Analytical Services May 19, 2009 Gretchen Grover –Lab Manager and Analyst Christal Wade - Principal Cryptosporidium

1. Analytical Services May 19, 2009 Gretchen Grover –Lab Manager and Analyst Christal Wade - Principal Cryptosporidium Analyst Jonathan Oglesby-Cryptosporidium Analyst Rick Fowler – Environmental Biotechnologist, Inorganic Analyst Jana Fattic – CWRS Associate Director, WATERS Director, Lab Analyst

2. Other WATERS Employees • Office Associate: Kristy Mayes • Part Time: Liz Thomas (Cryptosporidium Technician), Samantha Kramer (Chemist) • Students: KrissLeftwich, Daniel Nedvidek, Catherine Skees, Jennifer Ludden (Gatton Academy), Ben Fowler (IC, AA student analyst)

3. Customers • Private • Total Coliform/E. Coli, Metals, Ions, Atrazine • Wastewater • BOD, pH, TRC, DO, TSS, Fecal Coliform, Ammonia Nitrogen • Storm water • COD, Oil and Grease, TDS, TSS • Drinking water • Total Coliform/E.Coli • Source water • Cryptosporidium • Oil Wells • pH, TDS, Specific Gravity, Chloride

4. Certifications • Drinking Water Certification • Inorganic Certification • Cryptosporidium

5. Drinking Water • KY Microbiology Laboratory Certification Program (KMLCP) • Renewed January 2009 • Currently the largest (KMLCP) Certified Analytical Staff in the State • Christal Wade • Jonathan Olglesby • Jana Fattic • Rick Fowler • Gretchen Grover • Liz Thomas

6. Inorganics Rick Fowler • Computer-controlled automated instruments • Operators: One staff supervisor, one student analyst • Ion Exchange Chromatography • Dionex DX600 system with background suppression and detection by conductivity or UV-Vis absorbance • Anions (F, Cl, Br, NO2, NO3, PO4, SO4) and cations (Li, Na, K, Mg, Ca, and NH4+), MDL <0.05-0.1 ppm • Atomic Absorption Spectroscopy • Varian AA240FS with graphite furnace and flame capabilities • Trace metals, MDL <0.0005-0.001 ppm Gretchen Grover • pH, alkalinity, turbidity, TOC, TDS, odor

7. Cryptosporidium • WATERS Lab received approval from EPA for the analysis of Cryptosporidium on October 18, 2006. • Long Term 2 Enhanced Surface Water Treatment Rule (LT2) • Currently approximately 70 clients from 8 states:KY, TN, WV, VA, OH, NC, LA, MD • Crypto Program bills out ~$25,000 each month • Accept outside samples under LT2 and provides field filtration apparatus • www.epa.gov/safewater/disinfection/LT2/lab_aprvlabs.html

8. Projects • Mammoth Cave • Flood Pulse Event • City of Bowling Green • River & Cave System monitoring • Watershed Watch • Upper Green River watershed monitoring (Turbidity and Atrazine) • TMDL • Analyzing samples for this project • University Research • Support WKU faculty as well as other university projects • Cave monitoring (atrazine, solids) • Wastewater treatment/nanoparticles (TOC) • DOW • Data Collection for Nutrient Criteria Development in the Karst Pennyroyal Region of KY • About 50 sites, 3 sampling events, in stream water quality data (pH, DO, temp) and lab analyses (TP, NH3, TKN)

9. Projects completed • Mammoth Cave sediments bacterial community analysis (2000-2008) • NCKRI artificial substrates bacterial community analysis (2002-2008) • Final report for both projects combined into one report published on the Karst Information Portal and available at www.wku.edu/~rick.fowler/mammoth • Information gained from these studies led to discovery of fecal contamination resulting in multimillion dollar infrastructure reformations at MCNP visitor center

10. Projects completed (continued) • Oregon Caves National Monument tourist trail impact on microbial communities (2002-2006) • Identified numerous bacteria, fungi, and protozoans and discovered sulfur bacteria later observed by other investigators now believed to be widespread in caves • Louisville Water Company • Ammonia Oxidizing Bacteria in distribution system • Acanthamoeba in eyewash stations • KSU Aquaculture Research Center • DNA analysis of bacteria, algae, and fungi used to evaluate conditions for optimal shrimp production

11. New projects • USDA Animal Waste Research Station • Develop methods for identification of animal origins of fecal contamination from human and agricultural sources • Study transport of pathogens from the surface into subsurface aquifers due to agricultural practices on karst landscapes • Institute research and training programs at the newly founded Crump’s Cave preserve near Mammoth Cave National Park

12. Environmental Biotechnology • DNA extraction from water, soil, membrane filters, pure cultures, swabs, etc. • Yield of total DNA measured with Qubitfluorometer, sensitivity 0.2 x 10-12 g • Quantitative Real-Time PCR • Determines concentration of DNA from specific organisms in a mixture, sensitivity <1 ppt • DNA cloning and sequencing • Cloning permits segregation of individual species comprising environmental microbial communities • DNA sequencing yields unique gene sequences for identification of organisms

13. Fungi Parasites Bacteria Amoebae Algae Zooplankton Organisms analyzed with DNA

14. Scientific Outreach, Presentations, and Workshops • National Cave and Karst Research Institute (NCKRI) • 2007-present (Advisory board member) • China Environmental Health Project • 2007 (International Karst Workshop, ChongQing) • Crump’s Cave Farm Day • 2009 (Agricultural practices outreach and education) • International Karst Research Institute • 2009 (17th International Karstological School, Postojna, Slovenia) • International Congress of Speleology • 2009 (Kerrville, Texas) • Career Day for Middle School Students

15. Important Purchases Microbiology • Microcentrifuge Chemistry • Hach LBOD • Konelab • Hach Spec (COD, Surfactants, TRC) • GC MS

16. Future Capabilities • Unregulated Contaminant Monitoring Rule (UCMR2) • Organics (Work toward a GC MS/MS and HPLC) • Direct the Chemical Laboratory Certification Program for Kentucky • Voluntary Wastewater Certification • Pick up service • Consultation services • Sample collection services