HPLC STUDIES ON SOME ORGANOPHOSPHORUS AND CARBAMATE PESTICIDE RESIDUES IN THE ENVIRONMENT

Abstract

The widespread occurrence of pesticide residues in every agri cultural commodity in India and the growing resistance in vectors has been accepted as an unrefutable fact. Growth of population is making increasing demands on natural resources resulting in their exploitation in an unsustainable manner. Awareness on the deposi tion of pesticides outside the target area and the potential health effects due to this chemical trespass is nevertheless growing amongst the general public and regulatory agencies. The science of plant protection and pesticides is undergoing change and emphasis is now being laid on biodegradable, photo-sensitive and environmen tally safe pesticides. A limited number of analytical procedures, for identification and measurement of residues and their metabolites exist. Basic to evaluation of the environmental impact of residues is knowledge of their fate and persistence. Factors such as soil type, water quality, climate, agricultural practices and mode of application influence persistence. Therefore, it is important to have localized information on the occurrence of residues to be able to harness and direct their sensible use. A clear picture of the persistence pattern of residues in any area must be evolved for evaluating their impact on the ecosystem. So, before restricting use of certain pesticides, it is imperative to follow their persistence behavior. The highly versatile High Performance Liquid Chromatography (HPLC) technique has been explored for its adaptability to detect, separate and quantify pesticides and their residues in the

environment. Reversed-phase HPLC (with UV detector) has been chosen for these studies due to direct applicability to nonvolatile and thermolabile compounds, non-destructive nature, sufficient sensitivity and resolution of a wide spectrum of compounds including environmental samples. A C-18 yBondapack column has been used for the reversed-phase studies. Coupling RP-HPLC with a UV-Vis detector proves a versatile combination. In these studies, HPLC determination of selected organophosphates and carbamates in the Indian environment has been carried out. On the basis of a survey carried out in the surrounding Block Development Offices, which harness the supply and distribution of pesticides in each area, the six pesticides selected for study were : dimethoate, methyl-parathion, malathion (organophosphates), and carbaryl, carbofuran, carbendazim (carbamates). For clarity and convenience, the subject matter embodied in this thesis has been organized in the following chapters: 1. General Introduction 2. Selection of Detection Conditions 3. Determination of Pesticide Residues 4. Persistence of Pesticides in Soils 5. Persistence of Pesticides in Water 6. Persistence of Pesticides in Vegetables 7. Simultaneous Determination of Pesticides 8. Conclusion. Each experimental chapter constitutes a preliminary note followed by relevant literature, analysis protocol, results and discussion to aid unhampered scrutiny. The first chapter is of introductory nature, emphasizing the gravity of the pesticide pollution problem, importance of pesticide residue analysis, and the significance of use of reversed phase high performance liquid chromatography. In the second chapter, best conditions suited for analysis of standards and formulations were established. Pesticides were extracted from formulations using methanol, chloroform and acetone and the exact strength/percent purity of each was deter mined. The most appropriate detection parameters as well as optimum operating conditions were determined for each pesticide. Initially various solvents suitable for reversed-phase studies were experi mented with, finally settling on two solvent systems: acetonitrile- water and methanol-water in various proportions, in isocratic and gradient elution modes. An attemptwas made to minimize interference by co-extracted impurities from the formulation and to selectively elute the pesticide. Effects of change in flow rate at each mobile phase composition was determined. A variable wave-len gth detector was used to determine the usable wavelength ranges between 214-280nra. In general, lower wavelengths were favored. Injection volume was varied from 2-10yl in each case and injection volume fixed at 3vl for a 0.5ppm sample.. This gave adequate deflection (75% of full scale) at an attenuation which ignored vi minor impurity peaks. Formulation solutions were serially diluted to determine minimum detectable concentration for each pesticide. In the third chapter, extraction techniques were explored to evolve a single analytical procedure for each substrate-soil, plant and water, that could selectively determine the individual residues from the environment. Percent recovery was noted for the methods. The established methods were modified for application to the matrices and pesticides under study. Methanol-water extraction for soil samples, methylene chloride for water and methanol for crops in general proved efficient. All samples were partitioned into methylene chloride, dried over anhydrous sodium sulphate, suitably cleaned and analyzed by HPLC. Average recoveries ranged between 71-93%. The fourth chapter discusses studies related to persistence of residues in different soils. Three types of soils were first characterized according to their physico-chemical properties and each soil was spiked with the six selected pesticides separately. Residues were extracted according to the methods already estab lished. Degradation of pesticides was monitored over several weeks. Change in pH of soil along with degradation was measured continu ously. Half-life was determined in each case. Persistence was followed till 95-97% dissipation. The fifth chapter deals with the study of pesticide degradation in water in the presence of bottom sediment. Water samples along with sediments were collected from three different sources; each was spiked separately with each pesticide under study. After suitable extraction, persistence was studied separately in water and in water with sediment. Degradation was studied in pre and postvii monsoon waters. Three crops, potato (Solanum tuberosum) , egg plant (brinjal) (Solanum briniales) and cabbage (Brassica oleracea> were selected for field studies in the sixth chapter. Pesticide formulation was sprayed in appropriate concentrations and the crops harvested at various time intervals. Pesticides were extracted from all 18(3x6) samples using methanol. The extracts from potatoes were directly injected into the HPLC system after partitioning with methylene chloride, pre-filtration with 0.45 ym filters, trace enrichment and partial clean-up on Sep-Pak cartridges (C-18). Extracts from cabbage and brinjal, due to high pigmentation, were back flushed using a Waters Automated Valve Station(WAVS) and Guard Column. Zero-th day readings for plants (washed) were insignificant but residues built up by the 2nd day and then started decreasing in all three cases. Organophosphates did not persist beyond 9-11 days. All the studies were conducted till levels below those specified as maximum residue limits by FAO/WHO. The seventh chapter describes separation of a mixture of pesticide standards followed by multi-residue analysis from soil. Various mobile phase mixtures were investigated, in the presence and absence of a buffer (KH,PO,, pH 7.0). Flow rates were varied between 0.40 to 1.25ml/min. Both gradient and isocratic elution were applied and the best wavelength for simultaneous determination was established. Detection limits were specified. A concave gradient of acetonitrile-water (50:50 to 70:30) (aqueous phase constituting lOmM Phosphate buffer, pH 7.0) in 15 minutes (flow-rate varying from 0.75 to l.OOml/min) and conditions remaining constant for 5minutes, was found most suitable. All six pesticides were resolved within 20 minutes and could be measured at concentrations of

0.02ppm. Carbaryl and carbofuran could be detected upto 0.005ppm. With a sufficient understanding of the impact of pesticide residues and their potential hazards, it may be possible to continue using them in the years to come. It remains imperative to assess carefully the extent of ecological imbalance resulting from their misuse. The concluding chapter discusses the need for further studies and possible areas of research to gauge the environmental impact of pesticide residues.