H O W- T O - D O - I T OPTICAL BRIGHTENERS IN LAUNDRY DETERGENTS Help Us Determine the Source of Bacterial Contamination NEIL GLICKSTEIN B acterial loading in water from anthropogenic sources is of common interest in both student research projects and to regulatory agencies. The presence of fecal coliform bacteria in natural waters can be the deciding indicator for how the bodies of water are classified for use. Since current tests are not specific, it is difficult to determine whether the source is human or from any of a variety of endothermic organisms that also discharge coliforms in their waste. Since the harvest of intertidal filter-feeding shellfish is closely tied to the overlying water quality, there has been an effort in Gloucester, Massachusetts to distinguish between human and animal discharge of coliforms. The analysis employs detection of optical brightening agents and has been effectively used to locate point sources of untreated human effluent into the local coastal water (Sargent & Castonguay, 1998). The technique is simple, inexpensive, and easily adaptable to student research projects focused on biological sources of pollution. Primer on Fecal Coliform The coliform group is made up of many genera belonging to the Enterobacteriaceae which are mostly harmless bacteria that live in the gut of animals as well as in the soil and water. In environmental samples, they are known as total coliform bacteria. The fecal group reside in the intestines of homeothermic animals and are present in large quantities in their feces. Characterized by the ability to grow at elevated temperatures, the most common of these is Escherichia coli. Most fecal organisms are not pathogenic but are easily cultured, and their detection presumes the presence of other pathogenic bacteria, since disease-causing organisms are often present in smaller concentrations and would be unlikely candidates for culturing and identi- NEIL GLICKSTEIN teaches science at the Waring School and is an Adjunct Professor at Endicott College, both in Beverly, MA 01915; e-mail: nglickstein@hotmail.com. 296 THE AMERICAN BIOLOGY TEACHER, VOLUME 68, NO. 5, MAY 2006 fication. Diseases which might be contracted from contact with water demonstrating high fecal counts include typhoid fever, hepatitis, gastroenteritis, dysentery, and ear infection (EPA, 2000). Though these single cell bacteria can be seen under the high power magnification of a microscope, it is a tedious process to search for them. As they multiply, they form colonies that can be counted by eye. The assumption is that each bacterial cell in a culture is the ancestor of an individual colony; and thus, the number of cells in the original sample may be determined. Methods used to grow these colonies include the most probable number (MPN) method and the membrane filter (MF) method (Murphy, 2002). The current Commonwealth of Massachusetts’ recommendations for water quality based on E. coli cultures is found in Table 1. It is impossible to tell from cultured coliform procedures whether the source of elevated coliform counts in natural waters is due to runoff leached from a domestic, farm, or wild animal source or whether it has come from a failing septic system or storm water tie-in of an illegal domestic system. Persistent high counts indicate that corrective action is required. Clearly, corrective strategies for an agricultural source will differ from those for a domestic sewage problem. The former requires remediation but isn’t the same potential pathogenic threat to humans as the entry of domestic sewage into the environment. However, often spikes in bacterial counts following precipitation serve to indicate the existence of a problem of unclear origin. After a significant rain event, increased coliform counts in flowing waters could be due as much to a recent visit from a flock of snow geese as to elevated groundwater levels affecting a failing septic tank. There is little one can do to change coliform input by wild animals and the pathogenic threat from the source is not as great as a domestic sewage input. Clearly, the importance of the source becomes paramount to those charged with monitoring our environment for human health risks. Optical Brighteners Table 1. Commonwealth of Massachusetts Fecal Coliform The key to distinguishing whether Water Quality Standards (DEP 1997). fecal coliform counts indicate potentially hazardous presence of human pathogens USE COUNT/100 mL or are the result of natural runoff from Drinking water <1 homeothermic animals is to find a reliable anthropogenic tracer. One such tracer Shellfish harvesting <14 (10% of samples <43) exists in the group of chemical substances Shellfish harvesting with depuration <88 (10% of samples <260) used to enhance the brightening characteristics of domestic laundry detergents. Domestic water supply <20 (10% of samples <100) Optical brighteners (OBs) belong to a Domestic water supply with treatment <200 (10% of samples <400) group of fluorescent dyes that absorb in Body contact recreation <200 (10% of samples <400) the near-ultraviolet range and emit in the blue wavelengths. The blue emission looks Fishing and boating <1000 (10% of samples <2000) to the observer to have the quality of being intensely white, as the commercial goes, “whiter than white.” Of course this quality tic net bag weighted with stones can work in places where makes clothing look very clean, which is its major selling the flow rate is low. The materials necessary to begin a sampoint (Fay et al., 1995). pling program cost between $300 to $500 depending on the quality of the UV light purchased. A list of materials is Due to the ubiquitous use of optical dyes, they are available online (Sargent & Castonguay, 1998). commonly found in human waste waters that have laundry effluent as a component. Though these substances can be It is best to place the samplers in tributary waters, adsorbed by soil and organic molecules and are subject to because large bodies of water tend to dilute the dyes. photo decay, their presence in surface and ground water Nylon filament fishing line is handy to secure the cage to a indicates inefficient cleansing from the system. This propbranch or the mesh bag to a road grate. Exposure time can erty makes them very useful in tracing the course of inefvary in order to get clear positive results, but seven days fective human sewage disposal (Aley, 1991). seems to be adequate (Sargent & Castonguay, 1998). Of course, water flow and initial loading have a great effect on the quality and timing for readable results. On retrieval, The Technique pads are squeezed to remove excess water and placed in labeled ziplock bags. Essentially the procedure is to immerse clean, untreated cotton pads into bodies of water long enough for them to absorb traces of optical brighteners if present. Though experimental design and the sources of error must be considered carefully in planning a sampling program, the actual procedure for acquiring results is not difficult and is well suited to an inquiry-based approach to environmental science. Students must obtain sampling pads and samplers for the number of samples they would like to take. They need to determine where to place samplers in order to best “box in” sources of contamination. Attention must be paid to how samplers are placed and held in the water. They must consider the retrieval and storage of the samplers. And finally, students need to consider how to record their results and other pertinent information such as location and rainfall. Due to the potential for contact with contaminated wastewater, all investigators should wear rubber gloves when handling materials. Most cotton is treated with optical brighteners during production. However, untreated cotton pads may be obtained from V.W.R. Scientific or other vendors. The pads and all of the sampling equipment should be checked with a long wavelength ultraviolet light (365 nm) before use. Sampling requires a rigid sampling device to hold the cotton while allowing water to flow through unobstructed. A plastic- or vinyl-coated mesh square cage measuring 10 cm on a side with 1 cm openings has worked well. A 1 cm mesh plas- Acquiring useful data requires that a researcher identify and control the sources of error in an experiment. Students should be reminded that time spent considering sources of error before acquiring data can produce accurate, usable information without spending the time to repeat the procedure. Potential errors are inherent to OB sampling in addition to the design problems dealing with field placement. Most white paper contains optical brighteners and should not come in contact with the cotton samples. Also, students should not use laundry detergents within 24 hours of handling the pads. Any contact with a source of OBs could reduce the effectiveness of this technique. The analysis requires a very dark room and a 6 watt ultraviolet fluorescent light. Caution must be taken in working with UV light. Students should be reminded not to look directly at the light as it will cause damage to the eyes. Using a non-exposed cotton pad as a control, compare the exposed sample pads qualitatively for emission of visible light. A pad that definitely glows or fluoresces is a positive. If the pad is indistinguishable from the control, it is a negative. It is possible to have questionable results either because the sample does not look just like the control or because different readers see different results. It is sensible to put these in a retest pile. On retesting the sampled area for these questionable pads, it would be prudent BACTERIAL CONTAMINATION 297 to leave the cotton in place for a longer period of time or to wait for increased flow after a rain event. Fluorescent dyes, which include the group of optical brighteners, have been used for several years to trace the flows of surface and subsurface waters (Aley, 1991). The technique of analyzing sample waters testing positive for fecal coliform bacteria for the presence of optically active components of laundry detergents is useful in determining whether the bacterial contamination source is from human or other sources. The technique presented here is adaptable to most school situations, provides for low detection limits as well as ease and safety of use, and is economical. Acknowledgments Thanks are due for the patient development of this technique to Dave Sargent, formerly of the Gloucester Department of Public Health, co-author of An Optical Brightener Handbook, and currently the Gloucester Shellfish Constable; and to Robert “Stubby” Knowles (deceased), former Shellfish Constable for the City of Gloucester, Massachusetts, whose unfailing support made this project successful. Contact Information & Resources VWR International 1310 Goshen Parkway West Chester, PA 19380 Available online at:http://www.vwrsp.com/catalog/ page.cgi?tmpl=products. EPA Volunteer Stream Monitoring: A Methods Manual. The EPA guide for volunteer stream monitoring is an excellent primer for teachers unfamiliar with stream sampling or for students who are required to plan and implement their own sampling protocol. Available online at: http://www.epa.gov/volunteer/ stream/stream.pdf. References Aley, T. (1991). The Water Tracer’s Cookbook and Related Groundwater Tracing Information. Protem, MO: Ozark Underground Laboratory. Environmental Protection Agency (2000). Fecal Bacteria. [Online]. Available at: http://www.epa.gov/OWOW/monitoring/volunteer/stream/vms511.html. Fay, S.R., Spong, R.C., Alexander, S.C. & Alexander, C. (1995). Optical brighteners: Sorption behavior, detection, septic system tracer applications. Proceedings of the International Association of Hydrogeologists XXVI International Conference, Edmonton, Canada. Department of Environmental Protection. (1997). Massachusetts Surface Water Quality Standards. [Online]. Available at: http://www.state.ma.us/dep/bwp/iww/files/314004.pdf. Murphy, S. (2002). General Information on Fecal Coliform. [Online]. Available at: http://boulder.co.us/basin/data/ NUTRIENTS/info/EColi.html. Sargent, D. & Castonguay, W. (1998). Water Quality Sampling, An Optical Brightener Handbook. [Online]. Available at: http://www.naturecompass.org/8tb/sampling/index.html. A B T Goes E L E C T R O N I C ! 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