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

Alsehli, Bandar Rashed M. (2014) Behaviour of chlorpropham and its main metabolite 3-chloroaniline in soil and water systems. PhD thesis, University of Glasgow.

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.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Chlorpropham, 3-chloroaniline, soil and water systems, waste water, methanol-oxygen peak, HPLC, microbial degradation, adsorption-desorption.
• Subjects: Q Science > QD Chemistry
• Colleges/Schools: College of Science and Engineering > School of Chemistry
• Supervisor's Name: Flowers, Dr. Hugh
• Date of Award: 2014
• Embargo Date: 1 May 2021
• Depositing User: Dr. BANDAR ALSEHLI
• Unique ID: glathesis:2014-5529
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 04 Nov 2014 11:56
• Last Modified: 01 May 2020 08:18
• URI: https://theses.gla.ac.uk/id/eprint/5529