Historically, chlorine has been used as the disinfectant of choice to treat wastewater and most other products (e.g., food, utensils, clothing, surfaces) suspected of being contaminated with disease-causing microorganisms or pathogens. However, continued use of chlorine as a disinfectant is now being discouraged due to the many associated problems in the use of chlorine such as toxicity, formation of carcinogenic and toxigenic by-products which affect health of humans as well as aquatic organisms. In addition, the inherent danger to people during chlorine production, shipping, handling, use and storage has been well documented (Task Force on Wastewater Disinfection, 1996).

On January 15, 2006, the South Bay City Sewage Pumping Plant in Los Angeles County was forced to release approximately 1.5 million gallons of raw sewage onto beach sand at Manhattan Beach. To prevent sewage contamination of coastal waters, the Los Angeles County Sanitation Districts (LACSD) bermed the sand at the beach and this forced the sewage to percolate into the sand at depths of at least five feet. The submerged sewage and the sewage borne bacteria were detected in beach sand weeks after the spill event. LACSD used chlorine treatment to disinfect the sewage contaminated sand (LACSD, 2006). However, chlorine treatment was not effective in disinfecting the sewage borne microorganisms in sand. In addition, toxic chlorine in the atmosphere and in the sand were considered by individuals as well as environmental groups to be detrimental tohuman health and to the environment. These results led to public criticisms for the actions of LACSD that had to be addressed. As a result, LACSD held a one day workshop in LA on June 14, 2006 and invited the public, environmental groups and selected scientists. The objectives of the LACSD workshop were as follows: a). To review their mitigation plan. b). To review the actions and responses made by LACSD. c). To address the many criticisms levied against LACSD. d). To receive the comments, assessments and recommendations of the attendees. In this regard, the attendees were divided into five groups to focus on the following five sub-issues: 1). Spill Contingency Plan. 2). Spill Clean up Methods. 3). Measurements and Monitoring. 4). Spill Plan Presentation. 5). Public Notification. Several significant conclusions were made at that workshop. First, chlorine was not an effective means to disinfect sand. Second, the LACSD Spill Contingency Plan resulted in many criticisms by public groups and by scientists. Third, No alternative disinfection technology was identified as reliable and acceptable to treat sand contaminated with sewage.

The discussions presented at the LACSD workshop galvanized microbiologists who had been conducting research on microbial composition of beach sand to form a " Microbial Sand Group". This group of microbiologists pledged to work together to address three identified problems. First, to raise awareness that microbial sand contamination is a public health problem. Second, to convince federal (EPA) and state (Department of Health) agencies to take leadership roles in developing guidelines to assess the health risks of pathogens in beach sand. Third, to standardize the methods for sand analyses of microorganisms. As its first group action, the Microbial Sand Group organized a session at the 2006 EPA Beaches Conference (Niagara Falls, October 11-13, 2006). This session resulted in much discussion with the audience and EPA representatives. Denise Keehner, the leader of this EPA Beach Conference summarized the conclusions of this conference. One of her conclusions is summarized below:

Regarding Beach Sand:

"-EPA needs to consider leadership role in bringing people together to assess what we know, what we don't know, what the priority science/research needs are, AND, how we should move forward"

This conference conclusion by EPA was significant because this was the first official and public statement made by EPA on the subject of microorganisms in sand. Moreover, it directs EPA to take a leadership in assessing the public health significance of microorganisms in beach sand.

THE PROPOSED STUDY

A. The Identified National Sand Problem. Before the 2006 EPA Beach Conference, studies to assess microbial pollution of beach sand had been conducted primarily at Universities. For years, EPA and state governments ignored publications of microorganisms in sand because EPA had not identified this as an environmental problem. As a result, EPA had not developed guidelines or microbial standards for microbial pollution of sand. Therefore, in 2006, EPA was unprepared to provide guidance to LACSD when 1.5 million gallons of raw sewage from a pumping station were impounded behind sand berms and caused sewage contamination of sand at Manhattan Beach. When this pollution event occurred, LACSD quickly learned that there was no approved method to disinfect sewage contaminated beach sand. Moreover, standardized methods to assay sand for fecal bacteria had not been established. Since chlorine had been used to treat wastewater, chlorine was applied to treat the sewage contaminated sand. However, this treatment was not effective and resulted in toxic chlorine being released into the atmosphere surrounding that beach and to be detected in the treated sand. Due to public criticisms of using chlorine to treat sand, LACSD held a workshop to review the results of the sand contamination event at Manhattan Beach. The identified problems and needs raised by the LACSD workshop can be summarized as follows. 1) Another sewage contamination of beach sand is a likely event at many beaches in the nation. 2) There are no approved methods to assay for sewage borne microorganisms and pathogens in sand. As a result, test methods were not available to determine the extent of sewage contamination and moreover could not be relied upon to determine when sewage contaminated sand has been sufficiently treated. 3) Chlorine is not an effective means to treat sewage contaminated sand. 4) An approved sand treatment technology has not been identified. 5) There is a need to develop an effective and safe sand treatment technology.

B. Beach Sand in Hawaii differs from Beach Sand in Continental USA. Ideally an evaluation study of a sand treatment technology at one beach site can be effectively applied at all other beach sites in the USA. However, differences in sand composition can affect the effectiveness of the selected sand treatment technology. In this regard, the major source of Hawaii's white beach sand is biogenic and comprised of the structural remains of marine seashells and coral. The chemical composition of the natural white sand in Hawaii is calcium carbonate. It should be noted that the origin of some beach sand in Hawaii is terragenic and its color determines its source. The black sand is comprised of weatherized lava whereas the green sand is weatherized olivine crystals, a component of basalt lava. More recently, a man-made, white beach sand was used to create the beach surrounding the newly created Hilton Lagoon. Although this beach sand appears to be similar to natural beach sand from a distance, one can easily see that its particle size and shape differs from the natural beach sand. Hilton Hotel reported that the source of this new sand was from a quarry in the Nanakuli area of Oahu. The chemical composition of this quarry sand is primary limestone. As a result, the fate of sewage borne microorganisms which contaminate the different types of beach sand can be expected to differ.

In contrast, the source of beach sand found on the continental USA is primarily from the land (terragenic) as weathered rock particles and its primary chemical composition is silica or quartz. Due to these basic differences in sources and chemical composition of sand in Hawaii and in the continental USA, the sand treatment results may vary. The observed differences in the inactivation of microorganisms in different types of sand can often be explained by differences in the way the disinfectant will chemically react with the chemical composition of the sand. This is called the "matrix effect" or the interference of the environmental composition for that specific disinfectant. There is precedent for this kind of reaction based on studies using chlorine. Chlorine is known as a highly reactive chemical that reacts with almost everything it comes in contact. As a result, the effectiveness of chlorine is generally tempered by the matrix effect or the physical and chemical 'composition of the environment in which the contaminating microorganisms are found. For example, it well known that chlorine will react and form less reactive disinfectant species such as hypochlorite at elevated pH and will form several species of less effective disinfectants called chloramines after reacting with organic material (proteins, carbohydrates) or with nitrogenous chemicals such as ammonia. As a result, an additional identified problem is to determine the matrix effect of any new sand disinfecting technology.

C. CupriDyne^TM Technology: A Potential Effective and Safe Sand Treatment Technology. BioLargo, Inc. is a public company, which has patented a CupriDyne^TM technology that can generate free iodine (12) by dissolving two proprietary chemical compounds in water. Free iodine is an effective disinfectant and is soluble in water up to 337 ppm. BioLargo has reported that that free iodine will disinfect most microorganisms in the range of 20-80 ppm and at this concentration CupriDyne^TM and its products have been declared GRAS (Generally Regarded as Safe) by the US EPA 21 CFR. Moreover, at 80 ppb, this dose is equivalent to 1/25h the amount of chlorine in water. At this low dose, toxic by-products such as total trihalomethanes can be expected to be well below EPA limits. BioLargo also reports that most iodine is found in the world's oceans as an iodate and that phytoplankton and macroalgae are able to metabolize iodate to form iodine. Thus, there is a natural iodine cycle under most marine beach conditions and excess iodine is not expected to become a problem.

Two public domain reports show that CupriDyne^TM has been previously tested. The first report by Ingham (2007) used CupriDyne^TM as a solid reagent (prill) at a single dose (200 lb/acre) to disinfect soil containing nematodes. At this dose, this study did not show substantial reductions of nematodes at that single, low dose applied. The conclusion of that study was to repeat the study using higher doses of CupriDyne^TM . The second study by Jay (2007) was a bench scale study to determine the effectiveness of CupriDyne^TM as a disinfectant of sand contaminated with enterococci. In this study, an 80 ppm of CupriDyne^TM was prepared by dissolving the appropriate number of blue and white tablets provided by BioLargo and used to treat sand contained in beakers. The report by Jay (2007) concluded that CupriDyne^TM at 80 ppm dose effectively disinfected both naturally occurring and pure culture populations of enterococci in sand. The inactivation of enterococci in sand was observed after the minimum 5 minute treatment, indicating a rapid mode of action by CupriDyne^TM (iodine species). One advantage of these two completed studies is availability of data and methods used in the application of CupriDyne^TM The limitation of both of these studies was that a single dose of CupriDyne^TM was used in a single kind of matrix. As a result, the effectiveness of various concentrations of CupriDyne^TM and the impact of matrix effects in these samples were not assessed.

D. The Goals of this Proposed Study.

The first goal of this proposed study is to conduct a bench scale to evaluate the effectiveness of various concentrations of CupriDyne^TM to disinfect selected microorganisms (total bacteria, total aerobic spore forming bacteria, total coliform, E. coli, enterococci, C. perfringens and F+ coliphages) in selected water and sand samples The second goal of this study is to determine the matrix effects when the contaminating microorganisms are mixed in different kinds of environmental samples.

E. The Experimental Design and Methods to be Used for this Proposed Study.

Since this is bench scale study, the experiments conducted will primarily be completed under laboratory conditions at the University of Hawaii. Methods as used by Ingham (2007) and by Jay (2007) will be used as models for the experimental set up for this proposed study. However, this study will determine the disinfecting effect of various concentrations of CupriDyne^TM as a means of determining the minimum concentration of CupriDyne^TM required for effective disinfection. Measurements will also be made of the dose of CupriDyne^TM and the measured concentrations of free iodine in the dosed samples after various periods of time. These measurements will provide data on stability of the disinfecting effect of CupriDyne^TM .

This proposed study will assay for a variety of different microorganisms. The methods selected for use will be methods used in Standard Methods for the Examination of Water and Wastewater (APHA,AWWA, WEF 2005) or methods published separately by EPA. The following microorganisms were selected for analysis during this proposed study because they represent the numbers and variety of microorganisms found in water, sewage and sand contaminated samples:

1) Total bacteria: This heterogenous group of bacteria represents the greatest variety and concentrations of culturable bacteria in environmental samples. Analysis for this group of bacteria will determine the effectiveness of disinfection by CupriDyne^TM under the various conditions.

2) Total aerobic spore forming bacteria: This is the most resistant group of bacteria found in environmental samples. By analyzing for this group of bacteria, the relative disinfecting activity of CupriDyne^TM as compared with other disinfectants as published in the literature can be compared.

3) Total coliform, E. coli and enterococci: This group of fecal indicator bacteria is used by EPA to establish water quality standards. The effectiveness by which CupriDyne^TM disinfects this group of bacteria can be compared with published data for many types of disinfectants. Moreover, the guidelines used by EPA for public health assessments based on water quality can be used.

4) C. perfringens. This species of anaerobic, spore forming bacteria is specific to sewage and used to establish water quality standards in Hawaii. The resulting data can be compared with available data for chlorine and UV disinfection.

5). F+ coliphages: This group of naked viruses (coliphages), which infect fecal bacteria is considered the best surrogate for survival, persistence and movement of human enteric viruses under environmental conditions. The disinfection efficiency of this group of viruses will be used to predict the disinfection efficiency of CupriDyne^TM for human enteric viruses as compared to other disinfectants.

Finally, this study will evaluate the matrix effects of various environmental water and sand samples. Water samples will include fresh stream waters, estuary waters, coastal waters as well as sewage effluents and sewage contaminated waters. To assess the matrix effects of different water and sand samples, defined parameters (pH, salinity, temperature, turbidity, total solids, total organic carbon) of the various water and sand samples will be correlated with the degree of disinfection by CupriDyne^TM . To better define the disinfecting effect of CupriDyne^TM and the matrix effects, the disinfecting effect of CupriDyne^TM for the various microorganisms as suspended in water samples and as mixed in sand samples will be determined. In this regard, sand from different beaches will be used for this experiment. However, silica based sand representing the kinds of sand found on the continental USA will also be used for comparative purpose.