behaviour of chlorpropham and its main metabolite 3-chloroaniline in soil and water systems

CIPC removal from potato washing plant effluent PWPE was studied to determine the rate of CIPC degradation.. CIPC removal from synthetic potato wash water SPWW was studied to determine t

BEHAVIOUR OF CHLORPROPHAM AND ITS MAIN METABOLITE 3-CHLOROANILINE IN SOIL AND

WATER SYSTEMS

BANDAR RASHED M ALSEHLI BSc., King Abdulaziz University, Saudi Arabia, 2004

MSc., Loughborough University, United Kingdom, 2009

Thesis submitted for the degree of Doctor of Philosophy

February 2014

University of Glasgow College of Science and Engineering

Department of Chemistry Environmental, Agricultural and Analytical Chemistry Section

© ALSEHLI, B R M (2014)

Abstract

Chlorpropham, also known as isopropyl-N-(3-chlorophenyl) carbamate or CIPC is

a sprout suppressant and plant growth regulator of the chemical class derived from carbamic acid (NH2COOH) The substance was first developed as a pre-emergence herbicide, and it was quickly identified as a useful potato sprout suppressant for long-term tuber storage (Marth & Schultz 1952) Today CIPC is the major sprout inhibitor used in the potato industry (UK Potato Council 2013c)

As a consequence there is environmental concern about CIPC reaching the aquatic environment from potato washing plants

An RP-HPLC method for the analysis of CIPC and IPC in methanol solvent with an automatic integration method was developed and validated The correlation coefficients for CIPC and IPC regression lines at all calibration levels (0.001–100 mg/L) were (R2 >0.999) while IPC exhibited a slightly less linear calibration curve (R2 >0.98) at the lowest concentration range of (0.001–0.1 mg/L) An acceptable precision of 10% based on 10 injections was obtained at the limit of quantification of 0.001 mg/L for both analytes The 3CA was excluded at this stage as it overlapped with an extra peak which required extensive investigations The identification led to the conclusion that the artefact peak was a methanol-oxygen peak and elimination of the methanol-oxygen peak was not possible The evaluation of five different columns and conditions in separating the methanol-oxygen peak from 3CA in a mixture containing 3CA, IPC and CIPC was studied For the four peaks, the best separation at low eluant concentration was obtained at 55% methanol, but the run time was considerable

In contrast, the best separation at high eluant concentration was obtained at 75% methanol; however, the methanol-oxygen peak was still incompletely separated from the IPC peak due to the high size of the methanol-oxygen peak Further investigations were conducted to reduce the size of the methanol-oxygen peak by changing the mobile phase pH which had no effect Changing detection wavelength from 210 – 260 nm reduced the peak size, but considerable loss in sensitivity was observed Five different instruments were tried and at the end the Thermo HPLC system was chosen because it provided a smaller methanol-oxygen peak along with temperature control to enhance the methanol-

oxygen and 3CA peak separation at 60% methanol eluant, but the run time was still very long Therefore, to enable a compromise between baseline peak resolutions as well as high-throughput separations; two separate methods for 3CA and CIPC, including IPC were developed and validated The precision for both analytes at two levels of 0.01 and 1.0 mg/L based on 10 injections was ≤ 1%, the calibration curves at all levels were (R2 >0.999) and the limit of quantification was 0.001 mg/L Preparation of CIPC, IPC and 3CA standards in water from stock solutions in methanol and directly by dissolution in water was investigated The peak areas were not affected even at 0% methanol concentration and the peak shapes were sharper than that in methanol without affecting the peak area This validated the use of water as sample solvent to carry out the analysis by HPLC

To successfully prepare CIPC, IPC and 3CA in 100% water, it was necessary to develop methods for preparation and handling aqueous solution of CIPC, IPC and 3CA The solubility of CIPC and IPC were studied Both CIPC and IPC have low solubility in water while 3CA has higher solubility and dissolved quite rapidly The solubility time curve for CIPC showed a gradual concentration increase from initial time until day 3 stirring but after that the solubility was consistent and values of 106, 89 and 61 mg/L CIPC were obtained at 25°C, 22°C and 4°C respectively IPC exhibited similar solubility behaviour and the corresponding values were found to be 222, 200 and 140 mg/L at same temperatures respectively The solubility results agreed with the literature values Stock solutions and standards in aqueous solution were found to be stable on storage

at 4°C (refrigerator) and ~20°C (lab temperature) for up to 90 days For this work it was necessary to investigate possible CIPC, IPC and 3CA adsorption from aqueous solutions by glassware and filters All plastic glassware were avoided as they have measurable adsorption (20-40%) for the analytes, except high clarity polypropylene In contrast, glass materials particularly borosilicate and soda glass provided nearly zero adsorption for all three analytes Although it was possible to identify suitable glassware that did not adsorb CIPC, IPC and 3CA it was necessary to discard the first 25 mL of filtrate to overcome adsorption onto filters (Cellulose, Glass microfiber, PTFE and Nylon) The Glass microfiber, type GF/B filter, has a pore size of 1.0 µm and is often used as a prefilter However,

the 25 mL discarding from filtrate was suitable only for filtering sample larger than 25 mL For small scale filtration, a much smaller 0.2 µm PTFE filter in a 17

mm chemically resistant polypropylene housing disk attached to 3 mL BD syringe was used and only 1.5 mL of the sample was required to saturate the filter

A liquid-liquid extraction method with vortex mixer (LLE-Vortex) was successfully developed and validated for the extraction of CIPC and 3CA from dilute soil–water suspensions (0.001 g/mL) with a high recovery 98%–100% and RSD% less than 1.34% In addition, the method was reliable for extraction from high soil suspensions formed with 0.02 g/mL of soil and for 0.1 g/mL of soils with low adsorption capacity The average precision of extracting CIPC at 0.02 g/mL and 0.1 g/mL soil content was 1.6% and 3.2% while more precise extraction observed for 3CA of about 0.91% and 1.86%, respectively However, the extraction method did not work for soil suspension with the highest organic matter content and concentration equal or more than 0.1 g/mL

Investigations were carried out to examine the adsorption- desorption behaviour

of CIPC and 3CA from aqueous solutions onto different clay and sandy air dried soils The suitable contact time of two days using 1 g material size was determined At all temperatures, CIPC and 3CA were strongly adsorbed in clay soils while only slightly adsorbed in sandy soils A paired t-test was used to compare between the adsorption at 5°C and 30°C for CIPC and 3CA and concluded that there was a statistically significant difference between the two temperatures for both analytes (p-value < 0.05) The effect of pH was also studied and it was found that the soil pH had a negligible impact on the adsorption of CIPC, while for 3CA the adsorption at low and high pH was significant (p-value <0.05) The data was fitted to a Langmuir isotherm (R2=0.91-0.98) and adsorption maxima calculated The maximum adsorption capacities for CIPC in Downholland 1A, Downholland 2A, Midelney 2A, Midelney 1A, Midelney 1B, Dreghorn 1A, Dreghorn 1B, Quivox A and Quivox B were 1583, 668, 714, 927,

215, 325, 243, 355 and 194 µg/g respectively and for 3CA were 1024, 1104, 550,

651, 292, 278, 317, 239 and 162 µg/g respectively The main determining factor was soil organic matter Desorption for CIPC and 3CA from soils increased with reducing both carbon and LOI percentage In addition, investigations were extended to study the adsorption of CIPC and 3CA in oven dried plant waste

materials The data was also fitted to a Langmuir isotherm (R2=0.96-1.00) and adsorption maxima calculated The maximum adsorption capacities for CIPC in mixed bark, B&Q garden peat, Miracle-Gro compost, Pine needles, Scots pine bark and Birch bark were 3090, 2968, 2973, 3636, 3004 and 2581 µg/g respectively and for 3CA were 2914, 2724, 2953, 2787, 2358 and 2568 µg/g respectively

The removal of chlorpropham from two river water types was studied in laboratory incubation experiments at two temperatures and different treatments

of carbon, nitrogen, phosphorus, Fulvic acid and soil extracts The percentage of

a 10 mg/L addition of CIPC degraded over 40 days at 20°C in both River Kelvin water and Glazert Water water was less than 2% in all the treatments Increasing the river water incubation temperature to 30°C resulted in a slight increase in the degradation rate after 40 days No 3CA intermediate from the 10 mg/L CIPC spike was detected in any of the treatments of both the rivers

CIPC removal from potato washing plant effluent (PWPE) was studied to determine the rate of CIPC degradation CIPC completely removed after the first day with no detectable 3CA formation A second incubation experiment for PWPE removal was repeated after four months storage of the effluent at 4°C The result of CIPC removal showed a small initial drop about 14% within one day which might be interpreted as adsorption followed by a steady line with no further change in the concentration during the 22 days of incubation It is suggested that the cold storage killed off the bacteria and reduced the decomposition process Thus, having established that the microbial degradation was the predominant process with the fresh PWPE, the degradation kinetic order needs to be determined The analysis of degradation kinetics shows that the process corresponds to a first order model (R2=0.99) and the degradation rate was calculated to be 2.0 days−1 The half-life was 0.36 day

CIPC removal from synthetic potato wash water (SPWW) was studied to determine the rate of CIPC degradation CIPC completely disappeared after 1.2 days with no detectable 3CA formation An identical incubation experiment for SPWW was repeated after four months for potato tubers stored at 4°C The slowing of degradation might be explained by stressing of the potato surface‘s

bacteria due to the change from cold storage to 20°C causing one population to die and another to develop Thus having established that the microbial degradation was the predominant process with the fresh SPWW, the degradation kinetic order needs to be determined The analysis of degradation kinetics shows that the process corresponds to a zero order model (R2=0.98) and the degradation rate was calculated to be 7.3 mg/L/day

CIPC removal from suspensions of potato materials can be summarised as follows: CIPC adsorption process of potato materials lasts 1 day; it continues on the secondary adsorbent (starch) accompanied by slow microbial degradation and gradual microbial population growth Finally, microbial degradation finishes the process with a sharp decrease of CIPC concentration The 3CA intermediate from CIPC spike was undetected

The clarified synthetic potato washing water experiment supported the argument that the aim of excluding adsorption from the system worked and only the decomposition process was observed The 1 h sedimentation is sufficient to achieve removal of adsorption surfaces and the longer sedimentation time results in losses of decomposed microorganisms

Overall, the removal results suggested that there are two separate populations i.e CIPC decomposers and 3CA decomposers CIPC decomposing microorganisms and 3CA decomposing microorganisms are present in the effluent from the potato washing plant and on the surfaces or the soils of CIPC treated potatoes but not in the river water samples The numbers of CIPC and 3CA decomposing organisms decline on storage of the potatoes and the effluent at 4°C In addition, CIPC decomposition is inhibited by the addition of nutrients However, these removal studies were based on filtration Thus, to enable the amount adsorbed in soil suspensions to be measured and the microbial degradation rate

to be accurately evaluated, the application of (LLE-Vortex) for the simultaneous extraction of CIPC and 3CA from soil-water system was necessary

The microbial degradation of 10 mg/L CIPC and 10 mg/L 3CA at 20°C in the freshly prepared SPWW was simultaneously measured by PTFE filtration and LLE-Vortex methods to compare the methods The 3CA intermediate as a result of

CIPC degradation was also included The results showed that the degradation curves were similar for both analytical methods as the soil coating the potato tubers was very sandy and when the washes were generated in 2 L flasks and diluted, the content of soil in the suspension became negligible The microbial degradation of CIPC in SPWW was linear from start to the end with zero order degradation rate of 2.11 mg/L/day 3CA intermediate reached a maximum of 1.5 mg/L after day 1, and then degraded The 10 mg/L 3CA degradation was curved thus initial and final straight lines were fitted and the zero order degradation rates were found to be 0.74 and 2.82 mg/L/day, respectively The degradation for all was observed to be complete in less than one week

The incubation experiment at 20°C was repeated with the addition of 100 mg/L CNP nutrients from glucose, ammonium sulphate and monopotassium phosphate

to the spiked SPWW The addition of CNP nutrients suppressed 10 mg/L CIPC degradation and slightly delayed 10 mg/L 3CA degradation The 3CA intermediate was not detected The CIPC degradation rate calculation was impractical as 8 mg/L CIPC still remained in the suspension after 26 days and it was time dependent The degradation rate of 10 mg/L 3CA after the two days lag period was fitted and the zero order degradation rate of 3.36 mg/L/day was determined Degradation was observed to be complete for 10 mg/L 3CA sample

in less than one week which was similar to the unfortified finishing time

The SPWW suspension was incubated at four different temperatures of 5°C, 10°C, 15°C and 20°C to study the impact of these temperatures on the degradation rate The degradation of 10 mg/L CIPC increased with temperature with no lag phases; straight lines were plotted and the zero-order degradation rates were calculated as 0.52, 1.21, 1.83 and 2.13 mg/L/day at 5°C, 10°C, 15°C and 20°C respectively Analysis of 3CA intermediate formation shows that CIPC samples incubated at different temperatures demonstrated different 3CA formation trends and some of them reached 3.5 mg/L In contrast, the initial degradation rates of 10 mg/L 3CA at 5°C and 10°C could not be detected and the final rates were linear At 15°C and 20°C the graph was curved, forming an inconsistent trend between the initial and final stages Thus, at 5°C and 10°C the final rates were 0.28 and 0.53 mg/L/day respectively At 15°C and 20°C the initial rates were 0.35 and 0.71 mg/L/day, while final rates were 3.82 and 3.52

mg/L/day respectively Incubation of SPWW at different temperatures provided

an activation energy value of 63 kJ/mol for CIPC while the activation energy for 3CA based on initial and final rates were 99 and 130 kJ/mol, respectively

Fresh soils that had no history of CIPC application contained CIPC and 3CA degraders but they took 1–3 weeks to start The degradation was linear and zero order degradation rates were calculated for CIPC (4.20, 2.11, 2.62 mg/L/day) and 3CA (1.51, 2.62, 1.92 mg/L/day) in Darvel, Cottenham and Dreghorn 2A, respectively

Drying the soils killed bacteria but the suspension still contained small numbers capable of degrading CIPC and 3CA after a long incubation period

Table of Contents

Abstract 2

Acknowledgement 21

Author’s Declaration 22

List of Abbreviations 23

Chapter 1 - Main Introduction 25

1.1 Background related to the use of sprout suppressants in the potato industry 25

1.1.1 Current state of UK potato market 25

1.2 Potato storage 28

1.2.1 Chemical-free storage 28

1.2.2 Use of sprout suppressant chemicals 29

1.2.3 Toxicity of CIPC and its metabolites to humans 32

1.3 The environmental fates of CIPC and its metabolite 3CA 36

1.3.1 Environmental toxicity 36

1.3.2 Properties 37

1.3.3 Environmental fate of CIPC and 3CA 41

1.4 Analysis of CIPC and its metabolites in environmental samples 49

1.4.1 Extraction methods 49

1.4.2 Instrumental analysis 58

1.4.3 HPLC analysis 58

1.5 Validation of analytical method 75

1.5.1 Calibration linearity 77

1.5.2 Accuracy 79

1.5.3 Precision 80

1.5.4 Limit of detection and limit of quantification 80

1.6 Thesis objectives 84

Chapter 2 - RP-HPLC method development for chlorpropham, propham and 3-chloroaniline in methanol 86

2.1 Introduction 86

2.2 Materials and methods 89

2.2.1 Analysis of CIPC and IPC in RP-HPLC Shimadzu system A 89

2.2.2 Separation of the methanol-oxygen peak from 3CA 91

2.2.3 Effect of detection wavelength 92

2.2.4 Effect of different HPLC systems on the appearance of the methanol-oxygen peak 92

2.2.5 Effect of temperature control on the separation of the four peaks at 60% methanol in water 93

2.3 Results and discussion 94

2.3.1 Analysis of CIPC and IPC in RP-HPLC Shimadzu system A 94

2.3.2 Summary of CIPC and IPC analysis conditions by RP-HPLC Shimadzu system A 96

2.3.3 Identification of the unknown peak 100

2.3.4 Separation of the methanol-oxygen peak from 3CA 106

2.3.5 Effect of detection wavelength 110

2.3.6 Effect of different HPLC systems on the appearance of the methanol-oxygen peak 113

2.3.7 Effect of temperature control on the separation of the four peaks at 60% methanol in water 117

2.3.8 3CA method 120

2.3.9 CIPC method 122

2.4 Conclusion 125

Chapter 3 - Development of methods for preparation and handling aqueous solutions of CIPC, IPC and 3CA 126

3.1 Introduction 126

3.2 Materials and methods 129

3.2.1 CIPC, IPC and 3CA peak area comparisons 129

3.2.2 CIPC, IPC and 3CA standard preparation in 100% deionised water 131

3.2.3 CIPC, IPC and 3CA adsorption by labware 132

3.2.4 RP- HPLC measurement 134

3.3 Results and discussion 135

3.3.1 CIPC, IPC and 3CA peak area comparisons 135

3.3.2 CIPC, IPC and 3CA standard preparation in 100% deionised water 141

3.3.3 CIPC, IPC and 3CA adsorption by labware 147

3.4 Conclusion 168

Chapter 4 – Adsorption of CIPC and 3CA in soils and their economical removal by plant waste materials 169

4.1 Introduction 169

4.1.1 Importance 169

4.1.2 Adsorption in soil 171

4.1.3 Adsorption in plant and other waste materials 173

4.1.4 Objective 178

4.2 Materials and methods 178

4.2.1 Adsorption and desorption of CIPC and 3CA in soil samples 178

4.2.2 Removal of CIPC and 3CA by plant and other waste materials 183

4.2.3 Calculation 185

4.2.4 HPLC determination 186

4.2.5 XLfit ® software 186

4.3 Results and discussion 187

4.3.1 Chromatograms of CIPC and 3CA in soils and waste materials 187

4.3.2 Adsorption and desorption of CIPC and 3CA in soil samples 192

4.3.3 Removal of CIPC and 3CA by plant and other waste materials 212

4.4 Conclusion 226

Chapter 5 – Removal of chlorpropham from river and waste water 227

5.1 Introduction 227

5.2 Materials and methods 230

5.2.1 HPLC chromatographic method 230

5.2.2 Removal of CIPC from river water 230

5.2.3 Removal of CIPC from potato washing plant effluent 237

5.2.4 Removal of CIPC from synthetic potato washing water 237

5.2.5 Removal of CIPC from potato materials suspension 238

5.2.6 Removal of CIPC from clarified synthetic potato wash water 238

5.2.7 CIPC measurement in free solids samples 240

5.2.8 CIPC measurement in solids-water samples 240

5.3 Results and discussion 241

5.3.1 Representative chromatograms of CIPC in different environmental compartments 241

5.3.2 Removal of CIPC from river water 248

5.3.3 Removal of CIPC from potato washing plant effluent 254

5.3.4 Removal of CIPC from synthetic potato washing water 257

5.3.5 Removal of CIPC from potato materials suspension 261

5.3.6 Removal of CIPC from clarified synthetic potato wash water 264

5.4 Conclusion 269

Chapter 6 - Solvent extraction method development and application to degradation of CIPC and 3CA 270

6.1 Introduction 270

6.1.1 Overview of liquid extraction methods for pesticides recovery 270

6.1.2 EPA methods for CIPC and other pesticides 271

6.1.3 Extraction improvement techniques 271

6.1.4 Choice of a solvent for LLE 272

6.1.5 Simultaneous separation of several compounds 273

6.1.6 Modifications of LLE extraction for the present study 273

6.1.7 LLE method for CIPC and 3CA simultaneous extraction 274

6.2 Materials and methods 275

6.2.1 HPLC chromatographic method 275

6.2.2 Materials 275

6.2.3 Extraction of CIPC and 3CA from deionised water by immiscible solvents using separatory funnels 276

6.2.4 Extraction of CIPC and 3CA from deionised water by DCM using a LLE-VORTEX method 277

6.2.5 Validation of the optimum LLE-VORTEX method in waste water 278

6.2.6 Application of LLE-Vortex method to microbial degradation of CIPC and 3CA in synthetic potato wash water and soil suspensions 280

6.3 Results and discussion 282

6.3.1 Extraction of CIPC and 3CA from deionised water by immiscible solvents using separatory funnels 282

6.3.2 Extraction of CIPC and 3CA from deionised water by DCM using a LLE-VORTEX method 286

6.3.3 Summary of the optimum LLE-VORTEX method for simultaneous extraction of CIPC and 3CA from deionised water 288

6.3.4 Validation of the optimum LLE-VORTEX method in waste water 289

6.3.5 Summary on method development 298

6.3.6 Application of LLE-Vortex method to microbial degradation of CIPC and 3CA in synthetic potato wash water and soil suspensions 299

6.4 Conclusion 325

Chapter 7 – General discussion 326

7.1 Summary of experiments 326

7.2 Implications of the work 335

7.2.1 CIPC application 335

7.2.2 Storage 337

7.2.3 Washing water 337

7.2.4 Recommendations for future research 344

7.2.5 Recommendations for the potato processing industry 348

References 352

Publications 374

List of Tables

Table 1.1 - The detected residues in potato samples in the period 2000-2012 Adapted from

Pesticide Residues in Food, PRiF (2013) 35

Table 1.2 - Chlorpropham identification, physico-chemical and environmental properties 38

Table 1.3 - Propham identification, physico-chemical and environmental properties 39

Table 1.4 - 3-chloroaniline identification, physico-chemical and environmental properties 40

Table 1.5 - Sample preparation techniques Adapted from Beyer & Biziuk (2008) 50

Table 1.6 - Assessment of the linearity of a HPLC method 78

Table 2.1 - Five different columns used in developing and separating the four peaks of 3CA, methanol-oxygen peak, IPC and CIPC 91

Table 2.2 - LOD and LOQ based on 10 injections from three concentration levels 100

Table 2.3 - LOD and LOQ based on the lowest calibration curve (0.001 – 0.1 mg/L) 100

Table 2.4 - Peak areas of the unknown peak at different injection treatments using the Shimadzu A system with degassed 72% methanol eluant, 210 nm, 1 mL/min flow rate, Nemesis column at lab temperature, approx 20°C The injection solvent was a subsample from the mobile phase 103

Table 2.5 - HPLC Detectors accuracy and Bandwidth 116

Table 2.6 - System precision based on 10 injections from 0.01 and 1.0 mg/L 3CA in methanol 120 Table 2.7 - LOD and LOQ based on 10 injections from 0.01 and 1.0 mg/L 3CA in methanol 122

Table 2.8 - System precision based on 10 injections from 0.01 and 1.0 mg/L CIPC in methanol 123 Table 2.9 - LOD and LOQ based on 10 injections from 0.01 and 1.0 mg/L CIPC in methanol 124

Table 3.1 - Filter papers description 133

Table 3.2 - Solubility of CIPC in deionised water based on the mean of days 4-16 142

Table 3.3 - Solubility of IPC in deionised water based on the mean of days 4-16 144

Table 3.4 - Stability of stock and standard solutions at 4°C and 20°C 146

Table 3.5 - Recovery of 1.00 and 0.05 mg/L CIPC standards in deionised water from different containers 149

Table 3.6 - Recovery of 1.00 and 0.05 mg/L IPC standards in deionised water from different containers 150

Table 3.7 - Recovery of 1.00 and 0.05 mg/L 3CA standards in deionised water from different containers 151

Table 3.8 - Recovery of 1.00 and 0.05 mg/L CIPC standards in deionised water from different stoppers 152

Table 3.9 - Recovery of 1.00 and 0.05 mg/L IPC standards in deionised water from different stoppers 153

Table 3.10 - Recovery of 1.00 and 0.05 mg/L 3CA standards in deionised water from different stoppers 154

Table 3.11 - Recovery of 1.00 and 0.05 mg/L CIPC standards in deionised water from different syringes 155

Table 3.12 - Recovery of 1.00 and 0.05 mg/L IPC standards in deionised water from different syringes 155

Table 3.13 - Recovery of 1.00 and 0.05 mg/L 3CA standards in deionised water from different

syringes 155

Table 4.1 - Soil characteristics (Khan 1987; Bakhsh 1988; Mazumder 1992; Amin 1995) 180

Table 4.2 - Soil characteristics (Khan 1987; Bakhsh 1988; Mazumder 1992; Amin 1995) 181

Table 4.3 - Literature values for proximate composition of plant materials used in the study 184

Table 4.4 - Description of composts used in the study 184

Table 4.5 - Clay materials and descriptions 184

Table 4.6 - The maximum adsorption capacity (Xmax) of CIPC and 3CA in soils 204

Table 4.7 - Percentage of CIPC and 3CA desorption from clay and sandy soils 210

Table 4.8 - The maximum adsorption capacity (Xmax) of CIPC and 3CA in waste materials 222

Table 5.1 - Nutrients properties 232

Table 5.2 - Different concentration of clarified supernatant 239

Table 5.3 - The acidity of the potato washing plant effluent 255

Table 5.4 - Kinetic analysis of the removal process from the fresh PWPE suspension 255

Table 5.5 - The acidity of synthetic potato washing water 258

Table 5.6 - Kinetics analysis of the removal process from fresh SPWW suspension 259

Table 5.7- Characterization of CIPC degradation at various potato materials 262

Table 6.1 - Properties of organic immiscible solvents 275

Table 6.2 - Characteristics of two rotary evaporators 276

Table 6.3 - Soil characteristics (Khan 1987; Mazumder 1992; Amin 1995) 279

Table 6.4 - Extraction with ethyl acetate at different pH and in presence of sodium chloride 285

Table 6.5 - Extraction by dichloromethane from deionised water 286

Table 6.6 - Primarily extraction of CIPC and 3CA from deionised water by LLE-VORTEX 287

Table 6.7 - Optimum extraction of CIPC and 3CA from deionised water by LLE-VORTEX 287

Table 6.8 - Validation of CIPC and 3CA extraction from dilute waste water 296

Table 6.9 - Zero-order rate constants of 10 mg/L CIPC in unfortified SPWW at 20°C 302

Table 6.10 - Degradation rate constant of 10 mg/L 3CA in unfortified SPWW at 20°C 303

Table 6.11 - Degradation rate constant of 10 mg/L 3CA in fortified SPWW at 20°C 306

Table 6.12 - Degradation rate constant of 10 mg/L CIPC at different temperatures 309

Table 6.13 - Degradation rate constant of 10 mg/L 3CA at different temperatures 310

Table 6.14 - pH values of the incubated SPWW at different temperatures 312

Table 6.15 - Degradation rate constants of 10 mg/L CIPC in soil suspensions 320

Table 6.16 - Degradation rate constants of 10 mg/L 3CA in soil suspensions 321

Table 6.17 - The pH changes in the incubated three soil suspensions 321

Lists of Figures

Figure 1.1 - Consumption of carbohydrate meal occasions Data adapted from UK Potato

Council (2013a) 25 Figure 1.2 - Consumption of carbohydrate meal occasions Data adapted from UK Potato

Council (2013a) 26 Figure 1.3 - Potato production flowchart for Great Britain for June 2011 to May 2012 Taken

from Potato Council website (UK Potato Council 2013b) 27 Figure 1.4 - CIPC fate in environment Adapted from Führ (1991) 41 Figure 1.5 - CIPC degradation (blue line) and microbial population (red line) profiles for

primary (a) and secondary (b) metabolism (Linde 1994) 48 Figure 1.6 - HPLC scheme (Snyder, Kirkland & Dolan 2010) 59 Figure 1.7 - (a) Peak asymmetry and tailing factors definitions (A S and TF); (b) peak shape of

(AS and TF); (c) peak tailing effect on separation; (d) fronting; (e) overloaded tailing (Snyder, Kirkland & Dolan 2010) 66 Figure 1.8 - Influence of k, N and α on resolution (Ahuja 2003) 68 Figure 1.9 - Van Deemter curve presenting the relationship between HETP and average

linear velocity The Vopt = optimum velocity; Hmin = minimum plate height

(Dong 2006) 70 Figure 1.10 - HPLC chromatogram for pesticides analysis (Environmental Protection

Agency 2006b) Column: Ascentis column C18, 250 mm x 4.6 mm, 5 μm

Mobile phase: (%A) water, (%B) acetonitrile Column temperature: 30 °C UV detection: 210 nm Flow rate: 1.0 mL/min Samples were prepared in 16%

acetonitrile in water with an injection volume of 10 μL 74 Figure 1.11 - HPLC chromatogram for CIPC (David et al 1998) 74 Figure 1.12 - Calibration line of peak area versus concentration of an analyte 79

Figure 2.1 - Analysis of 3CA, IPC and CIPC using the Shimadzu A system with degassed 72%

methanol eluant, 210 nm, 1 mL/min flow rate, Nemesis column at lab

temperature, approx 20°C The standard was a 1 mg/L 3CA, IPC and

CIPC in methanol Peaks 1, 2,3 and 4 were 3CA, unknown peak, IPC and CIPC, respectively 94 Figure 2.2 - Analysis of IPC and CIPC using the Shimadzu A system with degassed 68%

methanol eluant, 210 nm, 1 mL/min flow rate, Nemesis column at lab

temperature, approx 20°C The standard was a 1 mg/L IPC and CIPC in

methanol Peaks 2, 3 and 4 were unknown peak, IPC and CIPC, respectively 95 Figure 2.3 - Shimadzu LC-Solution software integration method 97 Figure 2.4 - System precision at different concentration ranges for the analysis of IPC

and CIPC 98 Figure 2.5 - Calibration curve for the high range (10 – 100 mg/L) of CIPC and IPC in

methanol 99 Figure 2.6 - Calibration curve for the medium range (1 – 10 mg/L) of CIPC and IPC in

methanol 99 Figure 2.7 - Calibration curve for the low range (0.001 – 0.1 mg/L) of CIPC and IPC in

methanol 99 Figure 2.8 - Analysis of 3CA, IPC and CIPC using the Shimadzu A system with degassed 60%

acetonitrile eluant, 210 nm, 1.5 mL/min flow rate, Nemesis column at lab

temperature, approx 20°C The standard was a 1 mg/L 3CA, IPC and CIPC in acetonitrile Peaks 1, 3 and 4 were 3CA, IPC and CIPC, respectively 102

Figure 2.9 - Peak areas of the unknown peak in 15 different methanol batches using the

Shimadzu A system with degassed 72% methanol eluant, 210 nm, 1 mL/min

flow rate, Nemesis column at lab temperature, approx 20°C The injection

solvent was a subsample from the mobile phase 102

Figure 2.10 - Using the Shimadzu A system with 72% methanol eluant, 210 nm, 1 mL/min flow rate, Nemesis column at lab temperature, approx 20°C A blank from a mobile phase was 1) Inject without treatment, 2) Purged with air and 3) Degassed with Helium, using a) Degassed and b) Non-degassed mobile phase at 210 nm 104

Figure 2.11 - The effect of 72%-55% methanol eluant strengths on Nemesis column, using the Shimadzu A system, 210 nm, and 1 mL/min flow rate at lab temperature, approx 20°C The standard was a 1 mg/L 3CA, IPC and CIPC in methanol Peaks 1, 2, 3 and 4 were 3CA, methanol-oxygen peak, IPC and CIPC, respectively 106

Figure 2.12 - The effect of 72%-55% methanol eluant strengths on Columbus column, operated at same conditions 107

Figure 2.13 - The effect of 72%-55% methanol eluant strengths on Hypersil column, operated at same conditions 107

Figure 2.14 - The effect of 72%-55% methanol eluant strengths on a Spherisorb column, operated at same conditions 108

Figure 2.15 - The effect of 72%-55% methanol eluant strengths on a Sphereclone column, operated at same conditions 108

Figure 2.16 - The effect of 85%-72% methanol eluant strengths on Nemesis column, using the Shimadzu A system, 210 nm, and 1 mL/min flow rate at lab temperature, approx 20°C The standard was a 1 mg/L 3CA, IPC and CIPC in methanol Peaks 1, 2, 3 and 4 were 3CA, methanol-oxygen peak, IPC and CIPC, respectively 109

Figure 2.17 - The effect of 75% methanol eluant a) neutral and b) buffer using the Shimadzu A system, 210 nm, 1 mL/min flow rate, Nemesis column at lab temperature, approx 20°C The standard was a 1 mg/L 3CA, IPC and CIPC in methanol Peaks 1, 2, 3 and 4 were 3CA, methanol-oxygen peak, IPC and CIPC, respectively 110

Figure 2.18 - Effect of detection wavelength for analytes on Shimadzu A system at 72% methanol using Nemesis column Peaks 1, 2, 3 and 4 were 3CA, methanol-oxygen peak, IPC and CIPC, respectively 111

Figure 2.19 - UV spectrum for the four peaks using the Diode array detector, using 1 mg/L 3CA, IPC and CIPC standard in methanol 112

Figure 2.20 - Shimadzu system A with manual injection 114

Figure 2.21 - Shimadzu system A with autosampler injection 114

Figure 2.22 - Shimadzu system B with manual injection 114

Figure 2.23 - Thermo system with autosampler injection 115

Figure 2.24 - Diode array with autosampler injection 115

Figure 2.25 - The effect of 30°C column temperature on the separation of the four peaks using the Thermo system with degassed 60% methanol eluant, 210 nm, and 1.5 mL/min flow rate The standard was a 1 mg/L 3CA, IPC and CIPC in methanol Peaks 1, 2, 3 and 4 were 3CA, methanol-oxygen, IPC and CIPC, respectively 118

Figure 2.26 - The effect of 25°C column temperature on the separation of the four peaks using the Thermo system with degassed 60% methanol eluant, 210 nm, and 1.5 mL/min flow rate The standard was a 1 mg/L 3CA, IPC and CIPC in methanol Peaks 1, 2, 3 and 4 were 3CA, methanol-oxygen, IPC and CIPC, respectively 118

Figure 2.27 - The effect of 20°C column temperature on the separation of the four peaks

using the Thermo system with degassed 60% methanol eluant, 210 nm, and 1.1 mL/min flow rate The standard was a 1 mg/L 3CA, IPC and CIPC in methanol

Peaks 1, 2, 3 and 4 were 3CA, methanol-oxygen, IPC and CIPC, respectively 119

Figure 2.28 - The effect of 20°C column temperature on the separation of the three peaks using the Thermo system with degassed 60% methanol eluant, 210 nm, and 1.1 mL/min flow rate The standard was a 1 mg/L 3CA and IPC in methanol Peaks 1, 2 and 3 were 3CA, methanol-oxygen and IPC, respectively 120

Figure 2.29 - Calibration curve for (1 – 10 mg/L) 3CA in methanol 121

Figure 2.30 - Calibration curve for (0.05 – 0.8 mg/L) 3CA in methanol 121

Figure 2.31 - The effect of 33°C column temperature on the separation of the three peaks using the Thermo system with degassed 70% methanol eluant, 210 nm, and 1.4 mL/min flow rate The standard was a 1 mg/L IPC and CIPC in methanol Peaks 2, 3 and 4 were methanol-oxygen, IPC and CIPC, respectively 122 Figure 2.32 - Calibration curve for (1 – 10 mg/L) CIPC in methanol 123

Figure 2.33 - Calibration curve for (0.05 – 0.8 mg/L) CIPC in methanol 124

Figure 3.1 - IPC and CIPC peak shape in 100% methanol and 100% deionised water as injection solvent 136

Figure 3.2 - CIPC peak shape in 100% methanol and 100% deionised water as injection solvent 137

Figure 3.3 - 3CA peak shape in 100% methanol and 100% deionised water as injection solvent 138

Figure 3.4 - Comparison between the peak area of CIPC, IPC and 3CA in 1%, 5%, 20% and 100% methanol in deionised water 140

Figure 3.5 - The solubility of CIPC in deionised water till day 16 142

Figure 3.6 - The Solubility of IPC in deionised water till day 16 143

Figure 3.7- Dissolving speed of 3CA in deionised water 144

Figure 3.8 - Recovery of the 1st to 4th 5 mL aliquot of 1 mg/L standards in deionised water passed through different types of filter papers 158

Figure 3.9 - Recovery of five standards concentrations in ten sequential 5 mL aliquots passed through No.1 filter paper 160

Figure 3.10 - Recovery of five standards concentrations in ten sequential 5 mL aliquots passed through GF/B filter paper 161

Figure 3.11 - Recovery of five standards concentrations in ten sequential 5 mL aliquots passed through PTFE filter paper 162

Figure 3.12 - Recovery of five standards concentrations in ten sequential 5 mL aliquots passed through Nylon filter paper 163

Figure 3.13 - Recovery based on averaging aliquots 6-10 165

Figure 4.1 - Representative chromatograms of CIPC in soils at a concentration of 8.0 mg/L by Shimadzu A system 188

Figure 4.2 - Representative chromatograms of 3CA in soils at a concentration of 8.0 mg/L by Thermo system 189

Figure 4.3 - Representative chromatograms of CIPC in waste materials at a concentration of 8.0 mg/L by Shimadzu A system 190

Figure 4.4 - Representative chromatograms of 3CA in waste materials at a concentration of 8.0 mg/L by Thermo system 191

Figure 4.5 - 10 µg/mL CIPC and 3CA uptake within 30 days at 20°C and 30°C using 5g soil 194

Figure 4.6 – 10 µg/mL CIPC and 3CA uptake in clay and sandy soils within four days at 20°C using 1 g soil 195

Figure 4.7 - Temperature effect on 10 µg/mL CIPC and 3CA adsorption 196

Figure 4.8 - pH effect on 10 µg/mL CIPC and 3CA adsorption 198

Figure 4.9 - Adsorption isotherm of CIPC and 3CA in clay and sandy soils at 20°C 200

Figure 4.10 - Langmuir isotherm of CIPC in three soils 202

Figure 4.11 - Langmuir isotherm of 3CA in three soils 203

Figure 4.12 - CIPC experimental and theoretical adsorption isotherm for the three soils 205

Figure 4.13 - 3CA experimental and theoretical adsorption isotherm for the three soils 206

Figure 4.14 - CIPC and 3CA Xmax plotted against LOI in soils 207

Figure 4.15 - CIPC and 3CA Xmax plotted against total carbon in soils 207

Figure 4.16 - CIPC and 3CA desorption versus adsorption in clay soils at 20°C The 3CA desorption was not detected in Downholland 1A, Downholland 2A and Midelney 2A 209

Figure 4.17 - CIPC and 3CA desorption versus adsorption in sandy soils at 20°C 209

Figure 4.18 - Correlation between desorption and loss on ignition for CIPC and 3CA 211

Figure 4.19 - Correlation between desorption and total carbon for CIPC and 3CA 211

Figure 4.20 - The adsorption% of 10 µg/mL CIPC and 3CA on different sorbents 214

Figure 4.21 - Adsorption isotherm of CIPC and 3CA on waste materials 218

Figure 4.22 - Langmuir isotherm of CIPC in three waste materials 220

Figure 4.23 - Langmuir isotherm of 3CA in three waste materials 221

Figure 4.24 - CIPC experimental and theoretical adsorption isotherm for three waste materials 223

Figure 4.25 - 3CA experimental and theoretical adsorption isotherm for three waste materials 224

Figure 5.1 - River Kelvin water sampling location, Glasgow, UK 231

Figure 5.2 - Glazert Water water location, near Glasgow, UK 231

Figure 5.3 - Extraction of organic matter and fractionation of Fulvic acid 234

Figure 5.4 - Dissolved oxygen meter YSI Model 58 SN: 94 M27200 236

Figure 5.5 - Representative chromatograms of CIPC in Kelvin River water, a) blank, b) 10.0 mg/L CIPC Analysed by Thermo system; CIPC method in section 2.3.9 242

Figure 5.6 - Representative chromatograms of CIPC in Glazert Water water, a) blank, b) 10.0 mg/L CIPC Analysed by Thermo system; CIPC method in section 2.3.9 243

Figure 5.7 - Representative chromatograms of CIPC in potato washing plant effluent (PWPE), a) blank, b) 0.1 mg/L CIPC Analysed by Thermo system; CIPC method in section 2.3.9 244

Figure 5.8 - Representative chromatograms of CIPC synthetic potato washing water (SPWW), a) blank, b) 0.1 mg/L CIPC Analysed by Thermo system; CIPC method in section 2.3.9 245

Figure 5.9 - Representative chromatograms of 0.1 mg/L CIPC in potato materials suspension, a) chopped peel, b) blended peel, c) chopped tuber, d) blended tuber Analysed by Thermo system; CIPC method in section 2.3.9 246

Figure 5.10 - Representative chromatograms of 0.1 mg/L CIPC in 78% clarified synthetic potato wash water Analysed by Thermo system; CIPC method in section 2.3.9 247

Figure 5.11 - Degradation of CIPC in River Kelvin water fortified by nutrition at 20°C

(a) and 30°C (b) 252 Figure 5.12 - Oxygen consumption in River Kelvin water fortified by nutrition at 20°C

(a) and 30°C (b) 252 Figure 5.13 - Degradation of CIPC in Glazert Water water fortified by nutrition at 20°C

(a) and 30°C (b) 253 Figure 5.14 - Oxygen consumption in Glazert Water water fortified by nutrition at 20°C

(a) and 30°C (b) 253 Figure 5.15 - Removal of CIPC from potato washing plant effluent and 3CA formation fresh

(a), after 4 months storage at 4°C (b) 256 Figure 5.16 - Zero-, first- and second-order kinetic plots for PWPE from the one day data

of Fig 5.15a 256 Figure 5.17 - Removal of CIPC from SPWW and 3CA formation fresh (a), after 4 months

storage at 4°C (b) 260 Figure 5.18 - Zero-, first- and second-order kinetic plot for SPWW from the one day data

of Fig 5.17a 260 Figure 5.19 - Degradation of CIPC in solutions containing potato materials 261 Figure 5.20 - Degradation of CIPC (a) and formation of 3CA (b) in clarified suspensions

obtained after four days of calcium chloride sedimentation 265 Figure 5.21 - Degradation of CIPC (a) and formation of 3CA (b) in clarified suspensions

obtained after 1 h calcium chloride sedimentation 268 Figure 5.22 - Degradation of CIPC (a) and formation of 3CA (b) in clarified suspensions

obtained after 1 h sedimentation and fortified with CNP nutrition 268

Figure 6.1 - Evaporation of CIPC and 3CA standards by two rotary evaporators 283 Figure 6.2 - Extraction of 10 mg/L CIPC and 3CA from deionised water by four solvents 284 Figure 6.3 - Representative chromatograms of CIPC in synthetic potato wash water

(SPWW) at different trace levels of a) 0.66 mg/L, b) 0.066 mg/L, c) LOQ

and d) blank Analysed by Thermo system; CIPC method in section 2.3.9 290 Figure 6.4 - Representative chromatograms of 3CA in synthetic potato wash water

(SPWW) at different trace levels of a) 0.66 mg/L, b) 0.066 mg/L, c) LOQ

and d) blank Analysed by Thermo system; 3CA method in section 2.3.8 291 Figure 6.5 - Representative chromatograms of 0.66 mg/L CIPC in fresh soil extracts

a) Darvel, b) Cottenham and c) Dreghorn 2A Analysed by Thermo system;

CIPC method in section 2.3.9 292 Figure 6.6 - Representative chromatograms of 0.66 mg/L 3CA in fresh soil extracts

a) Darvel, b) Cottenham and c) Dreghorn 2A Analysed by Thermo system;

3CA method in section 2.3.8 293 Figure 6.7 - Representative chromatograms of 0.66 mg/L CIPC in dried soil extracts a)

Downholland 1A, b) Midelney 1A and c) Quivox B Analysed by Thermo system; CIPC method in section 2.3.9 294 Figure 6.8 - Representative chromatograms of 0.66 mg/L 3CA in dried soil extracts

a) Downholland 1A, b) Midelney 1A and c) Quivox B Analysed by Thermo

system; 3CA method in section 2.3.8 295 Figure 6.9 - LLE-VORTEX extraction of CIPC and 3CA from high soil suspension 297 Figure 6.10 - Microbial degradation of 10 mg/L CIPC and 3CA formation in unfortified

SPWW at 20°C, n = 2 300 Figure 6.11 - Microbial degradation 10 mg/L 3CA in unfortified SPWW at 20°C, n = 2 300

Figure 6.12 - Microbial degradation of 10 mg/L CIPC and 3CA formation in fortified SPWW

with CNP at 20°C, n = 1 304

Figure 6.13 - Microbial degradation of 10mg/L 3CA in fortified SPWW with CNP at 20°C, n = 1 305

Figure 6.14 - Microbial degradation of 10 mg/L CIPC at different temperatures 308

Figure 6.15 - Microbial degradation of 10 mg/L 3CA at different temperatures 309

Figure 6.16 - 3CA Intermediate formation at different temperatures 311

Figure 6.17 - CIPC linearity and activation energy data 314

Figure 6.18 - Initial 3CA linearity and activation energy data 315

Figure 6.19 - Final 3CA linearity and activation energy data 316

Figure 6.20 - Microbial degradation of 10 mg/L CIPC and 3CA intermediate formation in three soil suspensions 319

Figure 6.21 - Microbial degradation of 10 mg/L 3CA in three soil suspensions 319

Figure 6.22 - Microbial degradation of CIPC and 3CA formation in dried soils 323

Acknowledgement

I would like to express my sincere gratitude and appreciation to my supervisors

Dr T H Flowers and Dr H J Duncan for their combined supervision, inspiration, support and direction throughout the project

An acknowledgement is extended to Dr Geraldine McGowan and the staffs of Sutton Bridge Experimental Unit for providing the required CIPC-treated potatoes and the potato wash plant effluents that were used during degradation experiments

Special thanks to Dr John Dolan who is currently a principal instructor for LC Resources, California and a member of LCGC's editorial advisory board for his continuous valuable information regarding HPLC analysis and troubleshooting

I also wish to thank Dr John Forsythe of 1, 4 Group, USA, for providing information regarding CIPC application and unpublished potato wash treatments

I am also grateful to Isabel Freer, Michael Beglan, Stuart Mackay, Ibrahim Madi, Abdulmohsen Alsukaibi and other precious friends and colleagues at the Environmental, Agricultural and Analytical Chemistry Section

The financial support of Taibah University, Madinah, Saudi Arabia, that enabled this research is highly appreciated

Finally, and most importantly, I would like to express my sincere gratitude to my parent, wife and children for their help, patient and support Without you, it would have been difficult, if not impossible to carry out this work

Author’s Declaration

I hereby declare that this thesis is my own original research work and effort and that it has not been submitted anywhere for any award Where other sources of information have been used, they have been acknowledged Some of the results may have been published elsewhere (Doland & Alsehli 2012)

BANDAR RASHED M ALSEHLI

February 2014

LOD Limit of detection

LOQ Limit of quantification

psi Pound per square inch

rpm Revolutions per minute

RSD% Relative standard deviation (in Percentage)

SD Standard Deviation

Chapter 1 - Main Introduction

1.1 Background related to the use of sprout suppressants in the potato industry

1.1.1 Current state of UK potato market

Potatoes make up a significant part of the food market, and it is one of the feedstock for mass-consumption products, along with rice wheat and maize Currently, the demand for potatoes is growing (UK Potato Council 2013c)

According to the data provided by UK Potato Council (2013a), the shares of the main carbohydrate meal occasions for the four years ending November 2009-

2012 are illustrated in Fig 1.1

Figure ‎1.1 - Consumption of carbohydrate meal occasions Data adapted from UK Potato Council (2013a)

Bread, potatoes, pasta and rice were consumed in 17.1, 10.1, 2.6 and 1.5 billion in-home meal occasions, respectively The in-home potato meal occasions of 10.1 billion meal occasions, which means that each member of the population consumed almost 3.5 meals a week through different potato meal types The types of potato meal occasions are shown in Fig 1.2

In-home carbohydrate meal occasions

1 year end Nov 2009

1 year end Nov 2010

1 year end Nov 2011

1 year end Nov 2012

Figure ‎1.2 - Consumption of carbohydrate meal occasions Data adapted from UK Potato Council (2013a)

Fresh potatoes accounted for more than 69% of the in-home potato meal occasions, while frozen potatoes were less popular in all years and accounted for 29%

Fresh potatoes were used to prepare different meals The major proportion of fresh potatoes were boiled or mashed Roast and baked/jacket potatoes were less popular preparation methods

Home-grown potato production in Great Britain (GB) has to be sufficient to supply the markets with a high number of potatoes Fig 1.3 shows the potato production flowchart that has been recently updated on the UK Potato Council website (2013b) about crop production for the period of June 2011 to May 2012

The GB potato home crop production equalled 6090 thousand tonnes; the estimate supply for human food, stock feed and seed were 4303, 1473 and 360 thousand tonnes, respectively The total consumption was 5577 thousand tonnes: the GB retail consumption of fresh potatoes was 2418 thousand tonnes, while

3159 thousand tonnes (including a net import of 1357 thousand tonnes) were

distributed through the processed supply chain (UK Potato Council 2013b)

6.9

2.9 4.0

1.4 1.4

2.7

0 1 2 3 4 5 6 7 8

) In-home potato meal occasions

1 year end Nov 2009

1 year end Nov 2010

1 year end Nov 2011

1 year end Nov 2012

Figure ‎1.3 - Potato production flowchart for Great Britain for June 2011 to May 2012 Taken from Potato Council website (UK Potato Council 2013b)

The feedstock for most fresh potato meals are high-quality fresh-looking potatoes tubers, namely smooth, whole skin and properly coloured Since potatoes are a seasonal product, they require storage (UK Potato Council 2013c) Potato tubers that were spoiled during storage can be used (when the rotten or spoiled part of a tuber is cut off) for frozen potatoes products with some weight loss However, spoiled potatoes are always associated with profit loss

In the UK, potato crops are gathered in early autumn (Aksenova et al 2013) Thus, long-term preservation of potato tubers is a significant task for the potato industry (Booth & Shaw 1981) As potato consumption and demand is stable throughout the year yet the supply is seasonal, the storage of potatoes is required

of nutrients for them and the potato tubers start losing their consumer properties Thus, effective dormancy regulation is a challenge for storage purposes

Sprouting appears as a natural process at mild temperatures during long-term storage Sprouting reduces the quantity of marketable potatoes and is also a cause of potato dehydration (Slininger et al 2003), in addition to physical water evaporation from the potato surface This results in substantial weight loss and tubers can wrinkle and soften As a result, the marketing options are reduced since the crop can be used only as feedstock for French fries, crisps and potato flour, and is not suitable for fresh tuber supply Dehydration of potato tubers takes place at mild temperatures during the extended storage period Thus, temperature has to be kept as low as possible to prevent dehydration

However, low temperatures induce starch decomposition and the potato develops an excessively sweet taste (Sowokinos et al 1987) Light is another factor that affects the appearance of tubers (Sowokinos et al 1987) When crops are exposed to light, potato tubers develop chlorophyll, chlorogenic acid and glycoalkaloid (Dao & Friedman 1994) and the tubers become green These substances cause bitterness and some of them are toxic (Phillips et al 1996)

The green parts of the potatoes have to be removed for safety reasons as well as appearance This brings the same negative consequences as dehydration

In order to maintain frying quality and fulfil the demand of the processing industries, potato storage has to be done at a relatively warm temperature (8°C -10°C) with air circulation and humidity control (Singh & Kaur 2009) Nowadays, most stores are equipped with electronically controlled environment systems The potato dormancy length depends on the temperature and the potato variety, but generally at 5.5°C the dormancy period can last 100-175 days If potatoes are stored at 7.2°C they can only be stored for 85-145 days; storage at 8.8°C reduces the storage period to 80-130 days (Olsen 2013)

However, it is becoming increasingly difficult to ignore the need for longer storage periods to fulfil the tremendous demand throughout the year This can

be attained by the use of sprout suppressant chemicals

1.2.2 Use of sprout suppressant chemicals

Sprout suppressant chemicals are used to prevent potatoes from sprouting during the storage period Various sprout suppressants have been proposed, for example hydrogen peroxide (Afek, Orenstein & Nuriel 2000), bacteria (Slininger

et al 2003) and ethylene (Kleinkopf, Oberg & Olsen 2003) Novel inhibitors, for instance camptothecin, volatile monoterpenes, jasmonates, nonanol, abscisic acid, indole-acetic acid, dichlorbenil, dimethylnaphthalene and diisopropylnaphthalene have also been proposed (Afek, Orenstein & Nuriel 2000) 1,4dimethylnapthalene (DMN) was already registered in the USA but it is still not registered in Europe as its application requires some consideration due

to chemical evaporation and the lack of sprout control when applied for a long storage time

The search for natural alternatives is continuing as there is concern about using chemicals in the food industry Natural inhibitors that are extracted from plants are a new area that requires study Frazier, Olsen & Kleinkopf (2013) and Teper-Bamnolker et al (2010) indicated that mint oils (namely spearmint and peppermint oils) can be successfully applied as sprout suppressants However,

except for DMN, none of these substances have been commercially applied, mainly due to their high price and low efficiency in controlling the sprouting

Chlorpropham, also known as isopropyl-N-(3-chlorophenyl) carbamate or CIPC, is

a sprout suppressant and plant growth regulator of the carbamate class The substance was first developed as a pre-emergence herbicide and was quickly identified as a useful potato sprout suppressant for long-term tuber storage (Marth & Schultz 1952) Since then and up to this date, chlorpropham is considered to be the world-leader and is the major sprout inhibitor used in the potato industry (UK Potato Council 2013c; Slininger et al 2003; Frazier, Olsen & Kleinkopf 2013) There are no real alternatives to CIPC in terms of efficiency and sprout control It is also the preferred choice for the processing industry as almost 100% of the CIPC is removed when the potatoes are peeled, unlike maleic hydrazide that is distributed inside the whole potato tuber

CIPC sprouting inhibition, chemistry, impact and application methods have been well studied and reviewed (Michael, Thornton & Kleinkopf 2013) The sprouting inhibition by CIPC acts by interfering with cell division, namely spindle formation during active mitosis (Vaughn & Lehnen 1991)

Commercial application of CIPC in potato storage is performed by aerosol dispersion or emulsifiable concentrate (Michael, Thornton & Kleinkopf 2013) CIPC emulsifiable concentrate is applied during the grading and packaging process, while aerosol dispersion is more common for industrial storage applications (Agriculture and Rural Development 2013) The CIPC is distributed

by aerosol circulation through the potato bulk stores or box stores The circulation is performed by the ventilation system In some cases CIPC can be applied as a spray, delay-released granules or dust (Frazier, Olsen & Kleinkopf 2013) The use of a CIPC and isopropyl N-phenylcarbamate (IPC) mixture has been reported The advantage of the mixture is superior sprout control, particularly during the initial stages (Kleinkopf, Oberg & Olsen 2003)

Many application rates and distribution methods have been investigated to ensure efficient sprout control and low CIPC residue in the washed potato tuber (Brajesh & Ezekiel 2010) For instance, when an aerosol application was

performed at rates of 17-28 ppm (Kleinkopf, Oberg & Olsen 2003) the CIPC residue levels on the potato peel was much lower than the acceptable limits (Brajesh & Ezekiel 2005) For example, the tests on potatoes treated with CIPC during storage indicated that the CIPC concentrations in peeled and unpeeled tubers were 0.29-1.13 and 0.05-0.24 mg/kg, respectively (Brajesh & Ezekiel 2010) However, the high numbers of applications as a consequence of high demand resulted in a very substantial use of the chemical around the world to control millions of tonnes of potatoes in order to supply potato markets and processing industries with high quality products

According to the most recent survey by Garthwaite et al (2010), 49% of the 2010 harvest (2.03 million tonnes) in the United Kingdom received sprout suppressant treatments, and some potatoes were treated multiple times In 2010, thirty-six tonnes of sprout suppressant chemicals (Chlorpropham, ethylene, thiabendazole and imazalil) were used in the United Kingdom Among these, 85% was chlorpropham, which amounted to 30.6 tonnes Although the annual sprout suppressant chemical consumption in the UK reduced from 60 tonnes in 2004 to

36 tonnes in 2010 (Garthwaite et al 2010), the applied amounts are still substantial

Reducing the number of treatments by using proper application conditions can cut down the use of the chemical CIPC is considered a superior sprout suppressant and numerous treatment cycles may be not required; sometimes a single application provides long-term sprout inhibition (Frazier, Olsen & Kleinkopf 2013) The data provided by the Potato Industry CIPC Stewardship Group (2013c) indicates that usually a one-time application is enough and that the second treatment might be required only if there are visible signs of sprouting This is particularly important since the second treatment should be carefully considered

Since potato crops are treated with CIPC regularly, toxicity effects, particularly

on humans are of the greatest importance, especially due to the fact that CIPC can degrade to form 3-chloroaniline (3CA) which has a similar structure to the carcinogenic 4-chloroaniline compound 3CA might be formed via two processes: thermal degradation that occurs during CIPC hot fogging application, or when

the microbial degradation of CIPC takes place 3CA residues have been continuously detected in experimental and commercial potato stores The excessive use of the CIPC along with the likely formation of 3CA might be of greatest concern to human health and environment

1.2.3 Toxicity of CIPC and its metabolites to humans

Generally, CIPC is recognised as non-toxic to humans, in contrast to organochlorines (Smith & Bucher 2012) CIPC belongs to Toxicity Category III (slightly toxic); it is non-toxic through dermal exposure The acute CIPC effect is irritation of the eyes and skin (U.S National Library of Medicine 1995) As reported by Hartley & Kidd (1987), CIPC has a high LD50 (lethal dose, 50%): approximately 5000-7500 mg/kg At these doses reproductive effects were not observed (Hayes & Laws 1991) The compound is recognised as non-mutagenic or slightly mutagenic (U.S National Library of Medicine 1995)

According to an Environmental Protection Agency (1996a) classification, chlorpropham is placed on group E, which shows that there is evidence of non-carcinogenicity in humans However, the agency stated that its main metabolite, 3-chloroaniline (3CA), is similar in structure to 4-chloroaniline, which is a known carcinogenic compound The agency also stated that because there is no cancer data for 3CA, the potential risk of 3CA could be gauged appropriately using the cancer potency (Q1*) from 4-chloroaniline The agency believes, based on the compound‘s structure, that 3-chloroaniline possibly has a similar potency to 4-chloroaniline and is unlikely to be more potent than 4-chloroaniline

Although the World Health Organization (2003) acknowledged that chloroaniline is carcinogenic after long-term exposure, data for 3-chloroaniline toxicity in humans was not published (World Health Organization 2013) However, the organisation believed that all chlorinated aniline isomers in the positions ortho, meta and para (2, 3 and 4) showed some haematotoxic effects in mice and rats, and 4-chloroaniline was more severe Also, experiments in various systems indicated that 4-chloroaniline was genotoxic while 2 and 3 chloroaniline had weak genotoxic effects (World Health Organization 2003)

4-Nevertheless, governments and regulatory authorities have set guidance and regulations that can be followed to maintain safe practices when dealing with these chemicals In the UK, the expert committee on Pesticide Residues in Food (PRiF) annually monitors the chemical residues in different types of foods and drinks and assesses the results In 2006 they found that one potato sample contained chlorpropham at a level of 47 mg/kg (Table 1.1) However, the incident was not considered by the Advisory Committee on Pesticides (ACP) and other authorities as an accidental misuse of the chemical This resulted in some changes to the approval of CIPC to be used for stored potatoes (PRiF 2012a) The European Community (EC) reviewed CIPC in 2006 to ensure the safe use of the chemical and an MRL of 10 mg/kg for CIPC, based on washed potatoes, was set, which came into force on 21 April 2007

Maximum Residue Levels (MRLs) are defined as: the upper level of chemical concentration (mg/kg) that is legally allowed in food or drink commodities It is not considered to be a safety limit and exceeding the MRL does not directly imply hazard or toxicity, but it is assigned to primarily check that good agricultural practice (GAP) is followed

Good Agricultural Practice (GAP): "The nationally authorised safe uses of pesticides under conditions necessary for effective and reliable pest control (the way products should be used according to the statutory conditions of approval which are stated on the label) GAP encompasses a range of pesticide applications up to the highest authorised rates of use, applied in a manner which leaves a residue which is the smallest practicable Authorised safe uses are determined at the national level and include nationally registered recommended uses, which take into account public and occupational health and environmental safety considerations Actual conditions include any stage in the production, storage, transport, distribution and processing of food commodities and animal feed" (PRiF 2012b, p.41)

The European Food Safety Authority (EFSA) has requested information about residues of 3-chloroaniline, along with its parent chlorpropham, since 2008 However, in the most recent review by the EFSA (2012) they stated that due to insufficient information and significant gaps in 3CA studies, especially

genotoxicity or carcinogenicity, storage stability of 3CA, hydrolysis and validated analytical methods, only a tentative MRL proposal for 3CA could be proposed In the meantime, the residues of CIPC plus the residues of 3CA must not exceed the MRL of 10 mg/kg This was delivered because it was believed that only traces of 3CA were detected and therefore it was suggested to be added to CIPC residues However, a number of unpublished studies in Glasgow University laboratories have sometimes found that the level of 3CA in potato samples is significant (Harry Duncan, personal communication)

In the future further reductions might be expected as food industry safety regulations have a tendency to become stricter (Michael, Thornton & Kleinkopf 2013; European Food Safety Authority 2012; Kleinkopf, Oberg & Olsen 2003)

The safety limit is defined as an acceptable daily intake (ADI), which is the amount of chemical that can be taken on a daily basis for a lifetime without it causing any harm It is expressed by the unit (in mg) of chemical per kg of body weight per day The ADI is derived from the ‗no observed adverse effect level‘ (NOAEL), which is the greatest amount of a chemical that does not cause toxicity

to animals, including no alteration to growth, morphology, life span or development Because animals might be less sensitive to certain chemicals than humans, the NOAEL value is divided by 100 to give the safe ADI for humans (PRiF 2012b)

Accordingly, a limit of 0.1 mg/kg as an acceptable daily intake of CIPC for humans was temporarily adopted in the UK during the early 1990s (Department for Environment, Food and Rural Affairs 1993) Half this value was established as the limit in Australia and the USA during the same decade (National Registration Authority 1997)

Acute Reference Dose (ARfD) is comparable to the ADI but it is the amount that can be taken of a certain chemical in one day or one meal without any effect on the consumer‘s health It is normally calculated by applying a suitable factor (often 10) of uncertainty to the NOAEL value to take into account variation in sensitivity between individuals

Since the MRL has been set there have been incidents of it being exceeded (Table 1.1) In 2006, as previously indicated, there was one potato sample containing chlorpropham at a concentration of 47 mg/kg In 2007, 2008 and 2009 CIPC residues were detected on organic potatoes, which were believed to be as

a result of previous historic contamination of potato stores In 2010, three potato samples contained CIPC at concentrations of 14, 16 and 17 mg/kg, which are levels that exceed the MRL Two samples contained CIPC levels of 12 and 13 mg/kg in 2011 In the most recent updated report (PRiF 2012a), two potato samples were found to contain CIPC at levels of 15 and 16 mg/kg

Table ‎1.1 - The detected residues in potato samples in the period 2000-2012 Adapted from Pesticide Residues in Food, PRiF (2013)

Year source Potato Total number analysed Concentration (mg/kg) Number of samples

67

42

2

1.3 The environmental fates of CIPC and its metabolite 3CA

There are severe concerns about the fates of CIPC and 3CA when they are discharged to the environment (Ragnarsdottir 2000) Environmental exposure to CIPC mainly results from runoff from farmyards and outlets from sewage treatment plants (Holvoet, Seuntjens & Vanrolleghem 2007; Neumann et al 2002) The other common routes for pesticide entry are wastewater discharges and spills (Tiryaki & Temur 2010)

Thus, CIPC enters the environment mainly with a water stream and might be degraded to 3CA that can disperse well in water Since CIPC and its major metabolite are the potential long-term threat for groundwater, surface waters and soil environments, understanding their toxicity, properties and fate in the environment is crucial

1.3.1 Environmental toxicity

The effect of CIPC on aquatic organisms is recognised as toxic to moderately toxic, depending on the types of the organisms Hartley & Kidd (1987) reported moderate toxicity to freshwater fish For example, the LC50 (lethal concentration, 50%) was designated as 12 mg/L and 10 mg/L for bluegill sunfish and bass, respectively No toxic effects were observed for Streptomyces and nitrifying bacteria, but Maule & Wright (1983) reported toxic effects on cyanobacteria due to the inhibition of protein synthesis by blocking mRNA synthesis in cells A comprehensive review of CIPC toxicity was performed by Vonk & Smit (2011) It was reported that the LC50 ranged from 0.43 to 1.65 mg/L for algae, where the toxic effect had an impact on growth rate and cell density For Pisces and Amphibians, LC50 ranged from 4.9 to 20 mg/L Freshwater gram positive bacteria showed growth inhibition at 37°C (Vonk & Smit 2011)

In natural systems, CIPC metabolites are 4-hydroxychlorpropham, 3-chloroaniline and 3-chloroacetanilide (Carrera et al 1998), and 3-chloroaniline is the main metabolite (Kearney & Kaufman 1969) Maule & Wright (1984) indicated

inhibition of growth rate for cyanobacteria and algae in the presence of chloroaniline However, its toxic effect was less than for chlorpropham and the authors stated the same fact in their previous paper (Maule & Wright 1982) In some cases, a partial recovery of the population was observed

3-Sihtmäe et al (2010) performed research on aniline (including 3-chloroaniline) derivative toxicity to bacteria, protozoa and crustaceans The test showed that toxicity for crustaceans was more significant (10-100 times) than for bacteria and protozoa The LC50 of 3-chloroaniline for bacteria Aliivibrio fischeri was 43

mg/L The literature data allow making the conclusion that 3-chloroaniline is moderately toxic to aquatic organisms when compared to 4-chloroaniline (Könnecker, Boehncke & Schmidt 2003)

Given that CIPC is biologically active against seedling growth and tuber sprouting, it is also a reasonable concern that excessive environmental presence

of CIPC could reduce plant growth (Roberts 1965)

1.3.2 Properties

Chlorpropham (or CIPC) is considered to be the world leader and the major sprout inhibitor used in the potato industry There is no real alternative to CIPC

in terms of efficiency and sprout control Table 1.2 shows CIPC‘s properties

Propham (or IPC) used to be widely applied as an effective potato sprout suppressant and in some applications is mixed with CIPC to enhance sprout control Also, due to the similarity with CIPC‘s structure, propham can be an ideal internal standard that can be added with CIPC samples to correct for any analyte loss or variability Table 1.3 shows IPC‘s properties

3-chloroaniline (or 3CA) is an organochlorine compound that is structurally similar to 4-chloroaniline, which is a known carcinogenic compound It is frequently detected in potato samples and potato waste water streams as a result of CIPC thermal or microbial decomposition Table 1.4 shows 3CA‘s properties

Table ‎1.2 - Chlorpropham identification, physico-chemical and environmental properties

(IUPAC 2011; Vonk & Smit 2011; Hazardous Substances Data Bank 2011; European Commission Health & Consumer Protection Directorate 2003)

Name

Chlorpropham, CIPC Isopropyl 3-chlorocarbanilate Isopropyl m-chlorocarbanilate Isopropyl-N-(3-chlorophenyl) carbamate

Hydrolytic stability (DT 50 ) At pH 4-9, 20°C >1 year

Octanol/water partition coefficient (log P ow ) At pH 4-9, 20°C = 3.8

Table ‎1.3 - Propham identification, physico-chemical and environmental properties

(IUPAC 2011; Vonk & Smit 2011; Hazardous Substances Data Bank 2011; European Commission Health & Consumer Protection Directorate 2003)

Name

Propham, IPC Isopropyl carbanilate

or Isopropyl phenylcarbamate

Octanol/water partition coefficient (log P ow ) At pH 7, 20°C=2.6

Table ‎1.4 - 3-chloroaniline identification, physico-chemical and environmental properties

(IUPAC 2011; Vonk & Smit 2011; Hazardous Substances Data Bank 2011; European Commission Health & Consumer Protection Directorate 2003)

Octanol/water partition coefficient (log P ow ) At pH 7, 20°C= 1.72

The most important points that need to be highlighted from the cited tables are

as follows:

Chlorpropham is not sensitive to sunlight, so its photodegradation is not an important route in the environment It is not volatile from dry or moist soil surfaces due to its very low vapour pressure at 20°C; only at temperatures above 35°C was negligible volatility noticed CIPC solubility in organic solvents such as dichloromethane, ethyl acetate, acetone and methanol is higher than 1000 g/L

at 20°C In water, the solubility of CIPC ranges from 89-112 mg/L at pH 4-10 and 20°C

Propham is not sensitive to sunlight so its photodegradation is not an important route in the environment It is slightly volatile at temperatures above 35°C IPC solubility in organic solvent is similar to CIPC In water, the solubility of IPC ranges from 32-250 mg/L at 20-25°C