Grantee Research Project Results

2006 Progress Report: Cyanobacteria and Cyanotoxins in Water Supply Reservoirs – to Develop and Validate a Microarray to Test for Cyanobacteria and Cyanotoxin Genes in Drinking Water Reservoirs as an Aid to Risk Assessment and Management of Water Supplies

EPA Grant Number: R831627
Title: Cyanobacteria and Cyanotoxins in Water Supply Reservoirs – to Develop and Validate a Microarray to Test for Cyanobacteria and Cyanotoxin Genes in Drinking Water Reservoirs as an Aid to Risk Assessment and Management of Water Supplies
Investigators: Rublee, Parke , Henrich, Vincent C. , Burkholder, Joann M.
Current Investigators: Rublee, Parke , Henrich, Vincent C. , Burkholder, Joann M. , Glasgow, Howard
Institution: University of North Carolina at Greensboro , North Carolina State University
EPA Project Officer: Aja, Hayley
Project Period: November 1, 2004 through October 31, 2007 (Extended to April 30, 2008)
Project Period Covered by this Report: November 1, 2005 through October 31, 2006
Project Amount: $594,982
RFA: Microbial Risk in Drinking Water (2003) RFA Text |  Recipients Lists
Research Category: Drinking Water , Human Health , Water

Objective:

The objective of this research project is to develop and validate a gene microarray for the detection of cyanobacteria and cyanotoxin genes in drinking water reservoirs. The microarray can be used to monitor drinking water supplies as an aid to risk assessment and management.

Progress Summary:

During Year 2, we continued field sampling, including the evaluation of the phytoplankton and especially the cyanobacterial community and assessment of microcystin concentrations. Additionally, we generated limited additional clone libraries from targeted samples with high cyanobacterial diversity or blooms and additional probe and primers sets to both known and novel cyanobacterial taxa from our samples. We will also continue to enhance our probe and primer collection from newly described taxa/sequences derived from recent literature. Testing and validation was step up for the probe and primer sets and the first version of the array was printed and preliminary test were conducted.

Sampling

Intensive sampling of targeted water supply reservoirs continued during the summer of 2006. Collections were made monthly as in summer months in 2004 and 2005. Sampling included 11 potable water supply reservoirs (reservoir, raw, and finished water). We also obtained some samples from a number of additional recreational reservoirs, some of which feed into water supply systems.

Cyanobacterial Counts and Microcystin Analyses

Cyanobacteria counts were determined in selected reservoirs where blooms occurred during 2006. Examples from water supply reservoirs in High Point, NC (City Lake and Oak Hollow Lake) are given in Table 1. Microcystin concentrations were determined in all sampled reservoirs. During 2006, concentrations were nearly always less than 0.5 μg l^-1, although concentrations in one central North Carolina reservoirs rose to 0.271 μg l^-1 in August in City Lake which is a eutrophic lake know to have a Lyngbya problem (Table 1). Physical and chemical data were recorded for all samples, establishing our database to look for correlations among cyanobacterial taxa and toxin genes that will be indicated on the microarray and environmental parameters.

One of the difficulties in development of cyanobacterial microarrays is being able to monitor events in which cyanobacteria are clearly problematic. During 2006, we encountered one unique event, a snail kill in Tuckertown Reservoir (Fig. 1), that may be related to decomposition of algal blooms resulting in depressed dissolved oxygen concentrations. We will be responsive to any cyanobacterial blooms during 2007 by increased sampling in order to document bloom dynamics.

Table 1. Total Microcystin concentrations in NC water supply lakes. Only concentrations above limit of detection (0.147 μg/L) are reported.

Date	Lake Source	Cell bound	Free	Total MIC
		μg/L	μg/L	μg/L
6/28/06	High Rock - 2	0.217	0.046	0.263
7/24/06	University Lake r1	0.051	0.136	0.188
8/24/06	High Point - 1	0.181	0.090	0.271
8/24/06	High Point - 2	0.132	0.086	0.218
8/24/06	High Point - 3	0.157	0.085	0.241
8/24/06	Oak Hollow - 1	0.093	0.121	0.214
8/24/06	Oak Hollow - 2	0.143	0.073	0.216
8/24/06	Oak Hollow - 3	0.106	0.097	0.203
8/24/06	High Point WTP-Raw	0.005	0.163	0.168

   

Figure 1. Snail kill in Tuckertown reservoir.

Clone libraries, Sequences

Small subunit (SSU) DNA clone libraries were generated for selected samples using cyanobacteria-specific primers. Two hundred clones were generated in libraries from six different lakes suspected of high cyanobacterial abundance during 2006 based on high chlorophyll a values. Sequences of 400 - 700 bp have been generated for many of these amplicons, but due to instrument problems they have not all been sequenced yet.

We spent some effort developing a novel approach for rapid validation of array probes. The approach should result in considerable savings of resources (time and supplies) during the validation step; increased efficiency in probe development will allow both increased numbers of probes and increased redundancy (i.e. multiple probes to the same target) which will aid in QA/QC issues. To date, we have not perfected this methodology despite considerable effort.

Future Activities:

The major objectives for our third year are virtually the same as those described above for Year 2, primarily resulting from the delays encountered in perfecting the probe validation technique.

As described in our proposal, development of the gene microarray will provide: a research tool for aquatic scientists to identify factors that promote growth of different cyanobacterial species and their toxins; a prototype early warning system for the presence of potentially harmful cyanobacteria and their toxins which should be of use to water supply managers in order to reduce health risks; and the prototype will serve as the basis for development of a more “user-friendly” real-time (or near real-time) monitoring platform.

Journal Articles:

No journal articles submitted with this report: View all 33 publications for this project

Supplemental Keywords:

RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Water, Health Risk Assessment, Environmental Chemistry, Drinking Water, Environmental Engineering, Environmental Monitoring, microbial contamination, gene microarray assay, early warning, microbial risk assessment, monitoring, aquatic organisms, drinking water system, drinking water contaminants, other - risk assessment, cyanobacteria

Relevant Websites:

http://www.ncsu.edu/wq/ Exit

Progress and Final Reports:

Original Abstract

2005 Progress Report

2007 Progress Report

Final Report