Separation of pH, dilution, lonic strength and chemical matrix effects for biological monitoring of urines with the Microtox® test using nicotine, cotinine and reference urines

Abstract

The aim was to investigate the factors influencing light emission from Photobacterium phosphoreum in the Microtox test to interpret bioassay results for urine. Four reference urines were assessed as reference materials for the bioassay. Nicotine and cotinine were investigated as urinary markers for tobacco exposure. The optimum luminescence conditions were: 1.85%-3.25% NaCl, 0.33-0.58 mol/L ionic strength, and pH 5.8-6.7. Low pH values and high concentration of toxic trace metals were important factors in this study. Unexpected toxicity for a Standard Reference Material was attributed to zinc contamination. Nicotine and cotinine together exhibited antagonistic effects in 2% saline but this could not be observed in the urines because of substantial urine toxicity. Thus practical urinary biological monitoring with the Microtox test necessitates excretion of metabolites and compounds that are much more toxic than the urine components. Also, separation of the effects of physical factors like pH, ionic strength and dilution is essential before chemical toxicity effects can be assigned. This is the first report of Microtox EC50 values for nicotine and cotinine. The results have application to environmental samples since analyses are often uncontrolled relative to pH, ionic strength and dilution.

Mutagenesis and Acute Toxicity Studies on Saliva-Leached Components of Chewing Tobacco and Simulated Urine Using Bioluminescent Bacteria

“…The acute test dilution medium was 2.0% saline. Extensive testing (4) showed that the optimum bioluminescence at 5, 15 and 25 min occurred over the sodium chloride range of 1.9-3.3% (ionic strengths of 0.32-0.58 M) with EC 50 values of 1.3% and 5.0%. Thus sample sizes in toxicity testing should be kept small relative to growth medium.…”

“…The effective concentration at which the luminescence is 50% that of the control (EC 50 ) is the diagnostic parameter for the acute test. The 5 to 30 min EC 50 for the acute test (the Microtox test) is correlated to Draize Test results for eye irritation, the acute LD 50 for Salmo gairdneri, Daphnia, Spirillum, and the 14-day LD 50 for guppies (Poecilia reticulata) (4). The chronic test 24-h Lowest Observed Effective Concentration (LOEC) is correlated to 7-day Ceriodaphnia dubia data (3).…”

“…The reverse mutation assay of a dark mutant of the bioluminescent bacterium Vibrio fischeri to regain the bioluminescent state detects all these mechanisms sensitively (2). The same instrumentation is used to evaluate acute and chronic toxicity using bioluminescent Photobacterium phosphoreum (3,4). The effective concentration at which the luminescence is 50% that of the control (EC 50 ) is the diagnostic parameter for the acute test.…”

“…The aims of the present paper are to review the author's research with bioluminescent bacteria in assessing the acute toxicity of simulated biological media like urine and saliva in 5.0-mL volumes using Photobacteriumphosphoreum (4)(5)(6)(7)(8) and genotoxicity with a dark mutant of Vibrio fischeri in O.200 mL volumes (2). The measurements were performed on the Microtox Model 500 Analyser (Microbics Corp., Carlsbad, CA) at 15°C for the acute test, and at 27°C for the genotoxicity test.…”

Effect of Dust on the Viability of Vibrio fischeri in the Microtox Test

“…The bacterium is standardized by storing and transporting it as a freeze-dried powder that is reconstituted into a saline growth medium at 153C at the time of the analysis. Our research group has published on the variables that a!ect the test Que Hee, 1992, 1993;Chen and Que Hee, 1995) and on some applications (Chou and Que Hee, 1992, 1993, 1994aChen and Que Hee, 1995;Que Hee, 1997). The present study explores the dependence of light output on viable cell number, the e!ects of dust on the Microtox test and cell viability, how the viability of the cells is a!ected after spills, and how spills can be sampled.…”