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

Abstract

The standard Microtox test involving the bioluminescent bacterium Vibrio fischeri is a frequently used ecotoxicological bioassay whose EC50 values have been correlated to acute toxicity parameters of vertebrates, to irritancy measures, and to cytotoxicity indices. The aims were to explore the dependence of light output on viable cell number, with the latter estimated with the naked eye using a colorimetric tetrazolium salt method, the effects of dust on the bioluminescence and cell viability, how the viability of the cells is affected after spills, and how spills can be sampled. The lower limit of the linear dynamic range of the light-emitting bacterium was first defined to be 3.7×107 cells/mL, compared with 37×107 cells/mL in the Microtox assay. The effects of dust were then explored in the working range by the method of standard additions by adding 5-, 10-, and 20-mg amounts of Standard Reference Material Urban Dust 1649a. This simulated dust samples collected by a cordless vacuum technique involving a filter cassette. A mass of 20 mg dust totally inhibited the Microtox test at all times (5, 15, and 30 min). Masses of 5 and 10 mg dust lowered the luminescence significantly by 20 and 64%, respectively, after 30 min. However, the viability test was totally inhibited by 5 mg of dust. A spectrophotometric modification of the viability test using a wavelength of 508 nm was developed that was twice as sensitive as the naked eye test, and was as sensitive as the Microtox test. Mechanical shock involved with spilling and sampling bacterial reagent on hard surfaces killed the luminescent bacteria as shown by inhibition of luminescence. The optimum filter cassette for Microtox reagent collection was a 25-mm 1.00-μm PTFE filter in a 25-mm Delrin holder operated at 4.0 L/min, with a Tygon sampling probe.