–1 0.0001 150 0.9998 1.0–2.6 (Asghar, et al., Ru(bipy) 2+-DPA-CL 3 2013) 610 nm FI-CL 0.05–1.0 0.005 100 0.9987 1.0–2.6 (Waseem, et al., Luminol-O2-NaOH-CL 425 nm 2009)

FI-CL KBrO3-quinine-H2SO4- 0.001–20 0.0006 200 0.9998 0.94-1.76 This method CL 450 nm rFI = reverse flow injection, Spec. = spectrophotometry, CL = chemiluminescence.

o – The redox potential (E ) for BrO3/Br in mineral acid is 1.52 V, which shows its high oxidizing power. The usage of bromate as an oxidant in CL reaction accompanies an advantage of the lack of self-absorption of the emitted CL radiations – 4+ in visible region of electromagnetic spectrum. Other oxidants such as MnO4 , Ce , 3– Fe(CN)6 are colored in nature and are devoid of this advantage. H2O2 and N- bromosuccinimide are very unstable oxidizing agents compared to bromate (AC

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group, 1997). Bromate as an oxidizing agent has been used in various CL procedures for the quantitative analysis of steroids (Syropoulos, Sarantonis & Calorkerinos, 1990), bile acids (Psarellis, Deftereos, Sarantonis & Calokerinos, 1994), histamine (Sharipov, Kosareva, Fukalova & Karavaev, 1983), L-cysteine (Li, Zhang, Liu, & Xu, 2003) and formaldehyde (Li, Liu, Zhang & Xu, 2003). In this work, we propose a sensitive, simple and rapid procedure based on FI- CL technique, for the estimation of thiram in water samples. Thiram was oxidized with acidic potassium bromate and strong CL intensity was generated using quinine as a sensitizer. Spectrophotometric studies were performed for the elucidation of possible CL mechanism.

7.1 EXPERIMENTAL 7.1.1 Chemicals and solutions Analytical grade chemicals without further purification (BDH chemicals Ltd., Poole and Fisher Scientific, Loughborough, UK) and deionized water were used for the preparation of solutions. Potassium bromate (KBrO3, Mw 167.01) solution (0.25 M) was prepared by dissolving 4.18 g in 250 mL volumetric flask, diluted to the mark with 0.5 M aqueous H2SO4 solution and for the complete dissolution of KBrO3 the solution was sonicated vigorously for about 10 minutes.

Quinine sulphate (C20H24N2O2)2·H2SO4·2H2O, Mw 782.96) stock solution (0.1 M) was arranged by dissolving 3.91 g of the compound in 50 mL of 0.25 M

H2SO4 and for working standard solutions, required volume of quinine stock solution was diluted with 0.25 M H2SO4. Stock solutions (100 ppm) of aldicarb, aldrin, aminocarb, amitraz, antu, asulam, atrazine, bendiocarb, benomyl, carbendazim, carbofuran, diazinon, digoxim, dodemorph, dinoseb, fenarimole, fubridazol, gabapentin, malathion, mancozeb, maneb, molinate, nabam, paraquat, permethrin, phoxim, propanil, resmethrin, ribavirin, simetryn, terbufos, thiabencarb, thiabendazole, thiram, tridemorph and trimethoprim (Dr. Ehrenstorfer GmbH, Germany) were prepared by dissolving 2.5 mg of individual pesticide in total volume of 25 mL of absolute ethanol, sonicated for 10 minutes and kept at 4 oC in brown bottles and dilute working standard solutions were prepared in ethanol (0.1%) when needed.

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Stock solutions (500 ppm) of Na+, K+, Mg2+, Ca2+, Zn2+, Cu2+, Fe3+, Fe2+, 2+ 2+ – – – 2– 3– Co , Mn , NO3 , HCO3 , Cl , SO4 , PO4 and organic compounds such as ascorbic acid, humic acid and phenol for interference studies were arranged by dissolving the required amount of their respective salts/compounds in deionized water and further dilutions were brought with ethanol solution (0.1% v/v).

7.1.2 FI manifold and procedure In the proposed study, a three channel FI-CL manifold used is shown in Figure 7.2. To propel reagents and samples/standards, a four-channeled peristaltic pump was employed. Thiram standards/samples were injected into 0.1% ethanol (v/v) sample carrier stream by means of a six-port injection valve and this stream was then merged with another stream containing 1 mM quinine dissolved in 0.25 M sulfuric acid (R1). The merged streams were then mixed with 0.25 M potassium bromate solution in 0.5 M sulfuric acid (R2). The CL intensity was monitored in a glass spiral flow cell (1.5 mm, id., 80 mm length) mounted in front of an end window PMT, operated at 1250 V connected with a power supply. FI-manifold was connected using PTFE flow tubings (0.8 mm i.d.). The PMT output was recorded on the chart recorder.

Figure 7.2 FI manifold for the CL based quantitative analysis of thiram in water samples of natural origin. Carrier = 0.1% v/v ethanol, R1 = 1 mM quinine in 0.25 M sulfuric acid solution and R2 = 0.25 M potassium bromate in 0.5 M sulfuric acid solution.

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7.1.3 Analysis of natural water samples Water samples, (tap water, irrigation water and lake water) were obtained and collected in Quetta valley from different locations in 10% HCl washed bottles made of high density polyethylene and filtered through cellulose made filter membrane (47 mm diameter, Whatman, pore size 450 µm, Maidstone, UK) for the removal of suspended particles. A series of thiram standard solutions in the range of 0.05 – 0.3 ppm were spiked in these water samples and extracted with a solid phase extraction (SPE) technique employing disposable Sep-Pak C18 cartridges (Water Associates, USA). The conditioning of the cartridges was performed by percolating 5.0 mL of deionized water and 5.0 mL of absolute ethanol and dried by passing air for about 2 minutes. At a flow rate of 10 mL min–1, the spiked water sample was passed through the preconditioned SPE cartridge followed by washing with an aliquot of 0.5 mL deionized water and dried by passing air through it for 10 minutes. The retained thiram from SPE cartridge was eluted with 5 mL ethanol and dried under a stream of nitrogen. Re-dissolution of the dried thiram residue was accomplished with 0.1% ethanol aqueous solution (v/v) followed by injection for analysis into the optimized proposed FI-CL manifold.

7.2 RESULTS AND DISCUSSION 7.2.1 Kinetic curve of CL reaction The proposed reaction kinetic studies were investigated by employing a batch method. A 1.0 mL of 250 mM of KBrO3 in 0.5 M sulfuric acid was taken in a quartz cuvette fitted in front of an end window PMT then 1.0 mL of 5.0 ppm thiram standard containing 1.0 mM quinine in 0.25 M sulfuric acid was added. The CL transient peak reached to peak maximum in almost 0.56 seconds and returned to base line in 2 seconds as shown in Figure 7.3. This shows that the CL reaction is very fast and can be used for the analysis of thiram samples.

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Figure 7.3 Kinetic curve of KBrO3-quinine-H2SO4-thiram system. Experimental conditions: KBrO3, 250 mM (1.0 mL) in 0.5 M H2SO4; thiram, 5.0 ppm (1.0 mL) in

0.1% ethanol containing quinine 1.0 mM in 0.25 M H2SO4; 1100 V PMT voltage and 5 mm/s chart recorder speed.

7.2.2 Chemical and physical parameters optimization The effect of chemical and physical parameters including concentrations of

KBrO3, quinine, and H2SO4 and flow rates, sample loop volume and PMT voltage were studied and optimized. Thiram standard of 1.0 ppm was used for performing all these studies. Potassium bromate was used as an oxidant and its concentration effect, in the range from 0.1 to 500 mM, was investigated. At lower concentration, KBrO3 did not show any CL intensity and as its concentration was increased up to its maximum solubility range i.e., 250 mM that gave maximum CL signals. Further increase in its concentration resulted in the appearance of solid crystals of KBrO3 at the bottom of the reagent bottle and thus CL intensity remained constant as shown in Figure 7.4a.

Therefore, KBrO3 concentration of 250 mM was employed subsequently.

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Quinine acts as a sensitizer in the proposed CL reaction system and its presence in the reaction further enhanced the intensity of CL signal. The effect of quinine concentration in the range from 0 to 2 mM was investigated. The increase in CL intensity was in linear correlation with the increase in quinine concentration up to 1 mM as shown in Figure 7.4b and further increase in quinine concentration resulted in quenching of CL signals intensity possibly because of self absorption. Therefore, quinine concentration of 1 mM was used for further studies.

KBrO3 and quinine sulphate solutions were prepared in aqueous sulfuric acid and the effect of H2SO4 for both streams were optimized by univariate approach. The effect of H2SO4 in the range from 0.1 to 1 M was investigated on the quantitative analysis of thiram. The optimum H2SO4 concentrations of 0.25 and 0.5 M were oberved as shown in Figure 7.4c and d. Therefore, H2SO4 concentrations of 0.25 and

0.5 M were employed for KBrO3 and quinine streams respectively.

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Figure 7.4 Effect of chemical variables on the quantitative analysis of thiram: (a) effect of KBrO3 concentration prepared in 0.1 M H2SO4; (b) effect of quinine concentration prepared in 0.5 M H2SO4; (c) effect of H2SO4 concentration of KBrO3 stream and (d) quinine stream.

The effect of ethanol aqueous solution, used as a sample carrier, was investigated in the range from 0 to 5% (v/v). Upto 0.1% (v/v) of ethanol, a slight enhancement in the intensity of CL signal was observed and further increase in its concentration resulted in a gradual inhibition of CL intensity. Therefore, ethanol solution of 0.1% was employed as carrier stream for subsequent experimental work. To achieve high sensitivity, sample throughput and economy of reagent consumption, various physical parameters such as flow rate, sample loop volume and PMT’s voltage were optimized (Table 7.2). The simultaneous effect of flow rate of three streams was examined from 0.5 to 6.0 mL min–1 on the quantitative analysis of thiram. A linear positive correlation was observed between increase in flow rate and thiram CL signal intensity upto 4.5 mL min–1 and an infinitesimal decline in CL signal intensity was achieved when flow rate was furhter increased. Therefore, a flow rate of 4.5 mL min–1 was employed subsequently. Sensitivity and dynamic linear range both are highly dependent on the sample volume and its effect was investigated from 60 – 360 µL. Maximum CL intensity was observed at sample volume of 240 µL

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and was used subsequently. The effect of PMT voltage from 900 – 1300 V was examined and an optimum CL intensity was observed at 1250 V which was used for further studies. Table 7.2 Optimization of physical parameters on the quantitative estimation of thiram

Parameter Range studies Optimum

Flow rate (mL min–1) 0.5–6 4.5

Sample volume (µL) 60–360 240

PMT voltage (V) 900–1300 1250

7.2.3 Analytical figures of merit Under the optimum chemical and physical parameters, a series of thiram standard solutions were injected into the proposed FI-CL manifold and a linear calibration curve over the range of 0.001 – 20 ppm was obtained (Figure 7.5). The R2, RSD (n = 4) and regression equation were 0.9999 (n = 11), 0.94 – 1.76% and y = 873.93x – 41.066 (y = CL intensity (mV) and x = concentration of thiram (ppm)) respectivly. The limit of detection was based on the concentration giving mean response equal to three times of peak to peak base line noise (S/N = 3), was 6 × 10–4 ppm and the injection throughput was 200 h–1.

Figure 7.5 Calibration curve for thiram under optimum conditions.

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7.2.4 Interfernce study The effect of different cations, anions, organic compounds and some pesticides was checked in the absence of thiram (on the blank) and on the quantitative analysis of thiram. The tolerable level for these chemical species was selected as the concentration which does not enhance or inhibit the CL signal intensity by more than + + 2+ 2+ 5% for thiram 0.025 ppm. No significant effect for 1000 fold Na , K , Ca , Mg , + – – 2– 3– – NH4 , NO3 , Cl , SO4 , PO4 , HCO3 , phenol, humic acid, aldicarb, aldrin, aminocarb, amitraz, asulam, atrazine, bendiocarb, benomyl, carbendazim, carbofuran, diazinon, digoxim, dodemorph, dinoseb, fenarimole, fubridazol, gabapentin, molinate, paraquat, permethrin, propanil, resmethrin, ribavirin, simetryn, terbufos, thiabencarb, thiabendazole, tridemorph and trimethoprim, 500 fold Fe2+, Fe3+, Mn2+ and Cu2+, and 10 fold nabam, antu, malathion, phoxim, maneb, mancozeb and ascorbic acid was found on the quantitative analysis of thiram.

7.2.5 Applications to water samples This method was applied for the quantitative estimation of thiram in natural water samples. The samples were spiked with thiram standard solutions and the results are given in Table 7.3 showing good agreement with the results achieved by a reported HPLC method (Aulakh, et al., 2005) with a recovery of 92 – 110%. Results of the two methods were not significantly different from each other when a paired

Student t-test was applied at a confidence level of 95% (tcalc = 1.61; ttab = 2.3026).

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Table 7.3 Recovery test of thiram in spiked water samples and its comparison with a reported HPLC method (n = 4).

Proposed FI-CL HPLC method Sample Spiked method (Aulakh et al., 2005) matrix (ppm) Found Recovery Found Recovery (ppm) % (ppm) % Tap water 0.05 0.046 92 0.505 101 0.10 0.106 106 0.095 95 0.30 0.295 98.3 0.310 103 Irrigation 0.05 0.055 110 0.054 108 water 0.10 0.099 99 0.109 109 0.30 0.312 104 0.320 107 Lake water 0.05 0.051 102 0.046 92 0.10 0.091 91 0.105 105 0.30 0.290 97 0.312 104

7.2.6 Possible CL reaction mechanism For the elucidation of possible CL reaction mechanism, UV-visible spectrophotometric studies were performed. For this purpose, UV-visible spectra were obtained for thiram (10 ppm in 0.5 M H2SO4) and KBrO3 (0.25 M in 0.25 M H2SO4) at 270 nm and 209 nm respectively. The spectrum for thiram almost disappeared when it was added to KBrO3 solution in aqueous H2SO4 solution. In addition, the color of the mixture started to become dark yellow with time due to the possible formation of bromine molecule and showed maximum absorbance centered at 380 and 222 nm.

Thiram was oxidized with acidic KBrO3 generating bromide ion which in * turn is oxidized with strong sulfuric acid generating excited SO2 (Holleman & Wiberg, 2001). The excited sulfur dioxide transfers its energy to quinine molecule and gets excited. Quinine is a very good fluorescent molecule with an emission maximum at about 450 nm (Lakowicz, 1999). The excited quinine on de-excitation emits intense light centered at about 450 nm which is indirectly related to thiram concentration. The possible CL mechanism can be written as follows;

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ox – Thiram + KBrO3 → thiram + Br H+ + Br– → HBr

2HBr + H2SO4 → Br2 + SO2* + 2H2O

SO2* + Quinine → SO2 + Quinine* Quinine* → Quinine + hv (450 nm)

7.3 CONCLUSION

The proposed FI–CL method based on acidic KBrO3 and quinine CL system was used for the quantitative analysis of thiram in water samples of natural origin with a LOD (S/N = 3) of 6 × 10–4 ppm. No significant interfering activity was observed from the inorganic cationic, anionic and organic chemical species commonly present in natural water samples; therefore, sample preparation does not involve any complex preliminary steps. Results of the two methods were not significantly different from each other when a paired t-test was applied at a confidence level of 95% (tcalc = 1.61; ttab = 2.3026).

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CHAPTER–VIII REVERSE FLOW INJECTION SPECTROPHOTOMETRIC DETERMINATION OF THIRAM AND NABAM FUNGICIDES IN NATURAL WATERS

8 INTRODUCTION Dithiocarbamates (DTCs) fungicides are the most commonly used synthetic and organic foliar fungicides and have activity of wide spectrum. They are employed as foliar sprays for the protection of ornamentals, vegetables, and fruits against variety of fungal diseases and as well as, for the protection of seeds (Tadeo, 2008). Thiram and nabam are DTCs fungicides used to protect seeds, vegetable, fruits, turf crops and ornamentalplants from a variety of fungal diseases (Malik, & Faubel, 1999). Thiram causes several toxic effects, which include skin lesions (Saunders, & Watkins, 2001), hepatic dysfunctions (Edwards, et al., 1991), (Han et al., 2003) and citotoxicity in rat (Cereser, et al., 2001). The European Union set a maximum residue limits for dithiocarbamates (expressed as carbon disulphide) in the range of 2 – 7 mg kg–1 (Ruiz, Lozano, Tomas, & Casajus, 1996). Table 8.1 describes chemical name, structure and physicochemical properties of thiram and nabam fungicides.

Table 8.1. Chemical structures, trivial and IUPAC names and physicochemical properties of thiram and nabam fungicides (Tomlin, 2000).

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Literature reports a variety of analytical techniques for the quantitative analysis of dithiocarbamates fungicides in different samples. These include spectrophotometry / colorimetry (Sharma, Aulakh & Malik, 2005; Filipe, Vidal, Duarte, & Santos, 2007; Rastegarzadeh, & Abdali, 2013; Tunçeli, Bag, & Turker, 2001; Rastegarzadeh, et al., 2013; Malik, Kaul, Lark, & Rao, 1998), fluorimetry (Ruiz et al., 1996; Garcia, & Aguilar, 1993), polarography (Karageogiev, 1981), electrophoresis (Malik, Seidel, & Faubel, 1999), electrochemical [Lin, & Wang, 2004), GC with electron capture, flame photometry and mass spectrometry detectors (de Kok, & van Bodegraven, 2002; Pizzutti, Vareli, Da Silva, & de Kok, 2008; Coldwell, Pengelly, & Rimmer, 2003), HPLC with UV, FL and MS detectors (Gupta, et al., 2012; Aulakh, et al., 2005; Moral, Sicilia, & Rubio, 2009; Brewin, Miller, & Khoshab, 2008), and CL (Waseem, et al., 2009 & 2010). The most commonly used methods are spectrophotometers. They are simple, rapid, accurate, precise and economic as compared with other technique e.g. chromatography and electrophoresis (Rojas, & Ojeda, 2009). Sharma, Aulakh and Malik (2003) reported a comprehensive review on thiram degradation, applications and analytical methods applied for its analysis in commercial formulations, synthetic mixtures in grains, vegetables and fruits. This chapter gives information about a single channel rFI–spectrophotometric method for the quantitative analysis of thiram and nabam in natural water samples. Iron(III) was reduced with thermally degraded products of thiram/nabam in aqueous acidic medium at 60 oC. Potassium ferricyanide reagent was then injected into the incubated reaction mixture stream which resulted in the formation of iron(II)- ferricyanide complex monitored at 790 nm.

8.1 EXPERIMENTAL 8.1.1 Reagents and solutions All plastic and glassware were cleaned in a 2% v/v detergent (Nutrient free, Neutrocon, Decon Laboratories, UK) and after keeping in this detergent overnight, they were thoroughly rinsed with UHP water of conductivity of 0.067 µS cm–1 followd by storing and soaking in 10% v/v HCl for about 24 hours and again rinsed with UHP water several times to ensure complete removal of HCl. All of the reagents

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were of analytical grade, obtained from Merck (Darmstadt, Germany), unless detailed or else, and all solutions were arranged in UHP water. Iron(III) stock solution (0.1 M) was arranged when 4.822 g of

NH4Fe(SO4)2·12H2O was dissolved in HCl solution (0.15 M). A working solution (0.02 M) was arranged when the stock solution’s 20 mL was diluted upto 100 mL with 0.15 M aqueous HCl solution. The 0.1 M stock solution of potassium ferricyanide was arranged when 3.293 g of K3[Fe(CN)6] complex was dissolved in UHP water. A 0.02 M working standard solution of the complex was arranged when 20 mL of the stock solution of the complex was diluted upto 100 mL with 0.15 M aqueous HCl solution. 100 ppm stock solutions of thiram and nabam fungicides were arranged when 10 mg of each fungicide compounds in powdered form were dissolved in 100 mL of absolute ethanol (HPLC-grade) in glass bottels of brown color and stored in dark at −4 °C. Working standard solutions of these fungicides were arranged when the required aliquots from fungicide stock solutions were diluted on daily basis in

NH4Fe(SO4)2·12H2O solution (0.02 M) containing HCl (0.15 M). The stock solutions of 100 ppm of different pesticides including antu, asulam, diazinon, malathion, maneb, phoxim, terbofos, thiabendazole and thiobencarb were arranged for checking their interferent activities when 10 mg of each compound was dissolved in 100 mL of absolute ethanol (HPLC grade). Cation and anion stock + + 2+ 2+ 2+ 2+ – 2– solutions (250 ppm) including Na , K , Ca , Mg , Co , Mn , NO3 , SO4 and 3– PO4 ions were prepared in UHP water and ensuing standard arrangements of each were set up by serial diluting of stock solutions with HCl (0.15 M).

8.1.2 FI manifold and procedure The rFIA manifold used in this work is revealed in Figure 8.1. A peristaltic pump was used to propel the incubated mixture (ammonium iron(III) sulfate (0.02 M) in HCl (0.15 M) containing thiram and nabam standards, temperature 60 oC) at a flow –1 rate of 2.8 mL min . A rotary injection valve was used to inject K3[Fe(CN)6] solution (0.02 M, 60 µL) in to the incubated mixture stream. The stream was then passed through a reaction coil (20 cm) forming a soluble prussian blue complex that was monitored at 790 nm with a spectrophotometer (Jenway 6505, UK) equipped with a 1 cm path length glass flow through cell (80 µL, Hellma Analytics, Germany). The

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detector output was noted by means of a flatbed strip chart recorder. All manifold tubing was connected using PTFE tubing (0.8 mm i.d.).

Figure 8.1 Reverse FI manifold for the quantitative analysis of thiram and nabam. R1 = Ammonium iron(III) sulfate solution (0.02 M) in HCl (0.15 M) containing thiram o and or nabam standard, temperature 60 C; R2 = K3[Fe(CN)6] solution (0.02 M, 60 µL); PP = peristaltic pump (flow rate 2.8 mL min–1); RC = reaction coil (0.8 × 200 mm).

8.2 RESULTS AND DISCUSSION 8.2.1 Chemical and physical parameters optimization The optimum experimental conditions were established for quantitative analysis of thiram and nabam fungicides. The key variables on the peak absorbance were examined using one step approach. These were Fe(III), HCl, K3[Fe(CN)6], ethanol and various surfactants concentrations (Figure 8.2), flow rates, sample volume, coil length and reaction mixture temperature (Table 8.2). Thiram and nabam working standard solutions having strength each of 0.25 ppm were employed for the performance of these studies and each result was the mean of three measurements. The effect of Fe(III) concentration in the incubation mixture (thiram and or nabam standard solution (0.25 ppm) in ethanol (1% v/v) containing HCl (0.15 M) and temperature 60 oC) was examined over the range of 0.001 – 0.05 M and the maximum absorbance was achieved at 0.02 M (Figure 8.2a) and therefore was selected as an optimum for further studies. The pH is a key factor for the hydrolysis of

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dithiocarbamates and releasing of CS2 and the optimum HCl concentration for hydrolysis was 0.15 M (Figure 8.2b), and at higher acid concentrations, the absorbance decreased. K3[Fe(CN)6] solution (prepared in optimized HCl solution (0.15 M)) was injected via rotary injection valve into the incubated mixture steam for iron(II)-ferricyanide complex formation. The effect of K3[Fe(CN)6] concentration was examined over the range of 0.001 – 0.05 M. Maximum peak height absorbance was achieved at 0.02 M (Figure 8.2c) and therefore was selected as an optimum for subsequent studies. The effect of ethanol (0.1 – 15%, v/v) and various surfactants including CTAB, SDS, Brij-35 and Trion X-100 (0.001 – 1.0%, v/v or w/v) was examined on the quantitative analysis of thiram and nabam (results not shown). These reagents were added separately in the incubation mixture (NH4Fe(SO4)2·12H2O (0.02 M) in HCl solution (0.15 M), temperature 60 oC) and no increase in peak height absorbance was observed and therefore the use of ethanol and surfactants was abandoned subsequently.

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Figure 8.2 Variation of absorbance with; (a) ammonium iron(III) sulfate; (b) HCl and

(c) K3[Fe(CN)6] concentrations.

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The influence of physical parameters, i.e., flow rate, mixing coil length, reagent injection volume and temperature of reaction mixture were then investigated (Table 8.2). A flow rate of 2.8 mL min–1 gave maximum peak height absorbance with a steady baseline and reproducible signals and was therefore used for further studies.

A reagent ((NH4Fe(SO4)2·12H2O (0.02 M) in HCl solution (0.15 M)) volume of 60 L, a reaction coil length of 20 cm and wavelength of 790 nm gave almost maximum absorbance signals and were therefore used for all further studies. The effect of temperature was examined over the range of 20 – 80 oC by immersing the reaction mixture (NH4Fe(SO4)2·12H2O (0.02 M) in HCl solution (0.15 M) and analyte standard solution (0.25 ppm thiram and or nabam in ethanol (1% v/v)) in a circulating water bath (Type JB1, Grant Instruments, Cambridge, UK). The optimum peak height absorbance was achieved at 60 oC, above which non-reproducible signals and decrease in peak absorbance were observed probably due to the evaporation of reaction mixture.

8.2.2 Analytical figures of merit Under the optimized experimental conditions described above, calibration graphs for thiram and nabam were linear over the range of 0.01 – 8 ppm and 0.1 – 30 ppm with regression equations y = 0.2415x – 0.0013 (R2 = 0.9999, n = 8), and y = 0.0205x – 0.0018 (R2 = 0.9982, n = 10), [y = absorbance and x = concentration in ppm) after subtraction of the blank value and the RSDs (n = 3) was 0.8 – 1.6% over the range studied as shown in Figure 8.3A and B, respectively. The LODs (3σ blank) for thiram and nabam were 0.01 and 0.05 ppm respectively with a sample throughput of 60 h–1.

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Figure 8.3 Calibration curves for (A) thiram and (B) nabam under optimum conditions.

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Table 8.2 Checking the effect of different physical parameters for the quantitative analysis of 0.25 ppm (n = 4) of thiram and nabam each via the FIA– spectrophotometric manifold. The optimized variable was employed for the optimization of next parameters and at the optimum condition of a variable, peak absorbance was observed for other variables for thiram and nabam fungicides.

Variables Range studied Optimum value

Flow rate (mL min–1) 0.5–4.8 2.8

Reagent volume (µL) 30–300 60

Reaction coil (cm) 0–200 20

Reaction mixture temperature (oC) 20–80 60

8.2.3 Interference study The effect of some possible inorganic ions and pesticides such as antu, asulam, diazinon, malathion, maneb, phoxim, terbufos, thiabendazole and thiobencarb, at concentration level of 0.5 ppm (5 fold) and 1.0 ppm (10 fold) was examined on the blank and on the quantitative analysis of thiram and or nabam (0.1 ppm). The tolerable concentration of interference was defined as that giving a relative bias of ≤ ±5%. All the above-mentioned pesticides did not interfere on the blank and on the quantitative analysis of thiram and or nabam except antu >5 fold had enhancive effect on the peak absorbance. There was no effect from cations (Na+, K+, Ca2+, Mg2+, Mn2+ 2+ 3– 2– – and Co ) and anions (PO , SO4 and NO3 ) at 75 and 100-fold on the determination. Therefore, the developed method may find application in real samples containing the above foreign species.

8.2.4 Application to natural water samples The proposed method based on SPE and iron(III)-ferricyanide reaction was applied for analyzing thiram and nabam in tap, lake and irrigation water samples collected from various locations of Quetta valley in 10% HCl washed bottles made of high density polyethylene and filtered through cellulose made filter membrane (47 mm diameter, Whatman, pore size 450 µm, Maidstone, UK) for the removal of suspended particles. A series of thiram and nabam concentration each of the strength

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over the range of 5.0 – 3.0 ppm were spiked in these water samples for the performance of recovery experiments and extracted with a solid phase extraction (SPE) technique employing disposable Sep-Pak C18 cartridges (Water Associates, USA). Table 8.3 presents the recoveries results, which were in the range of 93  3 – 105  2% and 87  5 – 102  2% for thiram and nabam respectively and were in good agreement with a HPLC method (Aulakh et al., 2005). The conditioning of the cartridges was performed by percolating 5.0 mL of deionized water and 5.0 mL of absolute ethanol and dried by passing air for about 2 minutes (Aydin, Wichmann, & Bahadir, 2004). At a flow rate of 10 mL min–1, the spiked water sample was passed through the preconditioned SPE cartridge followed by washing with an aliquot of 0.5 mL deionized water and dried by passing air through it for 10 minutes. The retained thiram and nabam from SPE cartridge were eluted with 5.0 mL ethanol and dried under a stream of nitrogen. Re-dissolution of the dried thiram and nabam residue in the incubation mixture and was analyzed using the proposed rFIA-spectrophotometric manifold (Figure 8.1). The analysis of thiram and nabam was also carried out on LC–10AT liquid chromatograph (Shimadzu, Corporation, Tokyo, Japan) equipped with double pumps, Rheodyne injector with 20 L loop and an SPD–10A UV–Vis detector based on the work reported (Aulakh et al., 2005). An analytical column bonda pack C18 (250 × 4.6 mm i.d., 10 m) was used for analysis at 45 oC. Acetonitrile–water (70:30, v/v), at a –1 flow rate of 0.7 mL min was used as the mobile phase and the analytes were monitored at 254 nm. The retention time for thiram and nabam was found 2.6 and 6.1 min respectively.

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Table 8.3. Results of recovery tests of thiram and nabam in natural water samples (n = 4).

Recovery (%)

Spiked HPLC method Sample matrix Proposed method (ppm) (Aulakh, et al., 2005)

Thiram Nabam Thiram Nabam

Tap water 0.50 985 92±4 1005 96±4

1.00 943 102±3 987 98±3

1.50 962 101±2 924 94±5

Irrigation water 1.00 1052 99±3 1024 102±3

2.00 954 102±3 936 95±6

3.00 975 100±2 1052 97±4

Lake water 1.00 967 88±5 984 93±5

2.00 1025 90±4 1003 95±6

3.00 933 87±5 983 92±3

8.3 CONCLUSIONS The reported rFI-spectrophotometric method for the quantitative analysis of thiram and nabam in natural water samples is simple with low LODs (0.01 and 0.05 ppm respectively). The method has a high sample throughput (60 h–1) with RSDs (n = 3) of 0.8 – 1.6% in the range studied. The method was applied to natural water samples and the recoveries were in good agreement with a HPLC method. The summary of the comparison of the previously reported methods for the quantitative analysis of thiram with the present approach has been shown in Table 8.4. The proposed method is better in terms of LODs, good precision (RSD 1.6%) and high sample throughput all compare favorably with previously reported spectrophotometric methods.

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Table 8.4. Summary of the proposed approach with previously reported methods for the quantitative analysis of thiram fungicide.

Linear range LOD RSD Technique Matrix R2 value Ref. (ppm) (ppm) (%)

Spectrophotometry Commercial samples 0–24 0.3 0.9754 1.9 (Sharma et al., 2005)

Spectrophotometry Aqueous solution 0.5–2.5 0.33 NR NR (Filipe et al., 2007)

Colorimetry Natural waters and 0.048–2.404 0.041 0.9990 3.7 (Rastegarzadeh, plant seeds & Abdali, 2013)

Spectrophotometry River water 0–20 0.161 NR NR (Tunçeli et al., 2001)

Spectrophotometry Natural waters and 0.025–1.0 0.0115 0.9985 1.1–2.7 (Rastegarzadeh plant seeds et al., 2013)

Spectrophotometry Grains and vegetables 0.44–13.25 0.0147 NG 0.86 (Malik et al., 1998) rFI– Natural waters 0.01–8.0 thiram 0.01 0.9999 0.8–1.6 This work Spectrophotometry 0.1–30 nabam 0.05 0.9985 NR. not reported

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CHAPTER–IX DEVELOPMENT OF FLOW INJECTION SPECTROPHOTOMETRIC METHOD FOR ALPHA- NAPTHYLTHIOUREA USING SODIUM NITRITE AND SULPHANILIC ACID DIAZOTIZATION REACTION

9 INTRODUCTION 1-Napthylthiourea (ANTU) is a rodenticide that belongs to the class of organosulfur pesticide and it is in a form of white crystalline powder (Mackison, Stricoff, & Partridge, 1980; O'Neil, Smith, Heckelman, & Budavari, 2001; NIOSH, 2001; Richter, 1945). It is used as 10% active baits or 20% tracking powder (IARC, 1983). It is specifically used to control adult rattus norveqicus and 6 – 8 mg/kg is sufficient as toxic doses; however, other species of rattus are less toxic to it. The repetitive administration of rates with its sub-lethal doses caused them to develop a tolerance to it (Worthing, 1983). In general, domestic animals are safe from its toxic effects but the induction of vomiting in dogs and production of pleural effusion and pulmonary oedema in other experimental animals have been reported (Budavari, 1989). Human exposure to antu could be through ingestion, inhalation, or skin contact with the recommended occupational airborne exposure over an average of a ten-hour work shift is 0.3 mg/m3, while an exposure of 100 mg/m3 has been reported to be dangerous, both for human health and life (NIOSH, 2001). A 4000 mg antu/kg human body weight has been assigned as a lethal dose (Hazardous substance fact sheet alpha-naphthylthiourea, 2001). It does not degrade in the presence of sunlight and air. 1-Naphthylamine can cause coughing, breath shortness or fluid build-up in the lungs. It is the main degraded product of antu and considered to be a first-class carcinogen which can easily enter the fluvial systems in different ways (Hazardous substance fact sheet alpha-naphthylthiourea, 2001; Mar, Martinez, de Alba, Duran, & Martin, 2006). The chemical structure of antu is shown in Figure 9.1.

Figure 9.1 Chemical structure of antu (α-naphthylthiourea).

Various analytical procedures have been employed for the quantitative analysis of antu in food, biological and environmental samples. These include

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colorimetry (Bremanis, & Bergner, 1950; Vachek, 1960), fluorimetry (Sanchez, & Gallardo, 1993), voltammetry (Smyth, & Osteryoung, 1977; Stara, & Kopanica, 1984), LC based on different detectors (Diaz, Acedo, de La Pena, Pena, & Salinas, 1994; Wang, & Budde, 2001; Maizels, & Budde, 2001; Miles, & Moye, 1988) and stop FI-CL (Murillo, et al., 2005). A well-established technique known as FIA is employed for the analysis of diverse analytes in environmental, clinical, food and other areas that has the advantages such as high injections throughput, low sample and reagent consumption, high versatility and robustness (Ruzicka, & Hansen, 1988). This chapter reportes a simple FI–spectrophotometric method for the quantitative analysis and analysis of antu in water samples of natural origin. The approach was based on the conversion of nitrite into nitrous acid, which subsequently diazotized sulfanilic acid for the generation of diazonium salt. This salt was coupled with 1-naphthylamine which was the hydrolyzed product of antu under alkaline conditions to form 4-(sulphophenylazo)-1-naphthylamine (Mar, et al., 2006; Marczenko, 1976). The azo dye formed was monitored at 495 nm. The interfering effects of different pesticides, organic compounds, cations and anions were investigated. Antu from water samples was extracted using a solid phase extraction technique, analyzed by the proposed method and validated by the HPLC method.

9.1 EXPERIMENTAL 9.1.1 Reagents and solutions All chemicals used were of analytical reagent grade, provided by Merck (Darmstadt, Germany) and BDH Chemicals Ltd. (Poole, UK). Glassware and bottles were pre-cleaned in 10% HCl for three days, thoroughly rinsed with deionized water and stored in zip–locked plastic bags. A 0.1 M stock solution of sulphanilic acid was freshly prepared by dissolving 4.33 g in deionized water, sonicated for 5 min, diluted to 250 mL with deionized water and stored in a dark brown bottle. A 0.25 M stock solution of sodium nitrite was prepared by dissolving 4.3 g in deionized water and the volume was made up to 250 mL with deionized water. A 2.0 M stock solution of NaOH was prepared by dissolving 8.0 g in deionized water and the volume was made up to 100 mL with deionized water. Working standard solutions were prepared from these stock solutions in deionized water when required. Commercially available

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absolute ethanol was used for making different ethanol working standard solutions (% v/v) with deionized water. For the preparation of 250 ppm stock solutions of antu, asulam, diazinon, malathion, maneb, nabam, phoxim, terbufos, thiabendazole and thiram (Dr. Ehrenstorfer, Augsburg, Germany), the pre-weighed amounts of these pesticides were dissolved in absolute ethanol. All these solutions were sonicated and stored in dark brown bottles at 4 oC when not in use. These solutions were diluted serially to obtain lower concentrations using 0.25% (v/v) ethanol solution containing 75 mM NaOH. For the preparation of 2000 ppm stock solutions of cations such as Na+, K+, 2+ 2+ 2+ 3+ 2+ 2+ – – 2– 3– Mg , Ca , Fe , Fe , Mn and Co and anions such as Cl , NO3 , SO4 and PO4 , the required quantity of related salts were dissolved in deionized water and working standard solutions were prepared in 0.25% ethanol solution containing 75 mM NaOH.

9.1.2 FI manifold and procedure The proposed three channel FI manifold is shown in Figure 9.2. All the streams were propelled using a 4-channeled peristaltic pump at a flow rate of 0.5 mL min–1. The standards and samples of antu (300 µL) were injected via a rotary injection valve into the carrier stream of 0.25% (v/v) ethanol solution containing 75 mM NaOH. This resulted to form 1-naphthaylamine. For diazonium salt formation, the streams of 100 mM sulphanilic acid and 50 mM sodium nitrite were merged at a T- piece and then passed through a 200-cm reaction coil-I and thermostated at 30 oC. This stream was then merged with the carrier stream containing 1-naphthyleamine at a confluence point and passed through 150-cm reaction coil-II which was also thermostated at 30 oC. This resulted in the formation of 4-(sulphophenylazo)-1- naphthylamine formation, which was measured at 495 nm using a UV/Vis- spectrophotometer (Model 6505, Jenway Ltd., Feisted, Dunmow, Essex, UK), equipped with a glass flow through cell (path length 1 cm, volume 80 µL, Hellma Analytics, Germany). The output was recorded on a chart recorder. PTFE tubes (0.8 mm i.d.) were used to join all connections of FIA manifold.

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Figure 9.2 Optimized of FI-spectrophotometric manifold for the quantitative analysis of antu. Carrier, 0.25% (v/v) ethanol solution containing 75 mM NaOH; R1, 50 mM sodium nitrite solution; R2, 100 mM sulphanilic acid solution; PP, peristaltic pump; IV, injection valve; RC-I, reaction coil-I, 200 cm; RC-II, reaction coil-II, 150 cm; CR, chart recorder.

9.1.3 Collection of water samples Natural water samples such as tap, irrigation, and lake water from Quetta valley, were collected in 10% HCl washed high density polyethylene bottles and stored in the dark at 4 oC. For the removal of suspended solids, water samples were filtered through a cellulose membrane filter (Whatman, Madistone, UK) with the specifications: diameter 47 mm, pore size 0.45 mm and stored at 4 oC and analysed when required. The water samples were spiked with antu in the range from 0.5 – 3.0 ppm and extracted with SPE using disposable Sep-Pak C18 cartridges (Water Associates, USA). The cartridge was first conditioned with 5.0 mL deionized water, 5.0 mL ethanol and then dried by passing air through it for two minutes. Water sample was passed at a flow rate of 10 mL min–1 through the conditioned SPE cartridge, washed with 0.5 mL deionized water and dried by passing air through it for 10 min. Antu from the SPE cartridge was eluted with 5.0 mL ethanol and dried under a nitrogen stream. The dried antu residues were re-dissolved in 0.25% ethanol containing 75 mM NaOH and 0.1% acetic acid-acetonitrile (55:45, v/v) and both were analyzed by the

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proposed method and a previously reported HPLC method (Diaz, et al., 1994) respectively.

9.1.4 HPLC method The antu standards and samples were analyzed by the HPLC method, reported previously (Diaz, et al., 1994). In brief, a liquid chromatograph (Model LC-10AT, Shimadzu, Corporation, Tokyo, Japan), equipped with a UV-Vis detector (SPD-10A), set at 220 nm was employed. The samples were introduced through a six-port injection valve (Model 7725i, Rheodyne) with a sample loop volume of 10 µL. The chromatographic analysis was performed on an analytical column (bonda pack C18: 250 × 4.6 mm i.d., 10 µm) using 0.1% acetic acid - acetonitrile (55:45, v/v) as a mobile phase at a flow rate of 2 mL min–1. Under these conditions, the retention time for antu achieved was 2.1 min while a linear calibration graph was obtained by injecting the analyte standard solutions ranged from 0.1 – 5.0 ppm. The residues of analyte after SPE were dissolved in mobile phase and analyzed by the method.

9.2 RESULTS AND DISCUSSION 9.2.1 Optimization of physical and chemical parameters Various analytical parameters were optimized to enhance the peak height absorbance and sensitivity, to increase the linear dynamic range, sample throughput and to decrease the reagents consumption. All these studies were performed with 10 ppm antu standard solution. The spectrum of the final product of the reaction scheme (Scheme III) under the optimum conditions was scanned from 200 – 600 nm. The maximum absorption wavelength at 495 nm was obtained and was selected as an optimum wavelength for subsequent experiments in the flow mode. The effect of sulphanilic acid concentration in the range 0.5 – 200 mM was examined. At 0.5 mM concentration, no absorbance was observed; however, when the sulphanilic acid concentration was increased, the absorbance increased linearly. The maximum absorbance for sulphanilic acid concentration was observed at 100 mM as shown in Figure 9.3A. Beyond this concentration, the absorbance remained constant and bubbles were observed in reaction coil-II due to the possible degradation of the diazonium ion generating nitrogen gas. The maximum solubility of sulphanilic acid at

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30 oC is reported as 85 mM (Yalkowski, He, & Jain, 2010). Therefore, the optimum sulphanilic acid concentration of 100 mM was selected and used for subsequent studies. Sodium nitrite was used to generate nitrous acid in acidic medium and the effect of its concentration in the range 1 – 150 mM was examined. Maximum peak height absorbance was obtained at 50 mM as shown in Figure 9.3B. Further increase in the sodium nitrite concetration showed no appreciable increase in the absorbance and therefore, sodium nitrite conecntartion of 50 mM was selected and used for further studies. NaOH was used to hydrolyze antu into 1-naphthyleamine (Sanchez, & Gallardo, 1993) and as a reaction medium for the proposed chemistry as well. The effect of NaOH concentration in the range 1 – 150 mM was examined. Maximum peak height absorbance was observed at 75 mM, above which, the absorbance was decreased as shown in Figure 9.3C. Therefore, NaOH concentration of 75 mM with a waste of pH 10 was chosen for further studies. The solubility of antu in water is about 600 ppm, while in organic solvents, it has high solubility (e.g., 24300 and 86000 ppm in acetone and triethyleneglycol respectively) (O'Neil, et al., 2001). Hence, the effect of ethanol in the range 0 – 1% (v/v) was examined as a sample carrier stream. The absorbance was increased with an increase in ethanol concentration upto 0.25% above which the peak height absorbance was decreased as shown in Figure 9.3D. Therefore, ethanol concetration of 0.25% was selected and used as a sample carrier stream.

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Figure 9.3 Optimization of reagents concentrations. (A) Sulphanilic acid, (B) sodium nitrite, (C) NaOH and (D) ethanol (% v/v). Experimental conditions: flow rate 0.5 mL min–1, sample loop volume 300 µL, reaction coil-I length 200 cm, reaction coil-II length 150 cm, wavelength 495 nm, temperature 30 oC, and antu concentration 10 ppm.

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To achieve high sensitivity, linear calibration range and economy of reagents consumption, the effect of reagent streams flow rates, sample loop volume, reaction coils lenghts and the temperature was examined for the quantitative analysis of antu (Table 9.1). The effect of flow rates for each of the three streams in the range 0.2 – 1.2 mL min–1 was simultaneously examined. Maximum absorbance was observed at 0.5 mL min–1 with a steady baseline and reproducible peak height and therefore the flow rate of 0.5 mL min–1 was employed subsequently. The sample volume in the range 60 – 360 µL was examined and the absorbance increased with increase in sample volume up to 300 µL and therefore, was selected and used subsequently. The effect of reaction coil-I and coil-II lengths in the range 0 – 300 cm was examined on the diazotization process and formation of azo dye respectively. The maximum peak height absorbance was observed when the reaction coils lengths of 200 and 150 cm were used and a further increase in coils lengths did not show any appreciable increase in the absorbance. Therefore, the reaction coil-I and coil-II lengths of 200 and 150 cm were chosen and used respectively for further studies. The effect of reaction coils temperature in the range 10 – 50 oC was also examined. The maximum absorbance was obtained at 30 oC, above which appearance of bubbles in the reaction coils was observed probably due to possible dissociation of diazotized sulphanilic acid resulting in nitrogen gas. Therefore, the reaction coils temperature of 30 oC was fixed and employed for subsequent studies.

Table 9.1 Physical variables effect on the quantitative analysis of antu (n = 4).

Physical variables Range studied Optimum

Flow rate (mL min–1) 0.2–1.2 0.5

Sample injection volume (µL) 60–360 300

Reaction coil-I length (cm) 0–300 200

Reaction coil-II length (cm) 0–300 150

Reaction coil-I and II temperature (oC) 10–50 30

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9.2.2 Analytical figures of merit Under the optimum conditions described above, the calibration graph of absorbance versus antu concentrations was linear in the range 0.05 – 120 ppm. The R2, RSD (n = 3) and regression equation over the range studied were 0.9995 (n = 7), 1.8 – 3.6% and y = 0.031x – 0.018, (where y = absorbance and x = concentration in ppm), respectively. The LOD, based on the concentration response equal to three times of the peak to peak base line noise (S/N = 3) was 0.01 ppm. The injection throughput was 45 h–1. Table 9.2 reports the comparison of the analytical attributes of the proposed method with other reported methods used for the quantitative analysis of antu. Figure 9.4 illustrates the traces of chart recorder of basorbance intensity signals at 495 nm over the antu concentration range of 5 – 120 ppm and calibration curve in the inset for the same series of standards with the coefficient of quantitative analysis and regression equation.

Table 9.2 Summary of the comparison of the analytical attributes of the proposed analytical approach with the previously reported approaches for the analysis of antu.

Linear range LOD Technique Sample matrix R2 Ref. (ppm) (ppm)

FLa Wheat grain 0.05–1.00 0.01 0.999 (Sanchez, & Gallardo, 1993)

DPPb Urine 0.2–10 0.04 – (Smyth, & Osteryoung, 1977)

ASVc Cattle feed and urine Up to 0.81 8×10–5 0.9980 (Stara, & Kopanica, 1984)

HPLC-DAD River water – 1.2×10–3 0.9978 (Diaz, et al., 1994)

HPLC-MS Water 2×10–4–2.5×10–2 9.57×10–4 – (Wang, & Budde, 2001) sFI-CLd Water, wheat, barley 0.05–3 0.005 0.998 (Murillo Pulgarín, et and oat grain al., 2005)

FI-Spece Water 0.05–120 0.01 0.9995 This work aFlourimetry. bDifferential pulse polarography. cAdsorptive stripping voltammetry. dStop flow injection-chemiluminescence. eFlow injection-spectrophotometry.

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Figure 9.4 Traces of chart recorder of absorbance for antu different standards

9.2.3 Intrference study The influence of different cations, anions, organic compounds and some pesticides with and without antu was carried out. The tolerable level of these chemical species was selected as the concentration which did not enhance or inhibit the peak height absorbance for antu at 0.1 ppm by more than 5%. No significant effect of Na+, + 2+ 2+ 2+ 3+ 2+ 3– 2– – – K , Ca , Mg , Mn , Fe , Co , PO , SO4 , Cl and NO3 1000-fold, phenol and humic acid 10-fold, asulam, diazinon, malathion, maneb, nabam, phoxim, terbufos, thiabendazole and thiram 50-fold was observed. Therefore, the method could be easily applied for antu analysis in real samples in the presence of the above chemical species.

9.2.4 Application to water samples The present analytical approach was successfully applied for the quantitative analysis of antu in natural water samples. Table 9.3 reports the results of water

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samples spiked with antu standards which were in good agreement with the reported HPLC method (Diaz, et al., 1994) with recovery from 93 ± 1.9 to 110 ± 3.0%. There was no significant difference between the variances and mean results of the two

methods at 95% confidence level by applying the F- and T-tests (tcalc..= 0.884, ttab., v = 8,

p=0.05 = 2.31, Fcalc.= 1.262, Ftab. v1 = 8, v2 = 8, p=0.05 = 3.44).

Table 9.3 Percent recovery of antu in spiked natural water samples (n = 4).

Proposed Method HPLC method (Diaz et al., 1994). Spiked, Sample matrix Found Recovered Found Recovered (ppm) (ppm) % (ppm) %

Tap water 0.50 0.490.012 982.5 0.5050.011 101±2.2

1.00 0.930.018 931.9 1.020.032 102±3.1

1.50 1.440.040 962.8 1.480.027 99±1.8

Irrigation water 1.00 1.030.022 1032.1 1.100.033 110±3.0

2.00 2.090.050 1052.4 1.920.063 96±3.3

3.00 3.120.097 1043.1 2.810.048 94±1.7

Lake water 1.00 0.970.032 973.3 1.060.021 106±2.0

2.00 2.070.066 103.53.2 1.960.031 98±1.6

3.00 3.210.051 1071.6 3.130.10 104±3.2

tcalc..= 0.884, ttab., v = 8, p=0.05 = 2.31, Fcalc.= 1.262, Ftab. v1 = 8, v2 = 8, p=0.05 = 3.44

9.2.5 Possible reaction scheme In alkaline medium, antu was hydrolyzed and formed 1-naphthylamine (Scheme I) (Sanchez, & Gallardo, 1993). Nitrite was converted into nitrous acid in the acidic medium; subsequent diazotization of sulphanilic acid and formation of a diazonium salt (Scheme II) were achieved and finally this salt was coupled with 1- naphthylamine in an alkaline medium to form sodium 4-(sulphophenylazo)-1- naphthylamine salt (Scheme III) (Mar, et al., 2006; Hassan, Marzouk, & Sayour, 2003). The resulting azo dye showed maximum absorption wavelength at 495 nm. The possible reaction scheme is given below.

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S

HN NH2 NH 2 S OH H2O HO NH2 I

ANTU 1-Naphthylamine

O3SHC6H4NH2 II HO3S NH2 NO2 2H HO3S N N 2H2O

Sulphanilic acid Diazonium salt

NaOH III HO3S N N NH2 NaO3S N N NH2 H2O

Diazonium salt 1-Naphthylamine Sod. 4-(Sulphophenylazo)-1-naphthylamine (SSPAN)

9.3 CONCLUSIONS A simple rFI-spectrophotometric method was developed, enabling the quantitation of antu with low LOD (0.01 ppm) and good sample throughput (45 h–1). Anions and cations, and a number of pesticides had no influence on the quantitation of antu. The developed procedure was successfully applied for the quantitative analysis of antu in spiked natural water samples using SPE technique with recovery from 93 ± 1.9 to 110 ± 3.0%. There was no significant difference between the proposed rFI-spectrophotometric and HPLC method at 95% confidence interval by applying the tests of significance.

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CHAPTER–X GENERAL CONCLUSION AND FUTURE TRENDS

10 GENERAL CONCLUSIONS AND FUTRURE TRENDS 10.1 GENERAL CONCLUSION In the proposed study, seven FI-CL and FI-UV-Vis spectrophotometric methods were developed for the quantitative analysis of eight pesticides in water samples. The interfering effect of other pesticides belong to the other classes of pesticides, common organic and inorganic species present in fresh water samples were checked on the quantitative analysis of these pesticides. Generally, most of the established methods were free from the interfering effect of other chemical species except a few one. Those methods could be made selective for the quantitative analysis of respective pesticides by applying various separation, extraction and chemical techniques. The methods were able to determine low concentration of pesticides in water samples due to the inherent sensitivity of CL. The LOD can be extended further to lower concentration of pesticides (lower than 0.1 ppb) by applying SPE pre- concentration technique, as tried for the quantitative analysis of CYR. Thus, the sensitivity of the established FI-CL methods is comparable to GC or LC coupled with MS or tandem MS. Both, SPE cartridges and DLLME extraction technique, were successfully applied for the extraction of pesticides from water samples. The established methods were found to be very fast, as injection throughput per hour reached up to 200 for the quantitative analysis of thiram using acidic KBrO3 and quinine CL reaction. Transition metals in uncommon oxidation states such as Ag3+ and Cu3+ were successfully synthesized, characterized and coupled with CL reagents such as luminol 2+ and Ru(bipy)3 or used without any CL reagent in FI mode for the quantitative analysis of pesticides. The interfering effect of Cl– on the quantitative analysis of

CYR using DPA-H2SO4 CL reaction was eliminated by employing an inline amberlite column. As well as, interfering effect of metals such as Fe2+ and Fe3+ on the quantitative analysis of Mn2+ and its fungicides using lucigenin CL reaction was removed by employing an inline immobilized 8-HQ column. Quantitative analysis of 2+ thiram and aminocarb by Ru(bipy)3 –DPA CL reaction could be made selective by employing a non-ionic surfactant su ch as Triton X-100. A novel, simple, fast, sensitive and selective FI-CL method was established for the quantitative analysis of TBZ in natural water samples.

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The pesticides such as thiram and nabam were thermally degraded into CS2, which subsequently reduced Fe3+ into Fe2+ and made a Prussian blue colored and water soluble complex when reacted with ferricyanide. Thus, a sensitive and fast rFI- spectrophotometric method was established for their quantitative analysis in natural water samples. A rodenticide such as antu was hydrolysed in alkaline medium, subsequently reacted with diazotized sulfanilic acid and made a red colored dye. As a result, fast and sensitive FI-spectrophotometric method was established for the quantitative analysis of antu in natural water samples. All these methods were highly reproducible as the RSDs were lower than 5%. The variation and the mean results of the proposed methods were compared with the established methods by applying F and paired student T-tests and there were no significant differences at 95% confidence level. For the elucidation of possible CL reaction mechanisms, UV-Vis spectrophotometric and fluorospectrophotometric studies were performed. Based on the results of these studies and previous studies performed by various researchers, the possible CL reaction schemes were proposed. Kinetic studies were performed in batch and flow modes, which showed that the proposed CL reaction were fast and efficient for the quantitative analysis of pesticides in water samples. The comparison of the analytical figures of merit of the proposed methods with already established methods have been presented in the respective chapters.

10.2 FUTURE TRENDS As for 1997, over 900 pesticides were enlisted for registration in the United States for utilization in more than 20,000 diverse pesticide products. Every year new pesticides are introduced—for example, regularly new active ingredients (10 to 20) were registered every year from 1967 to 1997. Once the pesticides or their degraded products discharged into the environment, they might move through the hydrological framework and will reach to streams and ground water. When their concentration in water reaches to toxic level, then affect humans, oceanic life, or wildlife. The analysis of such a large number of pesticides at trace level needs selective, sensitive, economical and fast methods. FI-CL fulfills all these criteria, thus novel FI-CL methods must be developed for the quantitative analysis of pesticides in water samples, as well as in environmental, food and biological samples. Keeping in view

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the flexibility of CL reaction in batch or flow modes, a number of methods have been developed by coupling CL reaction with more selective analytical techniques (HPLC, capillary electrophoresis and immunoassays) for the quantitative analysis of pesticides in diverse samples. Thus, CL reactions have great future for the analyses of pesticides in water as well as other samples. New oxidants such as transition metals in uncommon oxidation states (Au, Ni, Co, Pt etc) can be synthesized and used for the CL quantitative analysis of pesticides. The number of CL reagents is very limited, novel CL reagents can be explored or synthesized to extend the applications of CL reactions. Solid phase micro-extraction such as a fiber coated with liquid polymer or solid sorbent and liquid phase micro-extraction such as single drop micro extraction can be used for the extraction of pesticides from samples and analyzed by FI-CL technique. As Pakistan is an agricultural country, selective FI-CL or FI-UV-Vis spectrophotometric methods can be used for the quantitative analysis of pesticides in various segments of environment and other samples.

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LIST OF PUBLICATIONS

PhD

1. Muhammad Asghar, Mohammad Yaqoob, Naheed Haque and Abdul Nabi, Determination of thiram and aminocarb pesticides in natural water samples using flow-injection with tris(2,2’-bipyridyl)ruthenium(II)- diperiodatoargentate chemiluminescence detection, Analytical Sciences, 29/11 (2013) 1061–1066. 2. Muhammad Asghar, Mohammad Yaqoob, and Abdul Nabi, Reverse flow injection spectrophotometric determination of thiram and nabam fungicides in natural water samples, Journal of Chemical Society of Pakistan, 36/3 (2014) 556-560. 3. Muhammad Yaqoob, Mohammad Asghar and Abdul Nabi, Determination of manganese- and manganese containing fungicides with lucigenin-Tween-20 enhanced chemiluminescence detection, Luminescence, 30/7 (2015) 950–961. 4. Muhammad Asghar, Mohammad Yaqoob, Nusrat Munawar and Abdul Nabi, Flow injection determination of thiabendazole fungicide in water samples using diperiodatecuprate(III)-sulfuric acid-chemiluminescence system, Analytical Sciences, 32/3 (2016) 337–342. 5. Muhammad Asghar, Mohammad Yaqoob, and Abdul Nabi, Flow injection determination of cyromazine in natural water samples using diperiodatoargentate(III)-sulfuric acid-chemilumiescence system, International Journal of Environmental and Analytical Chemistry, 97/3 (2017) 276–288. 6. Muhammad Asghar, Mohammad Yaqoob, and Abdul Nabi, Chemiluminescent determination of cyromazine in milk samples using Triton X-100-copper(III) chelate by flow injection analysis, Chemical Research in Chinese Universities, 33/3 (2017) 354–359. 7. Muhammad Asghar, Mohammad Yaqoob, and Abdul Nabi, Potassium bromate-quinine chemiluminescence detection of thiram in water samples using flow injection analysis, Journal of Analytical Chemistry, 74(4) (2019) 323–329. 8. Muhammad Asghar, Mohammad Yaqoob, Sami Ullah, Samar Ali, Nusrat Munawar, and Abdul Nabi, Development of flow injection spectrophotometric method for 1-napthylthiourea using sodium nitrite and sulphanilic acid diazotization reaction, Journal of Spectroscopy, Volume 2018, Article ID 3920289, 7 pages, https://doi.org/10.1155/2018/3920289. 9. Muhammad Asghar, Mohammad Yaqoob, and Abdul Nabi, Quantitative Analysis of Thiram in Water Samples Using Flow Injection Analysis Coupled with Diperiodatonickelate(IV)-Quinine CL System, Journal of Toxicological Analysis, 2018, Accepted 10. Muhammad Asghar, Mohammad Yaqoob, and Abdul Nabi, (2019). Carbofuran: analytical methods and its determination in natural water samples by flow injection DPA (III)–H2SO4 chemiluminescence. International Journal of Environmental Analytical Chemistry, 99(7), 692-705

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M.Phil

11. Muhammad Asgher, Mohammad Yaqoob, Amir Waseem and Abdul Nabi, Flow-injection determination of retinol and -tocopherol using surfactant- enhanced lucigenin chemiluminescence, Luminescence 26/6 (2011) 416-423. 12. Muhammad Asgher, Amir Waseem, Mohammad Yaqoob, and Abdul Nabi, Flow-injection chemiluminescence determination of retinol and -tocopherol in blood serum and pharmaceuticals, Analytical Letters, 44/1-3 (2011) 12–24. 13. Muhammed Asgher, Mohammad Yaqoob, Amir Waseem, Abdul Nabi, Ghulam Murtaza and Faridullah, Flow injection photo-sensitized chemiluminescence of luminol with Cu(II)-rose bengal: Mechanistic approach and vitamin A and C determination, International Journal of Analytical Chemistry, Volume 2014 (2014), Article ID 109592, 6 pages: http://dx.doi.org/10.1155/2014/109592.

Others

14. Lubna Rishi, Muhammad Asgher, Mohammad Yaqoob, Amir Waseem and Abdul Nabi, Enzymatic determination of vitamin A in pharmaceutical formulations with spectrophotometric detection, Spectrochimica Acta, Part A: 72/5 (2009) 989–993. 15. Lubna Rishi, Mohammad Yaqoob, Muhammad Asghar, S. Haider Shah and Abdul Nabi, Determination of vitamin A in pharmaceuticals and infant milk powder using flow injection cerium(IV)–sodium sulfite chemiluminescence, Analytical Letters, 45/14 (2012) 2037–2052. 16. Lubna Rishi, Mohammad Yaqoob, Muhammad Asgher, Abdul Nabi and Nusrat Munawar, Flow injection determination of lactate using immobilized lactate dehydrogenase enzyme with tris(2,2′-bipyridyl)ruthenium(III) chemiluminescence detection, Analytical Letters, 49/5 (2016) 654–664. 17. Manzoor Ahmed, Muhammad Asghar, Mohammad Yaqoob, Samar Ali, Nurat Bibi and Abdul Nabi, Determination of hyoscine butylbromide in pharmaceuticals using Ce(IV)-Na2SO3-CL system with flow injection analysis, Journal of Analytical Chemistry, 2018, Vol. 73, No. 11, pp. 1098– 1104. 18. Manzoor Ahmed, Muhammed Asghar, Mohammad Yaqoob, Nusrat Munawar, Farkhanda Shahid and Abdul Nabi, Flow injection- chemiluminescence method for determination of hyoscine butylbromide using silver(III) as oxidizing agent, Analytical Sciences, 33 (2017) 1259–1263. 19. Zahoor Ahmad, Mohammad Yaqoob, Muhammad Asghar, Abdul Kabir Khan Achakzai, and Abdul Nabi, Flow injection determination of cysteine and glutathione based on lucigenin-Cu(III) complex chemiluminescence detection, Journal of Analytical Chemistry, (2017) under review. 20. Fatima Khan, Mohammad Yaqoob, Muhammad Asghar, Iqbal, S., Samar Ali, Amir Waseem and Abdul Nabi. Surfactant enhanced flow injection

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chemiluminescence method for vitamin D3 determination in pharmaceutical formulations. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 208 (2019), 150-156 21. Zahoor Ahmad, Muhammad Asghar, Samar Ali, Nusrat Munawar, Abdul Nabi and Mohammad Yaqoob, Flow injection determination of asparagine and histidine using diperiodatocuperate based on spectrophotometric inhibition detection in amino acid supplements, Indo American Journal of Pharmaceutical Sciences (IAJPS), 4(11), (2017) 4112–4120. 22. Habibullah Khan Achakzai, Muhammad Younas Khan Barozai, Abdul Kabir Khan Achakzai, Muhammad Asghar and Mohammad Din, Profiling of 21 Novel Microrna Clusters and their Targets in an Important Grain: Wheat (Triticum aestivum L.), Pakistan Journal of Botany, 51(1) (2019), 133-142. 23. Habibullah Khan Achakzai, Barozai, M. Y. K., Baloch, I. A., Achakzai, A. K. K., Din, M., and Muhammad Asghar, The identification of eighteen precursor mirna clusters and their targets in barley (Hordeum vulgare L.). Pakistan Journal of Botany, 51(2), (2019), 469-477. 24. Muhammad Shoaib Khan, Muhammad Asghar, and Mohammad Yaqoob. Lansoprazole Determination in Pharmaceutical Formulations by Flow Injection Coupled with Acidic KMnO4–Quinine Chemiluminescence System. Analytical Sciences, (2019), 19P039. 25. Muhammad, A., Attiq-Ur-Rehman, N. K., Muhammad Asghar, Baqi, A., & Mohammad Hussain, H., Spectrophotometric determination of phenolic antioxidants in four varieties of apples (Pyrus malus) from Balochistan, Pakistan. Pure and Applied Biology (PAB), 8(1), (2019), 768-779.

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