Summary: While being a reliable tool for drinking water disinfection, UV light is difficult to measure for effectiveness. In fact, no method provides precise results. Yet, by using a bioassay, relatively accurate UV doses can be assigned, which result in a better comprehension of the method's dynamics.

Ultraviolet (UV) light is known to be an effective disinfectant of most microorganisms by damaging an organism's DNA thus preventing the organism from multiplying, and making it incapable of causing infection in a host. Due to varying absorbance characteristics of different organisms and abilities of organisms to repair their DNA following exposure to UV, the amount (dose) of UV required to cause irreversible damage varies among species.

UV dose is expressed as millijoules per square centimeter (mJ/cm2), which is equal to milliWatt-seconds per square centimeter (mWsec/cm2) or 1,000 microWatt-seconds per square centimeter (uWsec/cm2). For example, to achieve 4-log (99.99 percent) inactivation of vegetative bacteria, such as Escherichia coli, 5-to-10 mJ/cm2 are necessary. To achieve the same level of inactivation of spore forming bacteria and viruses, 25-to-50 mJ/cm2 are needed. Recent studies have shown that protozoa (Giardia and Cryptosporidium) are quite sensitive to UV, so that 2-to-10 mJ/cm2 would provide at least 4-log inactivation.[1,2]

James Bolton and Larry Henke provided an excellent review of UV disinfection in a 1999 WC&P article.[3] It's useful to reiterate two of their points. First, UV is a physical process. Photons of germicidal UV (those roughly in the 260-to-265 nanometer [nm] range) impact the DNA of target microorganisms. The result, or photoproduct, of this particular interaction is existing bonds within the DNA are broken and new ones (thymine dimers) are formed. As the DNA strands are now altered, they're incapable of proceeding through steps necessary for replication, and thereby the organism is no longer infectious. Second, UV dose is defined as the amount of UV light, or irradiance, reaching an object multiplied by the exposure time, in seconds. In UV reactors, the primary factors that influence UV dose are the output of the UV lamp(s), the spatial relationship of the lamp(s) to the chamber, retention time, and water quality (especially parameters contributing to UV absorbance). Particulate matter and/or turbidity can reduce UV performance so effective pretreatment may be required in some instances.

Need for a UV tool
The present discussion focuses on the need for and means of determining what UV dose is applied to the water containing microbiological contaminants as it flows through a UV reactor. Water treatment system designers, operators, regulators and customers need to have a measure of the level of disinfection applied to a water stream. When commonly used chemicals such as chlorine or ozone are applied for water disinfection, their presence, or residual, can be measured. The water treatment support industry has provided a wide range of analytical procedures-from quick, inexpensive colorimeters to on-line analyzers-so that interested parties can obtain precise information on concentrations of chemical disinfectants in a water system. Unlike chemical disinfection, however, the physical process of UV leaves no residual.

Once the photons have been absorbed by the water, particles, microorganisms, or the reactor walls, they're gone without a trace. A grab sample of the UV reactor effluent will yield no clues as to the amount of UV light to which the water has been exposed. To address this gap, UV reactor manufacturers have, for all but the most basic units, incorporated in-line UV sensors designed to measure the amount of UV reaching a distinct point(s) on the reactor wall. From this reading(s) and the UV absorbance and flow rate of water through the reactor (which determines average residence time) and knowing the distance from the lamp to the sensor, a delivered dose can be calculated.

To translate the readings of one or a few sensors to an estimated UV dose, it's necessary many assumptions be made. As a sensor measures UV at a single point on the reactor wall, some water is traveling near the lamp and some near the wall. Use of the sensor reading assumes a complete lateral mixing of water as it moves through the unit. Assignment of a UV dose value based on the sensor reading assumes all water is present in the reactor for the same amount of time. Baffles or diffusers may be employed to assure that mixing and consistent residence time occurs.

In an ideal reactor, with plug flow and complete lateral (radial) mixing, a properly functioning sensor can be relied upon to indicate average irradiance; however, in actual practice, the confidence one has with this indication may be compromised by several uncertainties: 1) A potential end-user may require validation a reactor performs as promised, 2) the sensor reading may lose sensitivity, and thereby not respond to fluctuations in lamp output or water quality, and 3) hydraulic dynamics may cause flow patterns outside the optimal, resulting in short-circuiting of some part of the water stream through the reactor. In these or any other situations where verification of reactor performance is necessary, some foolproof method is needed.

Bioassay
Because the overall goal of UV disinfection is to inactivate organisms to or beyond a pre-determined degree (usually some percent or log10 inactivation), a logical means of testing the system is to measure how well it's meeting its purpose. In drinking water, however, there are generally too few target organisms in the UV feed water to allow direct measurement of influent vs. effluent microbial levels. Therefore, it's necessary to challenge the system by spiking the feed with an amount of organisms sufficient to enumerate the orders of magnitude of inactivation based on the surviving number in the effluent. By challenging the reactor with organisms of a known response to UV, a UV dose corresponding to the level of inactivation achieved can be assigned. This is referred to as a bioassay, or biodosimetry.

Dose response curve
The key to a bioassay is in assuring the response of the organisms used to challenge the UV system has been fully characterized. To do so, a sample of test organisms to be delivered to the UV system is collected. Sub-samples are suspended in water in a petri dish and subjected to precisely quantified UV irradiance for a series of measured amounts of time. The test apparatus used for this is commonly referred to as a collimated beam device, wherein the majority of UV photons travel perpendicular to the surface of the petri dish, and a calibrated radiometer accurately measures the incident irradiance across that surface.

The exposure times are selected to give incrementing UV doses across the range of interest. Log inactivation of the test organism is plotted against UV dose to provide a dose response curve. A non-specific example is given in Figure 1. By comparing the inactivation achieved in an actual reactor against the plot of this collimated beam generated curve, the UV dose that was delivered by the reactor can be determined. In this example, if the reactor achieves 2.2-log inactivation, we find an equivalent of 30 mJ/cm2 was delivered.

Choice of organisms
The list of organisms available to use in a bioassay is greatly narrowed by limiting the candidate pool to those that are fairly easy to work with, have a log-linear response to UV, and respond consistently within the UV dose range of interest. It's desirable the organism chosen as a surrogate for specific target pathogens be of similar resistance to UV as the pathogen in question. Bioassays using organisms, which are significantly more resistant to UV than the target would seem to offer a conservative indication of reactor performance, but caution must be used when interpreting such results.[4]

A 2-log inactivation of a resistant surrogate might suggest greater inactivation of a UV sensitive organism could be achieved; however, if some condition such as poor reactor hydraulics or a failed lamp allowed 1 percent of the organisms to pass without receiving any irradiation, 99 percent or 2-log inactivation would be the limit for this reactor, regardless of how sensitive the target. Four organisms, which meet the above requirements, in the drinking water treatment UV dose range are E. coli, Saccharomyces cerevisiae, Bacillus subtilis spores and MS2 coliphage. Typical dose response curves for the latter three are given in Figure 2.

A comparison of these curves shows that E. coli, which is much more sensitive to UV than the latter two organisms, would be suitable for bioassay tests of a unit designed to provide only 4-to-10 mJ/cm2. To test systems in ranges more often applied to drinking water, the more resistant organisms are generally relied upon. For example, ANSI/NSF Standard 55 evaluates two types of UV drinking water treatment units with S. cerevisiae (point-of-use) and B. subtilis spores (point-of-entry) to verify the required 16 mJ/cm2 and 38 mJ/cm2, respectively, are delivered.[5] B. subtilis is also the surrogate used by both Germany and Austria, where extensive testing of UV systems is carried out to verify the required dose of 40 mJ/cm2 is applied.[4,6,7]

California guidelines
The state of California requires treatment of wastewater for reuse to meet specific virus and coliform limits. Guidelines developed by the state for validation of UV systems designed to meet these limits suggested bioassays using MS2 coliphage-an organism of sufficient UV resistance to serve as a surrogate for viruses.[8] Recently, those guidelines were updated and expanded to include a provision for application of UV to drinking water. Protocols for testing drinking water treatment systems also suggest the use of MS2. These California guidelines[9] offer significant detail to points covered in this discussion.

With the exception of the original California guideline (1993), a premise common to the above guidelines, protocols, or regulations regarding UV verification, is that each reactor style must be tested, and across the range of potential service flow rates. It's widely felt there can be little confidence in testing a small-scale pilot version and extrapolating results to larger units. A noted exception would be where a single reactor is validated, and the whole system scaled up, by introducing multiples of the tested unit in parallel.

Conclusion
The bioassay provides direct feedback for any UV reactor-inclusive of any weaknesses of design, operation or components-on how that reactor affects live organisms passing through it. As such, and in view of the lack of UV residual and shortcomings of UV sensors, it offers the only means of measuring delivered UV dose. To be assured, the undertaking of a bioassay requires adherence to strict protocols and quality control procedures. While there are limitations to this tool even when carried out properly, it affords a practical means of understanding the inactivation dynamics of UV reactors.

References
1. Clancy, J.L., et al., "Using UV to Inactivate Cryptosporidium," Journal of American Water Works Association, 92 (9) 97-104, 2000.
2. Shin, G., et al., Low Pressure UV Inactivation of Cryptosporidium parvum and Giardia lamblia Based on Infectivity Assays and DNA Repair of UV-Irradiated Cryptosporidium parvum Oocysts, AWWA WQTC Conference, November 2000, Salt Lake City, 2000.
3. Bolton, J.R. and L. Henke, "Ultraviolet Disinfection: A Basic Primer," WC&P, 34-38, April 1999.
4. Wright, H.B., and Y.A. Lawryshyn, An Assessment of the Bioassay Concept for UV Reactor Validation, Disinfection 2000: Disinfection of Wastes in the New Millennium, New Orleans, March 2000.
5. ANSI/NSF Standard 55, Ultraviolet Microbiological Water Treatment Systems, 1991.
6. Hoyer, O., "The Status of UV Technology in Europe," Abstracts and Proceedings, NWRI UV 2000, 35-41, 2000.
7. Sommer, R., et al., "Differences Between Calculated and Biodosimetrically Measured Fluences in UV Plants for Drinking Water Disinfection-Practical Experiences with the Austrian National Standard M5873-1," IUVA News, 2(5): 14-18, 2000.
8. NWRI, UV Guidelines for Wastewater Reclamation in California and UV Disinfection Research Needs Identification, Fountain Valley, Calif., 1993.
9. NWRI, AWWARF, Ultraviolet Disinfection: Guidelines for Drinking Water and Water Reuse, 2000.

About the author Tom Hargy is senior scientist at Clancy Environmental Consultants of St. Albans, Vt., which provides research and development services to the drinking water, wastewater and high purity water industries. He is a member of the International Ultraviolet Association, American Water Works Association and Water Quality Association. He is also a member of the WC&P Technical Review Committee. And he holds a bachelor's degree in geology from Macalester College in St. Paul, Minn. Hargy can be reached at (802) 527-2460, (802) 524-3909 (fax) or email: thargy@together.net