Determination of Geno- and Cytotoxicity of Groundwater and Sediments Using the Recombinant SWITCH Test

Abstract

The recombinant Salmonella typhimurium TA1535 strain carrying the SWITCH plasmid (combined construct of the SOS-Lux plasmid pPLS-1 and the LAC-Fluoro plasmid pGFPuv: pSWITCH) was treated with control substances for genotoxic (2-aminoanthracene) and cytotoxic (aureomycin) potency as well as with 18 environmental samples (groundwater, river water, sediments) provided at the SENSPOL Technical Meeting on Problems Related to Diffuse Pollution Sources (Characterization of Sediment, Dredged Material, and Groundwater) organized by the Federal Institute of Hydrology in Koblenz, Germany, during late October 2003. For metabolic activation the samples were treated with S9 mix (5% S9 fraction in cofactor mix, Aroclor 1254-induced rat liver microsomes, Moltox Inc., Mol, Belgium). Simultaneously determined cytotoxicity and genotoxicity data were derived through the Multilabel Counter 1420 Victor² (PerkinElmer, Boston, MA, USA), by sequential measurement of luminescence, absorbance, and fluorescence. The newly designed SWITCH test, as it was applied at the Koblenz meeting, displays a comparable sensitivity for test samples with known cyto- and genotoxic potential. Groundwater samples from the wells at the former gas plant site Kiel Canal and the agricultural area at Niederwerth expressed neither genotoxic nor cytotoxic responses of the bacteria for both metabolic conditions (+/−S9). Spiked groundwater samples from the Niederwerth well BW1 and the Urmitz well U12 located on the river Rhine were identified to be positive in terms of genotoxicity for the direct and the metabolic approach. Samples from the lake Tiefer See in the city of Potsdam showed a reduction of GFPuv expression as an indication for cytotoxicity, while luminescence output of incubated bacteria remained unaffected. This reflects the well-known presence of contaminations (especially cyanides) in the lake sediment as well as in the acetonic extract. The results obtained at the SENSPOL Technical Meeting show the SWITCH test to be of major relevance not only for the analysis of chemicals under laboratory conditions but also for environmental samples polluted by diffuse industrial sources.

Keywords:

• SOS response
• Bioluminescence
• Fluorescence
• Recombinant bacteria
• GFP
• Genotoxicity
• Cytotoxicity

ACKNOWLEDGMENTS

The authors wish to thank the BIOSET Concerted Action (EC Environment and Climate Program) for the promotion of the study. We especially thank Dr. Susan Alcock for supporting our participation at the SENSPOL Technical Workshop held in Koblenz in October 2003. We also thank the Federal Institute of Hydrology, Koblenz, Germany, for providing the environmental samples for the experiments. The method for the quantification of genotoxic agents by means of the SOS-Lux test as well as the plasmid pPLS-1 itself was patented by the German Aerospace Centre (DLR): (1) German Patent, N 19544374.8, 23.08.96 and (eq2) German Patent, N 19549417.2-41, 05.06.97.

Notes

*This paper was presented in the third workshop organized by SedNet working group on “Monitoring Sediment Quality at River Basin Scale” held at IPIMAR, Lisbon, Portugal, January 29–31, 2004.