A Respirometric Screening Test
For Toxic Substances
OWEN SLETTEN, Director
Division of Environmental Engineering
Nebraska State Department of Health
Lincoln, Nebraska
N. C. BURBANK, JR., Professor
Department of Civil Engineering
University of Hawaii
Honolulu, Hawaii
INTRODUCTION
This presentation is an abridgement of a reported investivation into the use of in vitro
techniques for evaluation of toxic materials and comparing results thus obtained with the
standard fish bioassay. The fish bioassay test involves the exposure of fish to various
concentrations of known or suspected toxicants to determine the median tolerance limit
(TLm), the concentration of material at which just 50 per cent of the test fish survive for a
specified period of exposure. The exposure period may be 24, 48, or 96 hours. Such
procedures, though well-accepted and effective, require significant time periods for
evaluation of toxicity. The present report describes a screening test intended to yield
findings similar to those one would expect in the standard fish bioassay.
The proposed in vitro method, employing manometric assay of tissue respiration, is a
new and non-traditional technique. It is inexpensive to operate and results may be obtained
in hours rather than in days.
The basis of the proposed in vitro technique is evaluation of the effect of chemical
pollutants on the rate of oxygen utilization by fish tissues in a Warburg respirometer.
A standard Warburg apparatus, with 18 manometers and specially-designed 15 ml
reaction flasks, had been set up to measure oxygen uptake in 3-hr runs and to compare
amounts between controls and samples. Various tissues, such as brain, heart, gill, and liver
from the bluegill and trout, minced or made into homogenates, were selected for controls.
The samples were various concentrations of phenol, o-clorophenol, o-nitrophenol, and
intact as well as fractionated components of carbon-absorbed extractables, exposed to
control replicates. The median tolerance limit in the manometric assay was defined as that
amount of chemical compound exposed to the tissue yielding half as much oxygen uptake
as the control.
IMPLEMENTS AND REAGENTS
Warburg respirometer
A Warburg apparatus, equipped with 18 manometers, manufactured by Gilson Medical
Electronics, Middleton, Wisconsin.
Warburg reaction flasks
The flasks were specially designed Pyrex erlenmeyers, fitted with ground-glass openings to
seat standard 14/20 tapered stopper or joint. Fused to the bottom of the flasks were
centerwells approximately 18 mm in height and 7 mm in diameter. The over-all capacity of
the reaction flasks was about 14.7 ml. Before use all were carefully calibrated for
determination of true volumes.
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