22    DEVELOPMENT AND PRELIMINARY VALIDATION OF THE
NEW, RAPID, TOXICITY BIOASSAY
DAPHNIAQUANT™
Douglas J. Fort, Director, Research & Technical Services
Enos L. Stover, President
Randall A. Atherton, Assistant Project Manager
Stover Biometric Laboratories, Incorporated
Stillwater, Oklahoma 74075
Sterling L. Burks, Professor
James T. Blankemeyer, Professor
Department of Zoology
Oklahoma State University
Stillwater, Oklahoma 74078
INTRODUCTION
Recent regulatory emphasis on integrated ecological hazard assessment has necessitated the development and validation of cost-effective alternative tools for estimating ecological impairment. Based
on new Clean Water Act legislation, the emphasis on ecological hazard assessment should increase
over the next 20 years. Since the costs required to maintain environmental compliance are escalating
due to both demand and the limited number of tools currently available to monitor aquatic toxicity,
the development of rapid, cost-effective, alternative toxicity monitoring techniques is imperative.
These inexpensive alternative assay systems may be used to monitor the potential ecological hazard of
effluents, manufactured products, hazardous waste sites, and regulated chemicals.
Because of this, DaphniaQuant™, an assay designed to detect the health of Daphnia sp. at the
cellular level was developed.1 The DaphniaQuant™ assay uses the uptake of fluorescent dye and
corresponding fluorescent measurement as an early indicator of toxicity. Following short-term exposure to a test chemical or mixture, fluorescence readings are collected, stored in a customized database
system in a standard IBM-compatible computer, and easily output for graphical observation or
statistical analysis. Preliminary results with several toxicants were encouraging and warranted further
validation of the assay to standard whole organism toxicity tests. Results of these studies with copper,
sodium chloride, and pentachlorophenate are presented in this report.
INVESTIGATIVE PROGRAM
General Description of System
The DaphniaQuant™ instrument (see Figure 1) effectively measures the electrical activity of cellular membranes as an alternative bioindicator of the sublethal effects of aqueous contaminants. In the
DaphniaQuant™ system, changes in cellular membrane potential of live, free-swimming Daphnia
were recorded following exposure to a given toxicant or complex mixture and compared to an
unexposed control treatment.2 Changes in the cellular membrane potential were measured by the
intensity of fluorescence of an electrochromic membrane bound dye, di-4-ANEPPS. Di-4-ANEPPS
was spiked into the test solution and passively adsorbed into the lipophilic portions of daphnid
membranes within minutes of exposure. Exposure to contaminants which affect membrane transport
kinetics causes rapid changes in intracellular ion concentrations. These changes resulted in a corresponding change in fluorescence of the dye. Changes in fluorescence as the result of exposure to
different concentrations of a given toxicant were determined by calculating a ratio of the fluorescence
change caused by changes in the membrane potential to the background fluorescence of the dye. Both
the fluorescence intensity changes caused by alteration of the membrane potential and the background fluorescence were corrected for nominal electronic noise of the instrument prior to the ratio
49th Purdue Industrial Waste Conference Proceedings, 1994 Lewis Publishers, Chelsea, Michigan 48118. Printed in
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