Evaluation of a microplate assay specific for heavy metal toxicity

Abstract

Abstract.A rapid, quantitative microbial assay, which is specific for heavy metal toxicity, has been developed. The assay (MetPLATE TM) is in a 96-well microtitration plate format and is suitable for determining toxicity characteristics such as median inhibitory concentrations. The sensitivity of MetPLATE TM to heavy metals [Cu, Zn, Cd, Pb, Hg, Cr(III)] was generally higher than Microtox and was of the same order as or better than Daphnia and fish bioassay. MetPLATE was insensitive to organic compounds at concentrations higher than those found in the environment. Six out of 10 industrial wastewaters or process waters surveyed were toxic. Heavy metal analysis of these waters confirmed the presence of heavy metals in the toxic samples. MetPLATE can be run concurrently with other assays for general toxicity to help determine the nature of chemicals causing toxicity.The deleterious effect of chemicals in the environment has traditionally been assessed with indicator organisms such as fish and invertebrates. Due to the large inventory of chemicals and the time-consuming nature of the traditional bioassays, microbioassays based on protozoa, microalgae, bacteria, and enzymes have become increasingly popular for the screening of environmental toxicants Dutka and Bitton, 1986). The trend in microbioassays is to package the assays in test kits, which facilitates application of toxicityscreening assays in the field or at smaller laboratories (Bitton and Koopman 1992).A limited number of test kits are presently available. The ECHA Biocide Monitor is a qualitative test based on inhibition of dehydrogenase activity and requires 18-24 h for completion (Dutka and Gorrie 1989). The Polytox kit includes a mixture of bacterial cultures, isolated from wastewater, which are used in respirometric tests (Elnabarawy et al. 1988). The Toxi-chromo Test is based on the inhibition of the biosynthesis of 13-galactosidase in Escherichia coli (Reinhartz et al. 1987). Both the Polytox and Toxi-chromo tests are quantitative tests, because they enable the determination of ECsos.Correspondence to: G. Bitton A common feature of currently available microbioassay kits is that they test for general toxicity, i.e., they respond to both heavy metal and organic toxicants. Apparently, hydrolases, particularly [3-galactosidase, are not sensitive to toxic organic compounds. In fact, researchers are still assaying for [3-galactosidase activity in the presence of sodium dodecyl sulfate, an organic chemical that is highly toxic to many of the test organisms used in toxicity testing protocols. [3-Galactosidase is also stable in pure acetone (Bitton, Kolar, and Koopman, unpublished data). This prompted investigation of the sensitivity of this enzyme to organic and inorganic toxicants. The results show that, indeed, [3-galactosidase is insensitive to all of the organic toxicants tested, while being sensitive to heavy metals (Dutton et al. 1988;Bitton et al. 1992aBitton et al. , 1992b.A qualitative assay that is specific for heavy metal toxicity and is not affect...

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“…Cenci et al (1987) also reported that phenol and some chlorophenols did not inhibit β-galactosidase activity of Escherichia coli even at high concentration. Similar insensitivity of β-galactosidase activity to phenol has also been confirmed through a number of investigations (Bitton et al, 1992a(Bitton et al, , 1992bBitton and Koopman, 1994). Similarly, Barnhart and Vestal (1983) reported that in vitro α-glucosidase activity was relatively insensitive to pentachlorophenol, phenol and sodium dodecylsulphate.…”

“…Inhibition of biosynthesis and activity of α-glucosidase and β-galactosidase in bacteria have been used to assess toxicity of inorganic and organic toxicants (Barnhart and Vestal, 1983;Dutton et al, 1988;Dutton et al, 1990;Guven et al, 2003;Bitton and Koopman, 1994;Codina et al, 1994, Nweke and. It was previously reported that bioassay based on α-glucosidase and β-galactosidase activity is insensitive to heavy metals (Barnhart and Vestal, 1983;Dutton et al, 1988Dutton et al, , 1990.…”

Toxicity of Zinc to Heterotrophic Bacteria From a Tropical River Sediment

“…In a MetPLATE ™ assay system (based on β-galactosidase activity), zinc IC 50 of 0.11 ± 0.001 mg/l (0.002mM) was reported for Escherichia coli [5]. In a growth inhibition test assessed via turbidity measurements, 7.15 mg/l Zn 2+ (0.12mM) inhibited the growth of Pseudomonas putida by 50 % [34].…”

A Physical and Chemical Assessment of the Maude Monroe Mine Site

“…welled METPlate and with E.coli as the indicator species (Bitton, et al, 1994). In the procedure a chromophore buffer was added, followed by a 90 minute incubation period.…”