1. Transport of Viruses, Bacteria, and Protozoa in Groundwater Joe Ryan Civil, Environmental, and Architectural Engineering Department University of Colorado, Boulder Environmental Engineering Seminar October 11, 2000

2. Acknowledgments Students University of Colorado: Jon Loveland, Jeff Aronheim, Annie Pieper, Becky Ard, Robin Magelky, Jon Larson, Theresa Navigato, Yvonne Bogatsu UCLA/Yale University: Jun Long, Ning Sun, Chun-han Ko Collaborators Ron Harvey, U.S. Geological Survey Menachem Elimelech, Yale University Funding National Water Research Institute U.S. Environmental Protection Agency Laboratory Assistance Chuck Gerba, University of Arizona Joan Rose, University of South Florida Field Assistance Denis LeBlanc & Kathy Hess, U.S. Geological Survey

3. Public Health Problem Waterborne Disease Outbreaks estimates for the United States 1 to 6 million illnesses per year 1000 to 10,000 deaths per year only 630 documented outbreaks 1971-1994 Milwaukee, Wisconsin, 1993 Cryptosporidium, the “hidden germ” about 400,000 illnesses, greater than 100 deaths DNA evidence: human, not bovine, origin

4. Public Health Problem Waterborne Disease Outbreaks acute gastrointestinal illness short duration, “self-resolving” for most people chronic, severe, fatal for some infants and elderly pregnant women immuno-compromised more serious illnesses heart disease, meningitis, diabetes (coxsackie virus) liver damage, death (hepatitus virus)

5. Public Health Problem Microbial Perpetrators viruses bacteria protozoa Where are they coming from? groundwater (58%), surface water point source, non-point source

6. Viruses Enteric replicate only in gut Size 20 – 200 nm Structure protein capsid RNA or DNA virushealth effect coxsackie “hoof and mouth” echo, adeno respiratory disease Norwalk, rota, calici, astro gastroenteritis hepatitis A hepatitis E jaundice, liver damage, death

7. Viruses Life Cycle ingestion drinking water within the gut adsorption penetration transcription replication assembly host cell lysis excretion from gut

8. Bacteria Enteric grow in gut (only?) Size 0.5 to 2  m Structure cell walls proteins phospholipids, fatty acids motililty flagellae cilia bacteriumhealth effect Escherichia coli, Shigella spp., Camplylobacter jejuni, Yersinia spp. gastroenteritis (arthritis, pneumonia, Guillain-Barre syndrome) Salmonella spp. enterocolitis (heart disease, meningitis, arthritis, pneumonia) Legionella spp. Legionnaire’s disease, Pontiac fever, death Vibrio cholera diarrhea, vomiting, death

9. Bacteria Life Cycle ingestion meat, vegetables, drinking water within the gut adsorption penetration growth release of toxins excretion from gut Vibrio Cholera adhering to rabbit villusE. coli adhering to calf villus

10. Protozoa Enteric grow in gut only Size 3 to 12  m Cyst Structure rugged protective membrane carries trophozoites protozoanhealth effect Cryptosporidium parvum diarrhea Giardia lamblia chronic diarrhea

11. Protozoa Life Cycle ingestion drinking water within the gut excystation parasitic growth cyst formation excretion from gut

12. Occurrence in Groundwater Viruses 38% positive by PCR 7% positive by cell culture Bacteria 40% positive for coliform bacteria 50-70% positive for enterococci Protozoa 12% Giardia and/or Cryptosporidium (5% in vertical wells)

13. Monitoring in Groundwater Maximum Contaminant Level coliform bacteria – 40 per liter viruses – 2 per 10 7 L (proposed, GWDR) Ground Water Disinfection Rule will require disinfection unless “proof” of adequate “natural disinfection” viruses nominated as target microbe Virus Transport Models predictions of travel time attachment and inactivation

14. Microbe Transport

15. Transport equation dispersion advection kinetic attachment/ release equilibrium attachment/ release growth or inactivation/ “die-off”

16. Microbe Attachment Attachment kinetic colloid filtration collision frequency  collision efficiency  release first-order (k det ) much slower than attachment equilibrium distribution coefficient linear, reversible time concentration tracer microbe time concentration tracer microbe

17. Microbe Attachment Surface Chemistry capsids, cell walls carboxyl – RCOO - amine – RNH 3 + net surface charge usually negative pH pzc ~3-4 for viruses, pH pzc can be estimated from protein content of capsid

18. Microbe Attachment Porous Media Surface Chemistry negative quartz, feldspars, etc. clay faces positive iron, aluminum oxides clay edges electrostatic interactions favorable deposition sites unfavorable deposition sites

19. Microbe Attachment Microbe Size small collisions caused by Brownian motion large collisions caused by settling Microbe Density Range 1.01 to 1.05 g cm -3 collisions caused by settling

20. Microbe Attachment Optimal Size for Transport about 1-2  m bacteria viruses collide by diffusion protozoa collide by settling protozoa also removed by straining

21. Microbe Attachment Target Organism collision efficiency about the same for all microbes variation in  comes from porous media collision frequency favors bacteria BACTERIA, but… adhesion favored for growth biofilms

22. Virus Attachment Bacteriophage PRD1 Cape Cod field experiments sewage- contaminated zone uncontaminated zone 100 L injections multi-level samplers

23. Virus Attachment Transport favored in contaminated zone PRD1 attachment sites blocked by sewage organic matter collision efficiency  fraction of favorable deposition sites

24. Microbe Growth/Inactivation Growth viruses – no replication outside gut bacteria – growth possible, but unlikely protozoa – no growth outside gut

25. Microbe Growth/Inactivation Inactivation viruses – mainly temperature- dependent bacteria – lysis? predation? protozoa – generally resistant to disinfection, so inactivation is slow?

26. Virus Inactivation Viruses inactivation in solution first-order decay inactivation on surfaces? effect of strong attachment forces

27. Virus Inactivation Bacteriophage MS2 Cape Cod sediment 32 P DNA 35 S protein capsid rapid loss of infectivity release of radiolabels

28. Summary Predicting microbe transport less difficult for viruses, protozoa cysts no growth, inactivation simpler more difficult for bacteria motility adhesion behavior motivated by growth, nutrients growth, die-off more complicated Bacteria should be target organism (?) least frequent collisions, motility may be complicated by longer-term adhesion strategies