2004 Progress Report: Effectiveness of UV Irradiation for Pathogen Inactivation in Surface Waters

EPA Grant Number: R829012
Title: Effectiveness of UV Irradiation for Pathogen Inactivation in Surface Waters
Investigators: Linden, Karl G. , Sobsey, Mark D.
Institution: Duke University , University of North Carolina at Chapel Hill
EPA Project Officer: Page, Angela
Project Period: August 20, 2001 through August 19, 2004 (Extended to August 19, 2005)
Project Period Covered by this Report: August 20, 2003 through August 19, 2004
Project Amount: $524,848
RFA: Drinking Water (2000) RFA Text |  Recipients Lists
Research Category: Drinking Water , Water


Ultraviolet ( UV) now is recognized to be an inexpensive and relatively easy means to achieve disinfection of Cryptosporidium parvum and does not appear to produce disinfection byproducts at practical doses. The germicidal effects of UV against emerging pathogens and challenges related to the application of UV disinfection for filtered and unfiltered surface waters needs to be assessed. The objectives of the research project are to:

  1. evaluate the susceptibility, repair potential, and resistance of select Contaminant Candidate List (CCL) pathogens and indicators to UV disinfection from low- and medium-pressure UV sources;
  2. and elucidate the relative germicidal effectiveness of different wavelengths of UV for these pathogens and indicators.

The extent to which microbes are associated with water treatment particles typical in unfiltered systems and the effects of this particle association and other water quality parameters on UV disinfection potential will be investigated. It is hypothesized that UV will be an effective means to inactivate CCL pathogens and that with proper system design, repair and reactivation and water quality challenges found in typical treatment scenarios will not compromise effective UV treatment.

Progress Summary:

Phase I. UV Dose Response for Pathogens—Basic Information

We have completed most of the work proposed in this phase. In this last period, we have confirmed our earlier work on inactivation of adenovirus and completed studies on Mycobacterium, wherein we determined a UV dose of 10 mJ/cm2 was capable of inactivating 2 log of Mycobacterium, with an overall inactivation rate constant of approximately 0.16 cm2/mJ. We did not identify any differences for inactivation of the microbes under study in laboratory waters or field waters. In the early phases of this research project, we observed that bacteriophage PRD-1 was very resistant to both low- and medium-pressure UV. A UV dose of more than 80 mJ/cm2 was required to achieve 4 log10 inactivation of this bacteriophage. Because t he required UV dose to achieve a 4 log10 inactivation of PRD-1 in our study was significantly higher than previously reported in the literature, we repeated this inactivation study. Although the results of the recent experiments were somewhat variable, the general trend was that PRD-1 was still very resistant to UV irradiation, and its resistance approached that of adenoviruses. Thus, it may be a better indicator of the survival of resistant viruses than bacteriophage MS2. A new plaque assay for the PRD-1 bacteriophage also was developed and tested. Instead of using the pathogen Salmonella as a host, which is current convention, a nontoxic Escherichia coli strain was integrated into the PRD-1 assay and successfully compared against the Salmonella host for assaying UV disinfection.

During this period, we began working with another enterovirus genus to substitute for the coxsackievirus. We now are using echovirus type 12, which also is a human enteric virus but does not cause human disease; thus, it is a safe model virus to work with and could be used in the laboratory as well as in the field in UV treatment systems. It also is possible to grow this virus to higher titers than was possible for coxsackievirus, and we have a robust and effective plaque assay for it. For these reasons, we believed that it was more important to work with than coxsackievirus. Pending time and resources, we also hope to complete work on Norwalk-like viruses (noroviruses).

Phase II. Wavelength Effectiveness

During this period, we developed a collaboration with the University of Vienna School of Veterinary Medicine and Institute for Medical Physics (Dipl.-Ing Alexander Cabaj) and were able to perform wavelength-dependent inactivation studies on two strains of Bacillus subtilis spores and MS2 coliphage. This work also was done with the assistance of professor Regina Sommer at the University of Vienna Institute for Hygiene and Medical Microbiology. In addition, we evaluated some adenovirus wavelength effectiveness data collected as part of another study by the principal investigator. These data collectively indicate that for viruses, the lower wavelengths (less than 230 nm) appear to be more effective for inactivation than UV 254 nm. These bench studies show the importance of the lower wavelengths, but implications for inactivation in UV reactors wherein these lower wavelengths will be filtered out in natural waters over long pathlengths need to be assessed. We also will test wavelength effectiveness for the echovirus type 12 with which we now are working.

Phase III. Pathogen Clumping and Particle Association

The work on particle clumping and association of microbes with particles was completed with Bacillus spores. As reported previously, we examined the UV dose response for indigenous and cultured strains of Bacillus spores and found that cultured strains were much more susceptible to UV. We identified hydrophobicity, surface charge, and particle size as important parameters relating to inactivation. In this period, efforts were focused on the effects of different coagulation conditions typically used in water treatment relating to the state of aggregation of the spores. Spores with and without addition of natural clay were examined. Aggregates were much more resistant to UV irradiation than dispersed spores. The measurement of absorbance in samples with high particles was found to be confounded when using conventional spectroscopy, but use of an integrating sphere absorbance method, which accounts for scattering resulting from particles in the water, corrected for this inaccuracy. This work was supplemented by some interesting microscopy work using scanning confocal, scanning electron, and atomic force microscopy to evaluate the state of aggregation of the spores.

In addition to the spore work, we have developed methods to prepare various cell-associated adenoviruses and echovirus, which were not included in the original proposal. Three different states of cell-associated viruses are being used to determine effect of cell association on UV disinfection.

Phase IV. Repair/Recovery of UV-Irradiated Pathogens

We have been utilizing DNA repair systems for both light and dark repair to determine the presence and extent of repair and recovery following UV irradiation. These systems have been verified with E. coli and were implemented with Mycobacterium spp. and Giardia cysts under the conditions typically found in clearwells, distribution systems, and aqueducts. Along with these repair systems, we also have established a molecular biology assay (endonuclease sensitive site assay) to better quantify the extent of DNA repair in the UV-irradiated microorganisms, including Mycobacterium and Giardia. We may try to implement this for Toxoplasma as well, pending success of the infectivity work.

Phase V. Development of Multibarrier Disinfectant Approaches Using UV and Chlorine

In the previous period, we utilized UV and chlorine laboratory exposure systems to evaluate sequential inactivation of adenovirus 2. Our results showed that, contrary to its response to UV irradiation, adenovirus 2 is highly sensitive to free chlorine. A significant inactivation (> 4 log10) of this adenovirus can be achieved by practical doses and contact time of UV irradiation and free chlorine. Recently, we have used this sequential disinfection setup to evaluate the inactivation of Mycobacterium spp. and also will be investigating echovirus and perhaps norovirus (depending on availability) and adenovirus type 41 in the coming period.

Future Activities:

We will look at the wavelength effectiveness of echovirus. Pending resources, we will try to examine two different UV wavelengths for Toxoplasma oocysts rather than the whole spectrum of wavelengths as originally proposed. We will continue and complete the study on the effect of cell association on UV disinfection against adenovirus and echovirus by cell culture. Also, we will complete the study on the effect of particle associat ion on UV disinfection against echovirus . We hope to finish the study to evaluate the presence and extent of repair and recovery of UV-irradiated Toxoplasma gondii oocysts under the conditions typically found in clearwells, distribution systems, and aqueducts. Infectivity assays will be employed in these studies. We will continue and complete sequential disinfection studies on the inactivation of adenoviruses, echovirus, Mycobacterium spp., T. gondii oocysts, and bacteriophage PRD-1.

Project Schedule

Although during this period we continued to experience a number of problems that minimized the progress of the Toxoplasma work, we again have oocysts on order and expect to continue this work in the coming months. All other work is being completed on or near schedule.

Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other project views: All 25 publications 12 publications in selected types All 12 journal articles
Type Citation Project Document Sources
Journal Article Brooks SS, Palmer MA, Cardinale BJ, Swan CM, Ribblett S. Assessing stream ecosystem rehabilitation: limitations of community structure data. Restoration Ecology 2002;10(1):156-168. R829012 (2004)
not available
Journal Article Ko G, Cromeans TL, Sobsey MD. Detection of infectious adenovirus in cell culture by mRNA reverse transcription-PCR. Applied and Environmental Microbiology 2003;69(12):7377-7384. R829012 (2004)
R829012 (Final)
  • Abstract from PubMed
  • Full-text:
  • Other: Applied and Environmental Microbiology PDF
  • Journal Article Mamane-Gravetz H, Linden KG. UV disinfection of indigenous aerobic spores: implications for UV reactor validation in unfiltered waters. Water Research 2004;38(12):2898-2906. R829012 (2003)
    R829012 (2004)
    R829012 (Final)
  • Abstract from PubMed
  • Full-text: Science Direct Full Text
  • Other: Science Direct PDF
  • Journal Article Mamane-Gravetz H, Linden KG. Relationship between physiochemical properties, aggregation and U.V. inactivation of isolated indigenous spores in water. Journal of Applied Microbiology 2005;98(2):351-363. R829012 (2004)
    R829012 (Final)
  • Abstract from PubMed
  • Supplemental Keywords:

    environmental engineering, UV inactivation, action spectra, aggregation, CCL microbes, ultraviolet, UV, Contaminant Candidate List, CCL, pathogens,, RFA, Scientific Discipline, Water, Environmental Chemistry, Health Risk Assessment, Ecological Risk Assessment, Ecology and Ecosystems, Drinking Water, Environmental Engineering, cryptosporidium parvum oocysts, pathogens, other - exposure, monitoring, CCL, chlorination, microbiological organisms, exposure and effects, disinfection byproducts (DPBs), exposure, UV treatment, Other - risk management, chlorine-based disinfection, cryptosporidium , public health, treatment, microbial risk management, water quality, DBP risk management, drinking water contaminants, drinking water treatment, Giardia, water treatment, contaminant candidate list, drinking water system, dosimetry

    Relevant Websites:

    http://www.cee.duke.edu/faculty/linden/index.php Exit
    http://www.unc.edu/~sobsey/ Exit

    Progress and Final Reports:

    Original Abstract
  • 2002 Progress Report
  • 2003 Progress Report
  • Final Report