Graduate Abstract

Stephen C Twomey
Spring 2002

The Study and Optimization of the Enzyme Inhibition Bioassay 96 the Detection of Organophospha te and Carbamate Compounds

A thesis submitted in partial fulfillment
of the requirements for the

Master of Science Degree

Department of Environmental Studies
Greenspun College of Urban Affairs

Graduate College
University of Nevada, Las Vegas
Spring 2002

by

Stephen C Twomey

Dr. Shawn Gerstenberger, Examination Committee Chair
Associate Professor of Environmental Studies
University of Nevada, Las Vegas

ABSTRACT

The assessment of human exposure to hazardous compounds (such as pesticides) has been of great concern for decades. One method of reducing uncertainties in the assessment of human exposure is to better characterize the compounds that may be present in our immediate environment. A major limitation to this approach, however, has been the sampling and laboratory analysis of contaminated environmental and biological samples. Traditional sampling techniques can be slow and expensive, therefore, limiting the number of samples that may be analyzed within given time and budget constraints. Faster, simpler, and more cost-effective field screening tools can increase the amount of information available concerning the location, source, and/or concentration of pollutants present in the environment. The Enzyme Inhibition Bioassay 96 (EIB 96) is one such field-screening tool.

The EIB 96 is an assay that can detect the presence of organophosphate and carbamate compounds, providing scientists with valuable information to focus their future sampling efforts. The assay is based on the inhibition of the enzyme acetylcholinesterase by organophosphate and carbamate compounds. The organophosphate and carbamate compounds that were used in this project were the pesticides carbaryl, chlorpyrifos, diazinon, dichlorvos, and parathion. The pesticides chlorpyrifos, diazinon, and parathion are organophosphorothioates, which are organophosphates with a P=S bond, and when these compounds are oxidized the compounds are converted to their oxygen analogs (P=O, substitution of a sulfur with an oxygen). Thus, this project was directed at determining the optimum oxidizing agent for use in the assay and then, to determine the oxidation products for the pesticide compounds used. The responses for the acetylcholinesterase in the presence of the oxidized and unoxidized pesticide compounds were measured using a V-max spectrophotometric kinetic plate reader. The analyses of the oxidation reactions were accomplished using an Agilent Technologies 6890 gas chromatograph/5973 mass-spectrometer (GC/MS).

The optimum oxidizing agent that was determined from this project was sodium hypochlorite (NaOCl), which oxidized the organophosphorothioates as well as bromine (Br2). Bromine was the initial oxidizer choice but, because of the relative difficulty in obtaining Br2 for use in the assay and its relatively higher cost as compared to NaOCl, NaOCl was determined to be the optimum choice. Also, the oxidative product for the model organophosphorothioates, parathion, was determined to be only paraoxon. Approximately 24±3% of the parathion was oxidized to paraoxon, and the non-organophosphorothioates, carbaryl and dichlorvos, were not damaged by the oxidation process using NaOCl and did not form any oxidation products.