Principal Investigator: Diana L. Bittner (Georgia Institute of Technology)
Principal Investigator: Greta Olsen (Georgia Institute of Technology)
Principal Investigator: Kenneth D. Hughes (Georgia Institute of Technology)
Sponsor: GWRI
Start Date: 1995-04-01; Completion Date: 1996-04-01;
Keywords: fluorescence, rapid toxicity assays, enzyme activity, selectivity
Description:
The use of microorganisms and small aquatic organisms as monitors of water and sediment quality is increasing and therefore the development of improved methodology and new technologies that facilitate the handling of these organisms is a high priority. Methodological changes that have occurred in the last several years have included a concerted effort to monitor changes in organism health at the molecular level. This is in stark contrast to assays that simply measure a single data point such as the LC50. The ultimate goal in these molecular level assays is to rapidly ascertain the very first sign of stress in an organism exposed to a water or sediment based toxicant. One of the first indicators of stress in many organisms is a perturbation in enzyme activity associated with metabolism or detoxification. Perturbations in enzyme activity may be monitored with high sensitivity by utilizing fluorescence techniques.
Design of rapid toxicity assays for accessing water and sediment quality demands simple experimental protocols that do not involve complicated fluorogenic substrate delivery mechanisms, time consuming steps for the separation and isolation of the resulting fluorophore, and expensive fluorescence intensity measurement instrumentation. Improvements in any one of these areas ultimately translate into more sensitive and reliable ecological data. Recently, fluorogenic substrates incorporating micron-diameter polymeric particles have been synthesized and investigated as a novel means of quantitating enzyme activity in the digestive tract of microorganisms. This project involved obtaining initial experimental evidence that this new technology (enzyme probes) can reduce, and in some cases eliminate, the limitations of using soluble enzyme substrates for toxicity measurements in whole organisms. Brachionus calyciflorous (rotifer) and Nannochloris (green algae) were used in these studies. It was clearly demonstrated that the enzyme probes based upon micron-diameter particles could be ingested by the rotifers, facilitating simultaneous measurement of ingestion rate and enzyme activity. These enzyme probes, however, were not accessible to the Nannochloris cells. These results provide evidence that enzyme probes based upon microndiameter particles will be useful in the targeting of rotifers and similar organisms in the presence of single cell organisms in aquaculture and field samples.