The State Water Board's Contract Manager shall be Chris Means of the San Diego Regional Water Quality Control Board. The Contract Manager shall be the day-to-day representative for administration of this agreement, and, except as otherwise specifically provided, shall have full authority to act on behalf of the State Water Board with respect to this agreement. The State Water Board's Executive Director, or designee, may also perform any and all acts which could be performed by the Contract Manager under this agreement. Except as otherwise expressly provided, all communications relative to this agreement shall be given to the Contract Manager.

The Contractor's Project Director shall be Vicki L. Wilson, Director, County of Orange, Public Facilities and Resources Department. The Project Director shall be the Contractor's representative for the administration of the agreement and shall have full authority to act on behalf of the Contractor. All communications given to the Project Director shall be as binding as if given to the Contractor.

The parties may change their Contract Manager or Project Director upon providing ten (10) days written notice to the other party.

2. Work To Be Performed

A. Scope and Objectives

The beach water at the mouth of San Juan Creek and the portion of the creek immediately upstream of the beach fails State bacteriological standards regularly. While there are suspected sources of bacterial contamination, no comprehensive study has been carried out to address this problem. In addition, no bacteriologic survey has been carried out for the remainder of the watershed so there is no information available on potential sources of pollution. This study has three purposes:

1. Provide a bacterial survey of the water quality of the San Juan Creek watershed in dry weather conditions and locate any areas with bacteriologic water quality problems. Survey problem areas in detail.

2. Determine the source of the bacteria found in the problem areas using bacterial source tracking.

3. Compare two different techniques of bacterial source tracking, Ribotyping and Antimicrobial Resistance Testing (ARA) to determine the accuracy of these techniques.

The San Diego Regional Water Quality Control Board (RWQCB) has identified San Juan Creek as an impaired water body for pathogens in the lower reach. The primary bacteriological contaminant that has led to the impaired designation is fecal coliform. Fecal coliform is a group of bacteria that is commonly associated with the digestive tracts of warm-blooded animals; including humans, domestic and wild mammals, and birds. The presence of fecal coliform within surface waters indicates the likely presence of fecal wastes from such warm-blooded animals. If the coliform is associated with human waste, it may indicate the presence of a wide variety of human pathogens (e.g., viruses, bacteria, etc.) that cause human disease. Although fecal contamination from non-human sources presents a reduced human health threat, some threat may still exist. Salmonella, Listeria, Campylobacter and Cryptosporidium (spp.) are some of the bacteria that can be found in animal wastes, can be carried via contaminated water, and can be contagious to humans.

The Orange County Health Care Agency (OCHCA) monitors beaches throughout Orange County to determine concentrations of various indicators of bacteriological and pathogenic water pollution. The OCHCA uses this monitoring data to determine if the water quality at the beaches is suitable for body contact recreation. The OCHCA will close beaches to body contact recreation when the concentrations of indicators exceed water quality objectives. Beach water quality is monitored for total and fecal coliform bacteria, and enterococcus. Sanitary surveys are conducted to determine the sources of pollution when the indicators exceed water quality objectives.

Based on the water quality monitoring conducted by OCHCA, and the history of closures of body contact recreation, the RWQCB has listed the lower reach of San Juan Creek (Creek) as water quality impaired in accordance with Section 303(d) of the Clean Water Act. The RWQCB determined that water quality in the Creek does not currently meet water quality objectives for fecal and total coliform established by the RWQCB for the protection of contact water recreation (REC-1).

The federal Clean Water Act requires the State of California (through the RWQCB) to identify impaired water bodies and to implement corrective measures to restore water quality areas that have been identified as impaired. These corrective measures include establishing Total Maximum Daily Loads (TMDLs) for contaminants and when necessary, implementation of enforcement actions such as a Cleanup and Abatement Order (CAO). Unless prior actions are taken to identify and reduce the bacteriological impairment of lower San Juan Creek, the RWQCB can be expected to ultimately utilize its enforcement authority.

When the State of California adopted Assembly Bill (AB) 411 (Wayne, 1997), it required county health officers to test recreational beach waters for three indicators of possible human pathogens. These include total coliform, fecal coliform and enterococcus. The AB 411 requires county health officials to post beaches if testing shows the presence of these indicators above certain standards. This sampling study will provide the County of Orange with valuable information on the relative magnitudes of the bacteria loadings and the sources of the bacteria. From such information, an effective correction program can be developed and implemented.

In late fall 1997, the City of Coronado attempted to determine the causes of high fecal coliform counts at the outfall of a dewatering system associated with their groundwater collection network. With high readings coming from the outfall area, the RWQCB adopted a cease and desist order that required the City to correct the high counts or stop the dewatering. The City contracted with Woodward Clyde to both engineer a source identification program and to study alternatives that would resolve the problem. After conducting DNA tests of input sources, the City was able to show that the fecal coliform came from a variety of human, animal and bird sources. Later Woodward Clyde helped design an ultraviolet radiation structure that treated the groundwater before release and that brought coliform counts down to acceptable levels for water recreation. Although the San Juan Creek storm drain system is much larger and more complex than the single dewatering outfall at Coronado, the same types of source identification can be expected to assist the County in meeting the standards of AB 411.

Although stream flows within the lower San Juan Creek watershed frequently contain concentrations of indicator bacteria that exceed bathing standards, health experts do not currently know the degree to which that bacteria may be associated with the presence of pathogens that are harmful to humans. All existing sampling has utilized methods that are not able to determine if the bacteriological contamination is from human sources. Identifying and stopping the bacteriological contamination will be much easier once the specific sources are known.

This project is designed to conduct a state-of-the-art study of existing bacteriological contamination within the San Juan Creek watershed. An extensive sampling program will be implemented to identify the relative contribution of bacteria loading from each of the major tributaries within the watershed. The sampling program will also utilize genetic fingerprinting to identify the most likely animal sources of the bacteria loading. The relative proportions of the bacteria loadings that are from humans, pets, shore birds, horses, or wildlife are needed in order to assess the level of human health threat that is posed by the contamination as well as to develop an effective strategy to reduce the loadings. In addition, this project will compare two different techniques of bacterial source tracking, Ribotyping and Antimicrobial Resistance Testing (ARA) to determine the accuracy of these techniques.

B. Work

The work to be performed under this contract includes the collection and analysis of water samples for total and fecal coliform, enterococcus, and Escherichia coli (E. coli). In addition, sample cultures of E. coli and Enterococcus will be analyzed using bacterial source tracking to qualitatively identify sources of fecal coliform found in the discharges. These analyses will determine whether there are human sources of bacteriological contamination, as well as identify the types of animals that contribute to the concentrations of bacteria in the discharges to the creek and beach. The specific number of samples and analyses will be determined within a sampling plan to be developed by the Technical Advisory Committee. The County will subcontract with the OCHCA, and/or other certified laboratories for the analysis of the water samples. For the ARA bacterial source tracking technique, samples will be collected by public health laboratory staff and any other organizations that can assist (animal control, sewage treatment plant personnel, volunteers, humane societies). Bacteria will be isolated and identified and frozen by the public health laboratory staff. Valerie Harwood, Ph.D., Assistant professor, University of South Florida, and Tampa, FL will subcontract with the public health laboratory to perform the ARA analysis. For the Ribotyping bacterial source tracking technique, samples that will be utilized are the same as ARA analysis. Bacteria will be isolated, identified and frozen by the public health laboratory staff. The ribotyping technique published by S. Parveen of the University of Florida (Parveen, 1999) will be utilized for analysis. The testing laboratory will be George Lukasik, Ph.D., Biological Consulting Services of North Florida, Gainsville, Florida or another acceptable laboratory.The contractor shall be responsible for the performance of work as set forth herein below and for the preparation of products and a final report as specified below.

Study Design

Phase 1: Bacteriological Survey of Watershed and Adjacent Beach Recreational Water

Phase 1 is designed to quickly survey the watershed to locate areas with bacterial pollution problems. Sites will be selected to sample tributaries, storm drains and to sample known or suspected problem areas and recreational water at the mouth of the creek. Approximately 30 sites will be sampled weekly for 10 weeks (300 samples).

Phase 2: Detailed Bacteriological Survey of Identified Problem Areas

Phase 2 is designed to further characterize locations with water quality problems identified in Phase 1. These sites will be tested in detail to determine maximum and minimum bacterial counts as well as temporal and geographic boundaries of problem. Existing data indicates that one known problem area is the very lower end of the creek, which is often prevented from flowing into the ocean by a sand berm. Samples will be taken from at least one known problem area and a second area geographically removed. E. coli and enterococcus isolates from a sub-set of samples will be saved to use in Phase 3.

Phase 3: Source Identification by ARA and Ribotyping: Library Preparation and Technique Accuracy Determination

Phase 3 is designed to build the necessary databases and determine the accuracy of two methods of bacterial source tracking, ARA (Antimicrobial Resistance Analysis) and Ribotyping. Overall, this will be done by constructing a library of bacterial isolates from known species, performing the source analysis testing to build or add to an existing database and determining the accuracy of the methods utilizing bacterial strains from known sources not included in the database.

ARA Technique:

ARA differentiates bacteria from different species of animals by comparison of their resistance to antibiotics. Fecal samples from known animal species and human sources are taken, E. coli and enterococcus bacteria are isolated and a collection of isolates is constructed. Each bacterial isolate is tested against 4 concentrations of 8 different antibiotics. Resistance patterns by species analyzed by discriminate analysis are used to classify individual strains into the most likely group (species). Quality assurance is carried out first by determining the initial accuracy of the technique by holdout analysis, a statistical study of the database. Additional quality assurance is covered under the Quality Assurance section below.

Several recent published studies done by several researchers on different watersheds have shown the utility of the ARA technique and have documented the accuracy as being between 75% and 85% (Wiggins, Hagedorn, Parveen, Harwood). In this study, dual analysis of both E. coli and enterococcus isolates is expected to improve the accuracy somewhat. In addition, isolates will be frozen so they can be re-analyzed by other techniques in the future. The technology is fairly simple resulting in lower cost per isolate and an increased probability that the technique can be rapidly imported into the Public Health Laboratory for future studies if it proves to be an accurate technique. This technique produces matches based on the local database of known isolates. This requires a large database be constructed. However, the database can be utilized for future bacterial pollution problems.

Ribotyping Technique:

Ribotyping differentiates bacteria by detecting changes in restriction enzyme cutting sites in the bacterial genome. Restriction fragments are separated by size on an agarose gel and the fragments containing r-RNA (ribosomal RNA) gene sequences are detected using a complimentary probe. The patterns of different sized fragments are compared between isolates. Isolates with the same patterns are considered related.

Similar to ARA, a library of E. coli strains from known animal species and human sources is utilized to identify the source of strains isolated from problem areas. Ribotype patterns, analyzed by discriminant analysis are used to classify strains into the most likely group (species). Unlike ARA, large isolate libraries, maintained by the testing laboratory have been traditionally utilized for matching along with the local library. However, a local isolate library is necessary to identify up to one third of strains. The accuracy of a geographically separated database is not established at this time. In the one published study utilizing ribotyping, an accuracy of 82% was achieved (Parveen 1999).

Quality assurance: Two types of quality assurance tests will be carried out:

1) Reproducibility. Twnety (20) strains of each bacterium will be submitted to the testing laboratory 3 different times blinded. The percentage of reproducibility will be determined for each strain and summed.

2) Accuracy. Final accuracy of each technique is determined by an analysis of 100 isolates from known species (which are not residing in the database) that are submitted to the reference laboratory blinded. These bacterial strains will be isolated from samples collected alongside the data base samples and held back. The analysis of rate of correct classification will be presented in a table similar to tables 1 and 2 of Harwood et. al. 2000. After this analysis adjustments can be made in the analysis technique to maximize accuracy if necessary. The same strains will be utilized for both typing techniques so a direct comparison can be made.

Phase 4: Source Identification by ARA and Ribotyping: Source Identification of Watershed Isolates.

Phase 4 is the analysis of bacterial isolates from problem areas that have been collected in phases 2 and 3 utilizing the two source tracking techniques and a comparison of the results of the techniques. Approximately 200 bacterial isolates (per species) from the problem areas will be provided to the two reference laboratories and will be compared to the library of isolates from known species to determine potential source. For ARA, a comparison is made between the results for the two different bacterial databases (E. coli and enterococcus) to determine a final result. For ribotyping the analysis will be done comparing the unknown E. coli strains to the local database and to the testing laboratory's database. The ARA results will be compared to that of the ribotyping analysis and written report issued. The report is also prepared for publication in a peer-reviewed journal.

Hagedorn, C., S. L. Robinson, J. R. Filtz, S. M. Grubbs, T. A. Angier, and R. B. Beneau. 1999. Determining sources of fecal pollution in a rural Virginia watershed with antibiotic resistance patterns in fecal streptococci. Applied and Environmental Microbiology. 65(12):5522 - 5531.

Harwood, V.J., J. Whitlock and V. Washington. 2000. Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis: use in predicting the source of fecal contamination in subtropical waters. Appl Environ Microbiol. 66:3698-3704.

Parveen, S., R. L. Murphree, L. Edmiston, C. W. Kaspar, K. M. Portier, and M. L. Tamplin. 1997. Association of multiple-antibiotic-resistance profiles with point and non-point sources of Escherichia coli in Apalachicola Bay. Appl. Environ. Microbiol. 63(7):2607-12.

Parveen, S., K. M. Portier, K. Robinson, L. Edmiston and M. L. Tamplin. 1999. Discriminant Analysis of Ribotype Profiles of E. coli for Differentiating Human and Non-Human Sources of Fecal Pollution", Appl Environ Microbiol. 65:3142-3147.

Wiggins, B. A., R. W. Andrews, R. A. Conway, C. L. Corr, E. J. Dobratz, D. P. Dougherty, J. R. Eppard, S. R. Knupp, M. C. Limjoco, J. M. Mettenburg, J. M. Rinehardt, J. Sonsino, R. L. Torrijos, and M. E. Zimmerman. 1999. Use of antibiotic resistance analysis to identify nonpoint sources of fecal pollution. Appl Environ Microbiol. 65:3483-6.