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HomeMy WebLinkAboutC-3479 - Water quality testing of storm drains and tributaries to Newport Bay for the presence of human viruses and bacteria9 0 PROFESSIONAL SERVICES AGREEMENT THIS AGREEMENT, entered into this 15th day of August, 2001, by and between CITY OF NEWPORT BEACH, a Municipal Corporation (hereinafter referred to as "City "), and The Regents of the University of California, on behalf of its Irvine campus (hereinafter referred to as "Consultant'), is made with reference to the following: RECITALS A. City is a municipal corporation duly organized and validly existing under the laws of the State of California with the power to carry on its business as it is now being conducted under the statutes of the State of California and the Charter of City. 1-: City desires to engage Consultant to provide staff services for \ BacterialNiral Source Tracking of Tributaries and Storm Drains Discharging into Newport Bay ('Research Project'). C. The principal investigator of Consultant is, for purpose of this Research Project, Dr. Sunny Jiang ('Principal Investigator "). D. City has reviewed the previous experience and evaluated the expertise of Consultant and desires to contract with Consultant under the terms and conditions provided in this Agreement. NOW, THEREFORE, it is mutually agreed by and between the undersigned Parties as follows: 1. TERM The term of this Agreement shall commence on the 15th day of August 2001, and shall terminate on the 14th day of August 2002, unless terminated earlier as set forth herein. 2. SERVICES TO BE PERFORMED Consultant shall diligently perform all the duties set forth in the Scope of Services, attached as Exhibit "A" and incorporated herein by reference. 3. COMPENSATION TO CONSULTANT City shall pay Consultant for the services in accordance with the provisions of this Section and the Research Project budget, attached as Exhibit "B" and incorporated herein by reference. No budget changes shall be made during the term of this Agreement without prior written approval of City. Consultant's compensation for all work performed in accordance with this Agreement shall not exceed the total contract price of Thirty -six thousand, one hundred seventy -nine dollars ($36,179). 3.1 Consultant shall maintain accounting records of its billings which includes the name of the employee, type of work performed, times and dates of all work which is billed on an hourly basis and all approved incidental expenses including testing supplies. 3.2 Consultant shall submit monthly invoices to City payable by City within thirty (30) days of receipt of invoice subject to the approval of City. 3.3 Consultant shall not receive any compensation for extra work without prior written authorization of City. 3.4 City shall reimburse Consultant only for those costs or expenses which have been specifically approved in this Agreement, or specifically approved in advance by City. Such cost shall be limited and shall include nothing more than the following costs incurred by Consultant: A. The actual costs of subconsultants for performance of any of the services which Consultant agrees to render pursuant to this Agreement which have been approved in advance by City and awarded in accordance with the terms and conditions of this Agreement. B. Approved computer data processing and reproduction charges. C. Actual costs and /or other costs and /or payments specifically authorized in advance in writing and incurred by Consultant in the performance of this Agreement. 3.5 Notwithstanding any other paragraph or provision of this Agreement, beginning on the effective date of this Agreement, City may withhold payment of ten percent (10 %) of each approved payment as approved retention until all services under this Agreement have been substantially completed. 4. STANDARD OF CARE 4.1 All of the services shall be performed by Consultant or under Consultant's supervision. Consultant represents that it possesses the professional and technical 2 9 0 personnel required to perform the services required by this Agreement, and that it will perform all services in a manner commensurate with the university's professional standards. All services shall be performed by qualified and experienced personnel who are not employed by City nor have any contractual relationship with City. Consultant represents and warrants to City that it has or shall obtain all licenses, permits, qualifications and approvals required of its profession. Consultant further represents and warrants that it shall keep in effect all such licenses, permits and other approvals during the term of this Agreement. 4.2 Consultant shall not be responsible for delay, nor shall Consultant be responsible for damages or be in default or deemed to be in default by reason of strikes, lockouts, accidents, or acts of God, or the failure of City to furnish timely information or to approve or disapprove Consultant's work promptly, or delay or faulty performance by City, contractors, or governmental agencies, or any other delays beyond Consultant's control or without Consultant's fault. _ 5. INDEPENDENT PARTIES City retains Consultant on an independent contractor basis and Consultant is not an employee of City. The manner and means of conducting the work are under the control of Consultant, except to the extent they are limited by statute, rule or regulation and the expressed terms of this Agreement. Nothing in this Agreement shall be deemed to constitute Consultant or any of Consultant's employees or agents, to be the agents or employees of City. Consultant shall have the responsibility for and control over the details in means of performing the work provided that Consultant is in compliance with the terms of this Agreement. Anything in this Agreement which may appear to give City the right to direct Consultant as to the details of the performance of the services or to exercise a measure of control over Consultant shall mean that Consultant shall follow the desires of City only with respect to the results of the services. 6. COOPERATION Consultant agrees to work closely and cooperate fully with Dave Kiff, Assistant City Manager, the City's designated Research Project administrator ( "Project Administrator "), and any other agencies which may have jurisdiction or interest in the work to be performed. City agrees to cooperate with the Consultant on the Research Project. 7. PROJECT MANAGER Consultant shall assign responsibility for the Research Project to a Principal Investigator, who shall coordinate all phases of Consultant's efforts on the Research Project. The Principal Investigator shall be available to City at all reasonable times during the Research Project term. Consultant has designated Dr. Sunny Jiang to be its 3 9 0 Principal Investigator. Consultant shall not bill any personnel to the Research Project other than those personnel identified in Exhibit "B ", whether or not considered to be key personnel, without City's prior written approval by name and specific salary rate. Consultant shall not remove or reassign the Principal Investigator designated in this Section or assign any new or replacement Principal Investigator to the Research Project without the prior written consent of City. City's approval shall not be unreasonably withheld with respect to replacement of the Principal Investigator. Consultant warrants that it will continuously furnish the necessary personnel to complete the Research Project on a timely basis as contemplated by this Agreement. 8. TIME OF PERFORMANCE Time is of the essence in the performance of the services under this agreement and the services shall be performed by Consultant in accordance with Exhibit A. The failure by Consultant to strictly adhere to the schedule may result in termination of this Agreement by City. Notwithstanding the foregoing, Consultant shall not be responsible for delays which are due to causes beyond Consultant's reasonable control. However, in the case of any such delay in the services to be provided for the Research Project, each party hereby agrees to provide notice to the other party so that all delays can be addressed. 8.1 Consultant shall submit all requests for extensions of time for performance in writing to the Project Administrator not later than ten (10) calendar days after the start of the condition which purportedly causes a delay, and not later than the date upon which performance is due. The Project Administrator shall review all such requests and may grant reasonable time extensions for unforeseeable delays, which are beyond Consultant's control. 8.2 For all time periods not specifically set forth herein, Consultant shall respond in the most expedient and appropriate manner under the circumstances, by either telephone, fax, e-mail, hand delivery or mail. 9. CITY POLICY Consultant will discuss and review all matters relating to policy and project direction with the Project Administrator in advance of all critical decision points in order to ensure that the Research Project proceeds in a manner consistent with City goals and policies. 4 0 0 10. CONFORMANCE TO APPLICABLE REQUIREMENT All work prepared by Consultant shall conform to applicable city, county, state and federal law, regulations and permit requirements and be subject to approval of the Project Administrator and City. 11. PROGRESS Consultant is responsible to keep the Project Administrator and /or his /her duly authorized designee informed on a regular basis regarding the status and progress of the work, activities performed and planned, and any meetings that have been scheduled or are desired. 12. HOLD HARMLESS Consultant shall indemnify, defend, save and hold harmless City, its City Council, boards and commissions, officers and employees from and against any and all loss, damages, liability, claims, allegations of liability, suits, costs and expenses for damages of any nature whatsoever, including, but not limited to, bodily injury, death, personal injury, property damages, or any other claims arising from any and all acts or omissions of Consultant, its employees, agents or subcontractors in the performance of services or work conducted or performed pursuant to this Agreement, but only in proportion to and to the extent such loss, damages, liability, claims, allegations of liability, suits, costs and expenses for damages are caused by or result from the negligent or intentional acts or omissions of Consultant, its officers, agents, or employees. City shall indemnify, defend, save and hold harmless Consultant, its trustees, boards and commissions, officers and employees from and against any and all loss, damages, liability, claims, allegations of liability, suits, costs and expenses for damages of any nature whatsoever, including, but not limited to, bodily injury, death, personal injury, property damages, or any other claims arising from any and all acts or omissions of City, its employees, agents or subcontractors in the performance of services or work conducted or performed pursuant to this Agreement, but only in proportion to and to the extent such loss, damages, liability, claims, allegations of liability, suits, costs and expenses for damages are caused by or result from the negligent or intentional acts or omissions of City, its officers, agents, or employees. Nothing in this indemnity shall be construed as authorizing, any award of attorneys' fees in any action on or to enforce the terms of this Agreement. 5 13. INSURANCE Without limiting consultant's indemnification of City, and prior to commencement of work, Consultant shall obtain and provide and maintain at its own expense during the term of this Agreement self- insurance or policy or policies of liability insurance of the type and amounts described below and satisfactory to City. Certification of all required policies shall be signed by a person authorized by that insurer to bind coverage on its, behalf and must be filed with City prior to exercising any right or performing any work pursuant to this Agreement. Except workers compensation, all insurance policies shall add City, its elected officials, officers, agents, representatives and employees as additional insured for all liability arising from Consultant's services as described herein. All insurance policies shall be issued by an insurance company currently authorized by the Insurance Commissioner to transact business of insurance in the State of California, with an assigned policyholders' Rating of A (or higher) and Financial Size Category Class VII (or larger) in accordance with the latest edition of Bests Key Rating Guide: unless otherwise approved by the City Risk Manager. A. Worker's compensation insurance covering all employees and principals of Consultant, per the laws of the State of California. B. Commercial general liability insurance covering third party liability risks, including without limitation, contractual liability, in a minimum amount of $1 million combined single limit per occurrence for bodily injury, personal injury and property damage. If commercial general liability insurance or other form with a general aggregate is used, either the general aggregate shall apply separately to this Research Project, or the general aggregate limit shall be twice the occurrence limit. C. Commercial auto liability and property insurance covering any owned and rented vehicles of Consultant in a minimum amount of $1 million combined single limit per accident for bodily injury and property damage. Said policy or policies shall be endorsed to state that coverage shall not be canceled by either party, except after thirty (30) days' prior notice has been given in writing to City. Consultant shall give City prompt and timely notice of claim made or suit instituted arising out of Consultant's operation hereunder. Consultant shall also procure and maintain, at its own cost and expense, any additional kinds of insurance, which in its own judgment may be necessary for its proper protection and prosecution of the work. Consultant agrees that, in the event of loss due to any of the perils for which it has agreed to provide comprehensive general and automotive liability insurance, Consultant shall look solely to its insurance for recovery. Consultant hereby grants to City, on behalf of any insurer providing comprehensive general and automotive liability insurance to either Consultant or City with respect to the services of Consultant herein, 3 0 0 a waiver of any right of subrogation which any such insurer of said Consultant may acquire against City by virtue of the payment of any loss under such insurance. 14. PROHIBITION AGAINST TRANSFERS Consultant shall not assign, sublease, hypothecate or transfer this Agreement or any for the services to be performed under this Agreement, directly or indirectly, by, operation of law or otherwise without prior written consent of City. Any attempt to do so without consent of City shall be null and void. 15. OWNERSHIP OF DOCUMENTS Each and every report, draft, work product, map, record and other_ document reproduced, prepared or caused to be prepared by Consultant pursuant to or in connection with this Agreement and delivered to City shall be the exclusive property of City. Documents, including drawings and specifications, prepared by Consultant pursuant to this Agreement are not intended or represented to be suitable for reuse by City or others on any other project. Any use of completed documents for other projects and any use of incomplete documents without specific written authorization from Consultant will be at City's sole risk and without liability to Consultant. Further, any and all liability arising out of changes made to Consultant's deliverables under this Agreement by City or persons other than Consultant is waived against Consultant and City assumes full responsibility for such changes unless City has given Consultant prior notice and has received from Consultant written consent for such changes. Consultant shall, at such time and in such form as City may require, furnish reports concerning the status of services required under this Agreement. 16. CONFIDENTIALITY Because the City deems the Scope of Services outlined in Attachment A to be of benefit to a wide variety of constituencies, the City and its Consultant apply no specific confidentiality limitations on any information which results from the services in this Agreement. Both parties reserve the right to retain any information as confidential if agreed to by both parties. 17. ADMINISTRATION This Agreement will be administered by the City Manager's Department. Dave Kiff, Assistant City Manager, shall be considered the Project Administrator and shall have the authority to act for City under this Agreement. The Assistant City Manager or 7 his authorized representative shall represent City in all matters pertaining to the services to be rendered pursuant to this Agreement. 18. RECORDS Consultant shall keep records and invoices in connection with the work to be performed under this Agreement. Consultant shall maintain complete and accurate records with respect to the costs incurred under this Agreement. All such records shall be clearly identifiable. Consultant shall allow a representative of City to examine, audit and make transcripts or copies of such records during normal business hours. Consultant shall allow inspection of all work, data, documents, proceedings and activities related to the Agreement for a period of three (3) years from the date of final payment under this Agreement. 19. WITHHOLDINGS City may withhold payment of any disputed sums until satisfaction of the dispute with respect to such payment. Such withholding shall not be deemed to constitute a failure to pay according to the terms of this Agreement. Consultant shall not discontinue work as a result of such withholding. Consultant shall have an immediate right to appeal to the City Manager or his designee with respect to such disputed sums. Consultant shall be entitled to receive interest on any withheld sums at the rate of seven percent (7 %) per annum from the date of withholding of any amounts found to have been improperly withheld. 20. CITY'S RIGHT TO EMPLOY OTHER CONSULTANTS City reserves the right to employ other consultants in connection with the Research Project. 21. CONFLICTS OF INTEREST A. The Consultant or its employees may be subject to the provisions of the California Political Reform Act of 1974 (the "Act "), which (1) requires such persons to disclose financial interest that may foreseeably be materially affected by the work performed under this Agreement, and (2) prohibits such persons from making, or participating in making decisions that will foreseeably financially affect such interest. B. If subject to the Act, Consultant shall conform to all requirements of the Act. Failure to do so constitutes a material breach and is grounds for termination of this Agreement by City. Consultant shall indemnify and [] 0 0 hold harmless City for any and all claims for damages resulting from Consultant's violation of this Section. 22. SUBCONSULTANT AND ASSIGNMENT Except as specifically authorized under this Agreement, the services included in this Agreement shall not be assigned, transferred, contracted or subcontracted without prior written approval of City. 23. NOTICES All notices, demands, requests or approvals to be given under this Agreement shall be given in writing and conclusively shall be deemed served whey delivered personally or on the third business day after the deposit thereof in the United States mail, postage prepaid, first class mail, addressed as hereinafter provided. All notices, demands, requests or approvals from Consultant to City shall be addressed to City at: City of Newport Beach City Manager's Office 3300 Newport Boulevard P. O. Box 1768 Newport Beach, CA, 92658 -8915 949/644 -3002 (phone) 949/644 -3020 (fax) All notices, demands, requests or approvals from City to Consultant shall be addressed to Consultant at: Attention: Doug Bickham Contracts Officer Sponsored Projects Administration University of California, Irvine 160 Administration Building Irvine, CA 92697- 1875 (949) 824- 2644 (phone) (949) 824- 2094 (fax) 24. TERMINATION In the event either party hereto fails or refuses to perform any of the provisions hereof at the time and in the manner required hereunder, that party shall be deemed in 0 9 0 default in the performance of this Agreement. If such default is not cured within a period of two (2) days, or if more than two (2) days are reasonably required to cure the default and the defaulting party fails to give adequate assurance of due performance within two (2) days after receipt of written notice of default, specifying the nature of such default and the steps necessary to cure such default, the nondefaulting party may terminate the Agreement forthwith by giving to the defaulting party written notice thereof. City or Consultant shall have the option, at its sole discretion and without cause, of terminating this Agreement by giving seven (7) days' prior written notice to the other party as provided herein. Upon termination of this Agreement, City shall pay to the Consultant that portion of compensation specified in this Agreement that is earned and unpaid prior to the effective date of termination. 25. COMPLIANCES _ Consultant shall comply with all laws, state or federal and all ordinances, rules and regulations enacted or issued by City. 26. WAIVER A waiver by either party of any breach, of any term, covenant or condition contained herein shall not be deemed to be a waiver of any subsequent breach of the same or any other term, covenant or condition contained herein, whether of the same or a different character. 27. INTEGRATED CONTRACT This Agreement represents the full and complete understanding of every kind or nature whatsoever between the Parties hereto, and all preliminary negotiations and agreements of whatsoever kind or nature are merged herein. No verbal agreement or implied covenant shall be held to vary the provisions herein. Any modification of this Agreement will be effective only by written execution signed by both City and Consultant. IN WITNESS WHEREOF, the Parties have caused this Agreement to be executed on the day and year first written above. 10 0 APPROVED AS TO FORM: By: R ert Burnham C' y Attorney for the City of Newport Beach ATTEST: By:�llh"1-e �1 / LaVonne Harkless City Clerk 0 CITY OF NEWPORT BEACH A Municipal Corporation By�w-t Garold B. Adams Mayor for the City of Newport Beach CONSULTANT u�cr3 c ': "9'vi The Regents of the University of California 11 EXHIBIT A Scope of Work and Study Description Determining the Relationship Between Fecal Indicators and Human Pathogenic Viruses in Newport Bay Watershed - What do Indicators Indicate? Phase Background and Objectives: The microbial quality of water is a prime public health concern in today's world. It is estimated that the risk of becoming ill as a result of microbial contamination of food and water is 100,000 times greater than the risk from pesticide contamination (WHO 1993). To protect public health, the World Health Organization and the U.S. Environmental Protection Agency have established microbial pollution indicator standards and recommended routine monitoring of water for fecal bacterial indicators (Dufour, 1984, U.S. EPA 1986,1994, WHO 1993). Coastal recreational water quality standards in California, and throughout most of the world, are based on the concentration of total coliform, fecal coliform, and enterococcus groups of indicator bacteria (Bartram and Rees 2000). However, the adequacy of current water quality standards to indicate the presence or absence of human pathogens is still questionable. For example, human viruses are more resistant to sewage treatment processes and environmental conditions than bacterial indicators and therefore may pose a substantial threat. It is now recognized that the absence, or a low concentration, of indicator organisms in water may not adequately reflect the absence of human viruses. In our previous study of southern California coastal waters, we found over 30% of coastal waters tested contained human viruses, and the presence of these viruses did not correlate with an elevated level of bacterial indicators (Jiang et al. 2000). Waterborne infectious viruses are suspected to be more important than current knowledge suggests because viral diseases are hard to identify by current diagnostic techniques. While the causative agent of nearly 50% of all acute gastrointestinal illnesses cannot be identified (Center for Disease Control, 1988, 1990, 1991, 1993) most of these illnesses are suspected to be caused by viruses. Therefore, viral quality of recreational coastal water is of particular importance and a rising public health concern. Newport Bay, California is an important estuarine system in Southern California. Tourists worldwide visit this area for boating, kayaking, sailing, swimming and other water recreational activities. To protect public health and the beneficial use of this valuable resource, the State Water Quality Control Board requires strategies to be developed and stepwise implemented to improve Newport Bay water quality to meet recreational standards (REC -1) by the year 2014. With the goal of understanding the relationship between fecal indicator bacterial concentrations and human pathogenic viral loads in this watershed, the specific objectives of this study are the following: 12 0 1. To determine the presence and distribution of human enteric viruses in various locations in Newport Bay and its tributaries using polymerase chain reaction (PCR) analysis; 2. To determine the abundance and distribution of F- specific coliphage in order to evaluate its adequacy as an alternative indicator of human viruses in the Newport Bay watershed. 3. To investigate the infectivity of human viruses present in the Watershed by combining PCR with tissue culture analysis. 4. To statistically determine the relationship between human viruses and fecal indicators in the Watershed. Project Approach: Results from the phase I & II Newport Bay water quality investigation conducted during the summer of 2000 indicated that the occurrence and distribution of human enteroviruses did not correlate with the abundance of total coliform, fecal coliform and enterococcus. However, only a few locations around the Bay were sampled repetitively to produce a statistically valid analysis of the relationship between human viruses and indicators. This proposed study will expand sampling locations and frequencies for human viral testing, guided by a desired statistical sensitivity and power. In addition, two more viruses will also be included in our detection effort because the presence of viral contamination in the watershed may shift with the human disease load and the populations harboring the disease (either symptomatic or asympotomatic) in a local community. Additional viruses that will be tested in this research are adenoviruses and hepatitis A viruses. Adenoviruses are widespread in coastal waters of Southern California according to a recent study (Jiang et al. 2000). Seasonal outbreaks of hepatitis A virus in Southern California are also documented by the Los Angeles Health Care Agency. The addition of these two viruses to the testing panel will increase the sensitivity and resolution of detection of human viral pathogens in the Newport Bay watershed. Sampling Plans This project will be conducted over a two -year period. The first year, the phase I part of the study, will primarily focus on a large -scale sampling effort from multiple locations in Newport Bay. The primary sampling locations will include 5 of the 6 sites collected in the previous year (Figure 1). The Backbay drain location will not be sampled this year due to the divergence of the flow. An additional location at Newport Dunes swimming lagoon will be added as the 6'" site due to high usage of this location by bathers. Samples will be collected from each location on a weekly basis starting on August 15`h for three consecutive weeks. Sampling times on each day will be determined based on the tidal chart to ensure all samples are collected during the ebb - tide period. This design will maximize the possibility of capturing urban runoff without being diluted with intruding seawater during the flood -tide period. The sampling frequencies at each site will be determined by statistical tests to achieve the desired resolution. 13 • Ten to twenty liter water samples will be collected from each site and split for detection of fecal bacterial indicators, human viruses and coliphage. Table 1. shows the proposed sampling schedule and parameters that will be determined at each location. Simultaneously, the temperature and salinity of the water will also be measured in situ to provide background information on basic environmental conditions. All data collected will be logged into a database on the same day of the sample collection. 14 Location: Six locations in Bay and tributaries -- similar sites as sampled in Summer /Fall 2000 Discussion: This effort would attempt to keep continuity with 2000's enteric virus /phagelbacti studies in that found the presence of virus particles in one (SA Delhi Channel) of the six sites tested. Dr. Sunny Jiang will test for phage plus viruses (using RT -PCR) and then save the concentrates for infectivity analysis to be completed later. OCHCA will take a split sample and test for the three bacteriological indicators. Sampling Sites: 43rd Street SA/Delhi - SA/Delhi - Univ SD Creek — SD Creek — Michelson Dunes East Mesa campus August 15, 22, August 15, 22, August 15, 22, August 15, 22, August 15, 22, August 15, 22, Dates: and 29 and 29 and 29 and 29 and 29 and 29 Time of Day: Morning Morning Morning morning Morning Morning Liter) I er Liter) Liter) Liter) Liter) Types of Samples Taken: 1 (3-0 Liter) 1 (3-4 Liter) 1 (3-0 liter) 1 (3-4 LileQ 1 (3-4 Liter) 1 (3-4 Liter) Testing for: FC/TC FC1TC FC/TC FCTC FC/TC FC/TC Ent Ent Ent Ent Ent Ent Enteric Virus Enteric Virus Enteric Virus Enteric Virus Enteric Virus Enteric Virus Infectivity Infectivity Infectivity Infectivity Infectivity Infectivity Concentrate Concentrate Concentrate Concentrate Concentrate Concentrate Coliphage Coliphage Coliphage Coliphage Coliphage Coliphage Bacti Tests /Day: 18 Viral TestsrDay: 6 Coliphage Tests/Day: 6 Viral Infect Con dDay 6 14 9 Upper n Sant Ana- elhi Channel \, Jamboree Road p A Main Dike-, B .. ---� -... iego ..'.Salty me 04 , \.Marsh s Dr. n ne =Umt I Sax The Nerrawa u,ddla De Mxe Marine Cm[awaya /^ Nedra IPlaonedi Ar s Drain PC a Brid¢a, Island e' Belboe Pednm7f \lp(},_ Newport Par Bay / � ' —Unit 11 Beam ughn's Launch ig Canyon Wash i J Shellmaker Island y Dc Pipe • ,o un stM,E,W,N Alvarado Mane Ba Isle lji Balboa Pier S St. Pacific Ocean x Ave. ark Ave. Grand Canal • B hia Corinthian don �ve. Harbor Patrol Beach Corona Del Mar Drain Beacl6 Rockv Pt. Beach Figure 1:Map of Newport Bay watershed, California. e indicates OCHCA Sampling Stations and 4indicates sampling locations of this study. Detection of Human Adeno -, Entero- and Hepatitis A Viruses A minimum of ten liters of water from each site will be concentrated using an ultrafiltration system as previously described (Jiang et al. 2000). The final concentrates will be split for determination of coliphage by plaque assay and human viruses by PCR. For human virus detection, the concentrates will either be used immediately for viral assay or frozen after concentration for analysis until the completion of the intensive sampling effort. 15 0 Viral nucleic acid extraction and purification will follow the method originally developed by Boom et al. (1990) with minor modifications. This method uses guanidinium thiocyanate (GuSCN) with Triton X -100 to lyse viral particles and silica beads to absorb nucleic acid, allowing effective purification of DNA and RNA at the same time. In brief, 50 µI of viral concentrate are lysed by 900 µd of GuSCN lysis buffer at room temperature for 10 min. Then 40 µd of silica particles are added and nucleic acids absorbed at room temperature for 10 min with gentle shaking. Silica beads are pelleted, washed and dried. The nucleic acid will then eluted from the beads using 50 PI . TE buffer at a temperature of 56 °C. The purified nucleic acid will be used for detection of human viruses by PCR or RT -PCR as described below. This method has been shown to be effective at purification of viral nucleic acid from seawater concentrates and sewage effluent. I do not expect a great deal of difficulty in purification of viral nucleic acids from Newport Bay waters. Primers specific for human enterovirus, hepatitis A virus and adenovirus will be used for amplification of the respective viral genomes (Table 1). The procedure for RT- PCR of enterovirus and hepatitis A virus will follow the protocol developed by Tsai et al. (1993) with a modification of the total reaction volume. Amplification products will be further confirmed by probing with internal oligonucleotide probes in a dot - blotting format. Probing will also increase the detection limit of the PCR amplification product. For adenovirus detection, a nested PCR protocol will be used as previously described by Pina et al. (1998). This procedure has been successfully used in my laboratory for detection of viruses in coastal waters. Table 1. PCR primers and probes for human virus detection Target Primer and probe sequences Amplicon and viruses target Pan- Upstream, 5'- CCTCCGGCCCTGAATG -3' 197 -bp highly enteroviru Downstream, 5'- ACCGGATGGCCAATC -3' conserved 5' s Probe, 5'- TACTTTGGGTGTCCGTGTTTC -3' untranslated Nested upstream,5'- GCCACCGAGACGTACTTCAGCCTG-3' Nested downstream 5'- TTGTACGAGTACGCGGTATCCTCGCGGTC-3' IT-9 region. Hepatitis Upstream, 5'- CAGCACATCAGAAAGGTGAG -3' 192 -bp VP 1 A virus Downstream, 5'- CTCCAGAATCATCTCCAAC -3' and VP 2 Probe, 5'- TGCTCCTCTTTATCATGCTATG -3' capside protein interphase. Adenoviru First upstream, 5'- 301 -bp Hexon s GCCGCAGTGGTCTTACATGCACATC -3' First downstream, 5'- CAGCACGCCGCGGATGTCAAAGT-3' 143 -bp Hexon Nested upstream,5'- GCCACCGAGACGTACTTCAGCCTG-3' Nested downstream 5'- TTGTACGAGTACGCGGTATCCTCGCGGTC-3' IT-9 10 0 Quantification of F- specific Coliphage by Plaque Assay The presence of F- specific coliphage will be quantified in each sample by a top agar overlay method using either freshly concentrated or unconcentrated water samples. One milliliter and 0.1 ml of sample are mixed with one ml of exponential E. coli HS (pFamp)R host in 1% soft agar, then overlayed over bottom nutrient agar containing 15 µg /ml of each ampicillin and streptomycin to prevent background growth of indigenous bacteria. E. coli HS (pFamp)R contains a plasmid coding for both ampicillin and streptomycin resistance and is a specific host for F- specific RNA coliphage. Plaques will be enumerated after 12 hours of incubation at 37 °C. Quantification of F- specific Coliphage by Culture enrichment In addition to direct plaque assay using concentrated viral samples, 1 -liter water samples from each site will be enriched with 50 ml sterile 11x TSB medium, 10 ml late log phase E. coli host and a final concentration of 15 mg /L of ampicillin and streptomycin. The enrichment method is expected to encourage the recovery of injured phage. Since this method was used in our previous study, enrichment will also be performed this year to provide consistency in sampling method. Five -tube enrichment will be used to determine the most probable number (MPN) of the phage. For MPN determination, five 100 -m1 water samples from each site were amended with 5 ml sterile 11 x TSB medium, 1 ml E. coli host and antibiotics. Negative controls will contain five tubes of 100 -m1 sterile DI water amended with nutrient medium, E. coli host and antibiotic as in regular sample assay. The enrichment cultures will be incubated at 37 °C for 24 hours before spot testing for the presence of F- specific coliphage. For replicate spot testing, 1 ml of log phase E. coli host will be mixed with top agar and overlaid onto TSB agar plates containing antibiotics to form an even bacterial lawn. Two microliters of each enrichment culture was spotted onto the freshly prepared bacterial lawn. Plates are incubated at 37 °C for 8 tol6 hours. Clearing spots are scored and the MPN calculated using Thomas' formula (Clesceri et al. 1998). Determine the Infectivity of Human Viruses by Integrated Cell Culture PCR For samples that test positive for human viruses by PCR assay, the frozen concentrate will be analyzed to determine the infectivity of these viruses by integrated cell culture PCR as described by Ballaster et al. (2000). This portion of the study will be conducted during the second year of the project once initial PCR screening have been completed. Due to the intensity of labor and time requirements for integrated cell culture PCR, analyses will focus on samples that are initially PCR positive for human viruses. A random selection of PCR negative samples will also be tested to ensure the correlation between the PCR results and infectivity analysis. The integrated cell culture PCR assay will be conducted by subcontracting with the Metropolitan Water District of southern California. Final results from these analyses will be compared with the results on presence of human viruses by direct PCR, standard fecal indicators, coliphage and basic environmental parameters. 17 i • Statistical Analysis to Determine the Relationship Between Human Viruses and Fecal Indicators Statistical analysis of the entire data set will be performed during the second project year when all tests for all samples are completed. Both linear regression analysis and multiple regression analysis will be conducted using the data set. The concentration of fecal indicators, human viruses and coliphage may be natural log transformed to achieve normal distribution. Human viruses will be used as the dependent variable and all other measured factors will be used as independent variables in the linear regression analysis. The relationship between independent variables will be examined by analysis of variance (ANOVA) to ruleout of the possibility of covariance. Other statistical analysis may also be used if considered necessary when data becomes available. All statistical analyses will be performed using the SAS or SPSS software packages (SPSS Inc. Chicago, IL). `3-'] a EXHIBIT B RESEARCH PROJECT BUDGET Budget Period: 8115/01 — 08/14/02 TOTAL A. PERSONNEL SERVICES - SALARIES AND WAGES Name: Position Title: Salary /hrly rate % or hrs S. Jiang Prin Investigator $5711 (summer) 0.5 $2,856 TBN Lab Asst III 2535x1.5mo; 2586x4.5mo 50% 6 mo. $7,720 Total Salaries And Wages $ 10,576 Staff Fringe Benefits: Sunny Jiang PI 12.7 %f1b x $2856 sal -$363 TBN Lab Asst III 23 %fb x $7720 sal 1.776 Total Fringe Benefits $ 2,139 Total Personnel Cost $12,715 B. OPERATING EXPENSES Supplies: sample bottles, plastic petridishes, pippets, flasks, and microbiological media Sample Preservation Expenses per sample for coliphage analysis @ $30x18 samples $540 Expenses per sample for enteric virus analysis @ $600x18 samples $10,800 Total Operating Expenses $11,340 Total Direct Costs $24,055 C. UNIVERSITY ADMINISTRATIVE OVERHEAD @ 50.4% $12,124 D. TOTAL COSTS K5,179 Breakdown of Costs per Sample: Direct Indirect Coliphage $ 100 $ 50 Human Viruses $ 1,236 $ 623 (Include entero -, hepatitus A- and adenovirus) IN 0 P F- SPECIFIC COLIPHAGE ANALYSIS AT UC IRVINE P.I. - S. JIANG CITY OF NEWPORT BEACH, CA BUDGET JUSTIFICATION PERSONNEL Principal Investigator: Sunny Jiang, Ph.D. will be in charge of the study design, data analysis for F- specific coliphage results. She will also train the research assistant for conducting the research, assist with sample collection, and sample analysis. Dr, Jiang will take compensation in the form of summer salary calculated at approximately 42% of one summer salary month for Phase I and Phase II of the study. Laboratory Research Assistant: Ms. Waxia Fu will be in charge of conducting the experiments and collection of the samples for F- specific coliphage analysis u. §ing the multi -tube enrichment method. She will conduct experiment for plaque assay and spot test to confirm the presence of coliphage. She will devote 5.5 hours per sample at the rate of $11.40 /hour. Fringe Benefits: Benefits for academic and staff personnel have been calculated at the University - established benefit rate of 9.2% for summer for academic personnel and 5.2% for casual staff. OPERATING EXPENSES Materials and supplies for this project are estimated to cost $30 per sample. These supplies include sample bottles, plastic petridishes, pippets, flasks, and microbiological media. Estimation is based on historical costs and listed catalog pricing. ADMINISTRATIVE OVERHEAD University Overhead (Facilities and Administrative costs) were estimated in accordance with UCI's current indirect cost rate agreement. UCI's indirect cost rate agreement was approved by DHHS, the Federal Cognizant Audit Agency for UCI on 4/10/97 and remains valid through June 30, 2001. 20 0 f ITEM 11 tlJ TO: Members of the Newport Beach City Council COUNCIL AGENDA FROM: Dave Kiff, Assistant City Manager 140. 14 SUBJECT: Contract with the University of California: Water Quality Testing in �'�� I Storm Drains and Tributaries to Newport Bay RECOMMENDED Authorize the Mayor to execute a $36,179 sole- source contract with the Regents ACTION: of the University of California for the testing of storm drains and tributaries to Newport Bay for the presence of human viruses and bacteria. BACKGROUND: While most of Newport Bay's waters routinely test "clean, " several locations in Newport Bay often show bacteria levels higher than allowed by State law (AB 411, Wayne, 1997) for water contact recreational activities like swimming and wading. These areas include: 43rd Street Beach (Channel Place Park) Arches Marina (PCH and Newport Boulevard) Santa Ana /Delhi Channel San Diego Creek at Jamboree APPROVED Big Canyon Wash f f\ U Y U Back Bay Drain/ East Side of Newport Dunes The Orange County Health Care Agency's Environmental Health Division (HCA) tests these six areas and about 30 more (see the map within this staff report) at least once a week. If the areas exceed AB 411 standards for total colifonn, fecal colifonn, or enterococcus, the County directs that the waters be posted as potentially unsafe for water contact recreation. A posting is separate from a closure. Per AB 411, the latter occurs when HCA suspects or knows of a sewage spill or leak. The sign on the left is a closure sign - the sign on the right is a posting sign. Beach closure KEEP OUT SEWAGE CONTAMINATED WATER oeacn WARNING! u Euo n!e brews` 1AVISO! oYEM:auLPlFkaNIWY!!5 � 0 0 The three bacteria listed above are known as indicator bacteria, meaning that they may indicate the presence of a human pathogen like a virus. The key word is may - for we do not know with certainty whether or not a virus is always attached to or near the indicator bacteria. Nor do we know the source of the indicator bacteria - the bacteria may be present due to bird droppings, pet waste, garden fertilizer, or other decomposing organic matter. Determining the source of the bacteria is an expensive challenge, with differing ways on how such sourcing can be done. These methods include: • Genetic Fingerprinting. This methodology takes unknown bacteria from the water, determines the bacteria's DNA pattern, and then attempts to match that pattern to a pattern from bacteria from a known source. • Antibiotic Resistance. This method takes unknown bacteria from the water, determines how the bacteria reacts to up to eight common antibiotics, and then attempts to match the reaction patterns with bacteria from known sources. Newport Bay Study Area Sant Ana- clhi Channel �� me.... a..d Z Ww M. B ego v:.5alti c: (, me } '.Marsh :, : - :.. sD ne -amp I Bum mt 11 D•m aPP" 1.1n4 ughn's Launch T"• "• ^•^ ig Canyon wash meal. aloe Northstar Beach D...nn a.nn. Doory ^^ Ar . onln `PI.on.al Y snalm.Y.r 1n..a (� \1 \4j y Dr. Pipe 43r Pcx Brles. la non, shore ac u s M,E,w,N ue Sth 3 SL 1a.ne s. Yoe lub 19th t each c St.$eac x Ave. Bale.. Plains . DB L B .IDw 1.na ark Ave. Ke"OrY Pl.r arnel Ave. rand Canal !p 6bne "ahi. l inNMn ..D.. Saphire ve.Abalo qVe. m.ne Harbor Patrol Beach r— Il l�tv lj AIV M a 6a Isle coron. bm M.r w.m Btl°•' ^° N St. Bear Rocky Pt. Beach Pacific Ocean 0 0 Better than merely using indicator bacteria, the City has embarked upon a limited testing program of actual viruses within Newport Bay. These tests are expensive, costing more than $1,000 per test. Last summer, the City contracted for viral testing with Dr. Rachel Noble of the Southern California Coastal Water Research Project (SCCWR), a Westminster -based joint- powers agency set up to do water quality research. Dr. Sunny Jiang of the University of California, Irvine, joined Dr. Noble in these efforts. Drs. Noble and Jiang tested the following areas for human enteric virus particles: 43rd Street Beach (Channel Place Park) Arches Marina Bayshore Beach Santa Ana Delhi Channel (near Mesa and near University) San Diego Creek (near Campus and near Michelson) Big Canyon Wash Back Bay Drain/ East Side of Newport Dunes During two distinct periods of testing last summer, Drs. Noble and Jiang's data showed no detectable levels of human enteric viruses in any area EXCEPT the Santa Ana Delhi Channel. Five of six tested samples from the Santa Ana Delhi Channel tested positive for the presence of human enteric viruses. The City paid for this testing using a grant from the State Water Resources Control Board. As Summer 2001 approached, the City planned additional testing efforts and received another $400,000 in State grants to do so. The City's Summer 2001 water quality testing program includes: • Antibiotic Resistance Testing in West Newport. A joint OC Health Care Agency -City effort (which began in August and will continue into October) will attempt to determine the source of chronic bacteria problems around Newport Island. • Swimmer Contribution Study. A City- Regional Board -OCSD effort (completed in late August) will attempt to determine if there is a link between swimmers and fecal coliform in a high -use swimming area. • Vessel Waste Study. A City- Regional Board -OC HCA effort (planned for this fall) will attempt to determine the extent of illegal Haste discharges from boaters into Newport Harbor. • Baywide Bacteria and Virus Testing. In an attempt to determine if contamination levels have changed since 2000, the City and the Regional Board have directed the completion of a 3 -week study in mid -to late- August that re -tests six locations in the Bay for human viruses, coliphage, and the three bacterial indicators. This Baywide Bacteria and Virus Testing effort, again conducted for the City by Dr. Jiang of UCI, followed the Scope of Work shown on the next page. Readers will see that Dr. Jiang tested six different sites in the Bay, all of which were tested during the 2000 studies. Dr. Jiang has added an infectivity component to her study in an attempt to determine to what extent, if any, virus particles detected in the Bay had the ability to infect humans. 0 0 Baywide Sampling Scope of Work - Summer 2001 A study design team oversees all four of the Summer 2001 studies -this team includes staff from OC HCA, the Regional Board, and Newport Bench resident Dr. Jack Skinner, and me. When the results are compiled by Dr. Jiang and reviewed by the Study team, the data shall become publicly available. The total cost of the Baywide Bacti /Virus Sampling is $36,179.00. This amount includes UCI's mandatory 50% charge for overhead. We did not anticipate this overhead charge when the Sampling protocol was originally envisioned, since we were told that it can be waived for certain contracts. UCI, however, denied this waiver for the City . As such, the contract once envisioned to be less than $30,000 and able to be authorized by the City Manager is now over $30,000 and subject to Council approval. The $36,179.00 is budgeted in the City's FY 2001 -02 Capital Improvement Program (CIP #7251- C5100596). Since very few entities in the nation have the ability to conduct these tests -and since Dr. Jiang is familiar with Newport Bay - th e Regional Board and the City have selected her to conduct this work. THIS AGENDA This Agenda Item authorizes the Mayor to execute a contract with the Regents of ITEM: th e University of California to perform baywide bacterial and viral sampling. The contract cost is $36,179.00. The sampling program is complete as of the date of this staff report, but the report - writing and data analysis may not be complete until October 2001. ATT ACHMENT: Attachment A - Contract with the Regents of the University of California I Baywide Ractiflifirus Sampling Location: six locations in Bay and tributaries — similar sites as sampled in SummerlFall 2000 Discussion: This effort would attempt to keep continuity with 2000's enteric vimslphagalbacti studies in that found the presence of virus particles in one (SA Delhi Channel) of the six sites tested. Dr. Sunny Jiang will test for phage plus viruses (using RT.PCR) and then save the concentrates for infectivity analysis to be completed later. OCHCA will take a split sample and test for the three bacteriological indicators. Sampling Sites: QNSeeel SNDem�. SALlelni -Univ MQSa SD Gcek— Cam us SD Leek— MicTelsm Dunes East Aupusl 15.22. AuBUSI 15, 22, Augmc 15, U. August 15, 22, August t5, Z. August 15, 22. Dates: antl 29 and 29 antl 29 and 29 antl 29 and 29 Time of Day: Mpmirp Mpni�g Mominp Mpnirg Morning morning er i r uteri er ier Types of Samples Taken: 1 (aa lii¢q t (34 kge0 t (].a Liter) t (].4 ulep t (a4 L'derj t f3a Lite,) Testing W. FGTC FGiC FClfC FCrtC FGRC FCrtC Ent Ent Em Ent Ent Ent Emetic Vims Enteric VlNS Enlenc Vims enteric Vims Enledc virus Enteric VlNs Infectivity Inlec4vilY InfecevilY infectivity Bacli TestslDay: Colipnage TeiuAviy'. Samples Collected by: Sunny Jiang. Mike Fennessey. CwuKeeper (2) Total p of Samples Collected: 36 Tests done by: vital (RTPCRj. mfediviry Conc(fro:en). Pnage— Sunny Jiang Bacti (MTFj — OC HCA Estimated Cost: A study design team oversees all four of the Summer 2001 studies -this team includes staff from OC HCA, the Regional Board, and Newport Bench resident Dr. Jack Skinner, and me. When the results are compiled by Dr. Jiang and reviewed by the Study team, the data shall become publicly available. The total cost of the Baywide Bacti /Virus Sampling is $36,179.00. This amount includes UCI's mandatory 50% charge for overhead. We did not anticipate this overhead charge when the Sampling protocol was originally envisioned, since we were told that it can be waived for certain contracts. UCI, however, denied this waiver for the City . As such, the contract once envisioned to be less than $30,000 and able to be authorized by the City Manager is now over $30,000 and subject to Council approval. The $36,179.00 is budgeted in the City's FY 2001 -02 Capital Improvement Program (CIP #7251- C5100596). Since very few entities in the nation have the ability to conduct these tests -and since Dr. Jiang is familiar with Newport Bay - th e Regional Board and the City have selected her to conduct this work. THIS AGENDA This Agenda Item authorizes the Mayor to execute a contract with the Regents of ITEM: th e University of California to perform baywide bacterial and viral sampling. The contract cost is $36,179.00. The sampling program is complete as of the date of this staff report, but the report - writing and data analysis may not be complete until October 2001. ATT ACHMENT: Attachment A - Contract with the Regents of the University of California Concentrate Concentmle COncentmte Concentrste Infectivity Cmcem2te InlecCVily Cmcentmle CdIPnaBe CpllPnage COIIIMage CdiPnag¢ Ddlpndge CdiPndge 16 Viml Tests/Day� Viral intact ConUDay 6 5 A study design team oversees all four of the Summer 2001 studies -this team includes staff from OC HCA, the Regional Board, and Newport Bench resident Dr. Jack Skinner, and me. When the results are compiled by Dr. Jiang and reviewed by the Study team, the data shall become publicly available. The total cost of the Baywide Bacti /Virus Sampling is $36,179.00. This amount includes UCI's mandatory 50% charge for overhead. We did not anticipate this overhead charge when the Sampling protocol was originally envisioned, since we were told that it can be waived for certain contracts. UCI, however, denied this waiver for the City . As such, the contract once envisioned to be less than $30,000 and able to be authorized by the City Manager is now over $30,000 and subject to Council approval. The $36,179.00 is budgeted in the City's FY 2001 -02 Capital Improvement Program (CIP #7251- C5100596). Since very few entities in the nation have the ability to conduct these tests -and since Dr. Jiang is familiar with Newport Bay - th e Regional Board and the City have selected her to conduct this work. THIS AGENDA This Agenda Item authorizes the Mayor to execute a contract with the Regents of ITEM: th e University of California to perform baywide bacterial and viral sampling. The contract cost is $36,179.00. The sampling program is complete as of the date of this staff report, but the report - writing and data analysis may not be complete until October 2001. ATT ACHMENT: Attachment A - Contract with the Regents of the University of California 0 0 PROFESSIONAL SERVICES AGREEMENT THIS AGREEMENT, entered into this 15th day of August, 2001, by and between CITY OF NEWPORT BEACH, a Municipal Corporation (hereinafter referred to as "City "), and The Regents of the University of California, on behalf of its Irvine campus (hereinafter referred to as "Consultant'), is made with reference to the following: RECITALS A. City is a municipal corporation duly organized and validly existing under the laws of the State of California with the power to carry on its business as it is now being conducted under the statutes of the State of California and the Charter of City. B. City desires to engage Consultant to provide staff services for BacterialNiral Source Tracking of Tributaries and Storm Drains Discharging into Newport Bay (`Research Project'). C. The principal investigator of Consultant is, for purpose of this Research Project, Dr. Sunny Jiang ('Principal Investigator "). D. City has reviewed the previous experience and evaluated the expertise of Consultant and desires to contract with Consultant under the terms and conditions provided in this Agreement. NOW, THEREFORE, it is mutually agreed by and between the undersigned Parties as follows: 1. TERM The term of this Agreement shall commence on the 15th day of August 2001, and shall terminate on the 14th day of August 2002, unless terminated earlier as set forth herein. 2. SERVICES TO BE PERFORMED Consultant shall diligently perform all the duties set forth in the Scope of Services, attached as Exhibit "A" and incorporated herein by reference. 0 0 3. COMPENSATION TO CONSULTANT City shall pay Consultant for the services in accordance with the provisions of this Section and the Research Project budget, attached as Exhibit 'B" and incorporated herein by reference. No budget changes shall be made during the term of this Agreement without prior written approval of City. Consultant's compensation for all work performed in accordance with this Agreement shall not exceed the total contract price of Thirty -six thousand, one hundred seventy -nine dollars ($36,179). 3.1 Consultant shall maintain accounting records of its billings which includes the name of the employee, type of work performed, times and dates of all work which is billed on an hourly basis and all approved incidental expenses including testing supplies. 3.2 Consultant shall submit monthly invoices to City payable by City within thirty (30) days of receipt of invoice subject to the approval of City. 3.3 Consultant shall not receive any compensation for extra work without prior written authorization of City. 3.4 City shall reimburse Consultant only for those costs or expenses which have been specifically approved in this Agreement, or specifically approved in advance by City. Such cost shall be limited and shall include nothing more than the following costs incurred by Consultant: A. The actual costs of subconsultants for performance of any of the services which Consultant agrees to render pursuant to this Agreement which have been approved in advance by City and awarded in accordance with the terms and conditions of this Agreement. B. Approved computer data processing and reproduction charges. C. Actual costs and /or other costs and /or payments specifically authorized in advance in writing and incurred by Consultant in the performance of this Agreement. 3.5 Notwithstanding any other paragraph or provision of this Agreement, beginning on the effective date of this Agreement, City may withhold payment of ten percent (10 %) of each approved payment as approved retention until all services under this Agreement have been substantially completed. 4. STANDARD OF CARE 4.1 All of the services shall be performed by Consultant or under Consultant's supervision. Consultant represents that it possesses the professional and technical 2 0 personnel required to perform the services required by this Agreement, and that it will perform all services in a manner commensurate with the university's professional standards. All services shall be performed by qualified and experienced personnel who are not employed by City nor have any contractual relationship with City. Consultant represents and warrants to City that it has or shall obtain all licenses, permits, qualifications and approvals required of its profession. Consultant further represents and warrants that it shall keep in effect all such licenses, permits and other approvals during the term of this Agreement. 4.2 Consultant shall not be responsible for delay, nor shall Consultant be responsible for damages or be in default or deemed to be in default by reason of strikes, lockouts, accidents, or acts of God, or the failure of City to furnish timely information or to approve or disapprove Consultant's work promptly, or delay or faulty performance by City, contractors, or governmental agencies, or any other delays beyond Consultant's control or without Consultant's fault. 5. INDEPENDENT PARTIES City retains Consultant on an independent contractor basis and Consultant is not an employee of City. The manner and means of conducting the work are under the control of Consultant, except to the extent they are limited by statute, rule or regulation and the expressed terms of this Agreement. Nothing in this Agreement shall be deemed to constitute Consultant or any of Consultant's employees or agents, to be the agents or employees of City. Consultant shall have the responsibility for and control over the details in means of performing the work provided that Consultant is in compliance with the terms of this Agreement. Anything in this Agreement which may appear to give City the right to direct Consultant as to the details of the performance of the services or to exercise a measure of control over Consultant shall mean that Consultant shall follow the desires of City only with respect to the results of the services. 6. COOPERATION Consultant agrees to work closely and cooperate fully with Dave Kiff, Assistant City Manager, the City's designated Research Project administrator ( "Project Administrator "), and any other agencies which may have jurisdiction or interest in the work to be performed. City agrees to cooperate with the Consultant on the Research Project. 7. PROJECT MANAGER Consultant shall assign responsibility for the Research Project to a Principal Investigator, who shall coordinate all phases of Consultant's efforts on the Research Project. The Principal Investigator shall be available to City at all reasonable times during the Research Project term. Consultant has designated Dr. Sunny Jiang to be its 3 0 0 Principal Investigator. Consultant shall not bill any personnel to the Research Project other than those personnel identified in Exhibit "B ", whether or not considered to be key personnel, without City's prior written approval by name and specific salary rate. Consultant shall not remove or reassign the Principal Investigator designated in this Section or assign any new or replacement Principal Investigator to the Research Project without the prior written consent of City. City's approval shall not be unreasonably withheld with respect to replacement of the Principal Investigator. Consultant warrants that it will continuously furnish the necessary personnel to complete the Research Project on a timely basis as contemplated by this Agreement. 8. TIME OF PERFORMANCE Time is of the essence in the performance of the services under this Agreement and the services shall be performed by Consultant in accordance with Exhibit A. The failure by Consultant to strictly adhere to the schedule may result in termination of this Agreement by City. Notwithstanding the foregoing, Consultant shall not be responsible for delays which are due to causes beyond Consultant's reasonable control. However, in the case of any such delay in the services to be provided for the Research Project, each party hereby agrees to provide notice to the other party so that all delays can be addressed. 8.1 Consultant shall submit all requests for extensions of time for performance in writing to the Project Administrator not later than ten (10) calendar days after the start of the condition which purportedly causes a delay, and not later than the date upon which performance is due. The Project Administrator shall review all such requests and may grant reasonable time extensions for unforeseeable delays, which are beyond Consultant's control. 8.2 For all time periods not specifically set forth herein, Consultant shall respond in the most expedient and appropriate manner under the circumstances, by either telephone, fax, e -mail, hand delivery or mail. 9. CITY POLICY Consultant will discuss and review all matters relating to policy and project direction with the Project Administrator in advance of all critical decision points in order to ensure that the Research Project proceeds in a manner consistent with City goals and policies. rd 0 10. CONFORMANCE TO APPLICABLE REQUIREMENT All work prepared by Consultant shall conform to applicable city, county, state and federal law, regulations and permit requirements and be subject to approval of the Project Administrator and City. 11. PROGRESS Consultant is responsible to keep the Project Administrator and /or his /her duly authorized designee informed on a regular basis regarding the status and progress of the work, activities performed and planned, and any meetings that have been scheduled or are desired. 12. HOLD HARMLESS Consultant shall indemnify, defend, save and hold harmless City, its City Council, boards and commissions, officers and employees from and against any and all loss, damages, liability, claims, allegations of liability, suits, costs and expenses for damages of any nature whatsoever, including, but not limited to, bodily injury, death, personal injury, property damages, or any other claims arising from any and all acts or omissions of Consultant, its employees, agents or subcontractors in the performance of services or work conducted or performed pursuant to this Agreement, but only in proportion to and to the extent such loss, damages, liability, claims, allegations of liability, suits, costs and expenses for damages are caused by or result from the negligent or intentional acts or omissions of Consultant, its officers, agents, or employees. City shall indemnify, defend, save and hold harmless Consultant, its trustees, boards and commissions, officers and employees from and against any and all loss, damages, liability, claims, allegations of liability, suits, costs and expenses for damages of any nature whatsoever, including, but not limited to, bodily injury, death, personal injury, property damages, or any other claims arising from any and all acts or omissions of City, its employees, agents or subcontractors in the performance of services or work conducted or performed pursuant to this Agreement, but only in proportion to and to the extent such loss, damages, liability, claims, allegations of liability, suits, costs and expenses for damages are caused by or result from the negligent or intentional acts or omissions of City, its officers, agents, or employees. Nothing in this indemnity shall be construed as authorizing, any award of attorneys' fees in any action on or to enforce the terms of this Agreement. 5 0 0 13. INSURANCE Without limiting consultant's indemnification of City, and prior to commencement of work, Consultant shall obtain and provide and maintain at its own expense during the term of this Agreement self- insurance or policy or policies of liability insurance of the type and amounts described below and satisfactory to City. Certification of all required policies shall be signed by a person authorized by that insurer to bind coverage on its behalf and must be filed with City prior to exercising any right or performing any work pursuant to this Agreement. Except workers compensation, all insurance policies shall add City, its elected officials, officers, agents, representatives and employees as additional insured for all liability arising from Consultant's services as described herein. All insurance policies shall be issued by an insurance company currently authorized by the Insurance Commissioner to transact business of insurance in the State of California, with an assigned policyholders' Rating of A (or higher) and Financial Size Category Class VII (or larger) in accordance with the latest edition of Bests Key Rating Guide: unless otherwise approved by the City Risk Manager. A. Worker's compensation insurance covering all employees and principals of Consultant, per the laws of the State of California. B. Commercial general liability insurance covering third party liability risks, including without limitation, contractual liability, in a minimum amount of $1 million combined single limit per occurrence for bodily injury, personal injury and property damage. If commercial general liability insurance or other form with a general aggregate is used, either the general aggregate shall apply separately to this Research Project, or the general aggregate limit shall be twice the occurrence limit. C. Commercial auto liability and property insurance covering any owned and rented vehicles of Consultant in a minimum amount of $1 million combined single limit per accident for bodily injury and property damage. Said policy or policies shall be endorsed to state that coverage shall not be canceled by either party, except after thirty (30) days' prior notice has been given in writing to City. Consultant shall give City prompt and timely notice of claim made or suit instituted arising out of Consultant's operation hereunder. Consultant shall also procure and maintain, at its own cost and expense, any additional kinds of insurance, which in its own judgment may be necessary for its proper protection and prosecution of the work. Consultant agrees that, in the event of loss due to any of the perils for which it has agreed to provide comprehensive general and automotive liability insurance, Consultant shall look solely to its insurance for recovery. Consultant hereby grants to City, on behalf of any insurer providing comprehensive general and automotive liability insurance to either Consultant or City with respect to the services of Consultant herein, 11 0 0 a waiver of any right of subrogation which any such insurer of said Consultant may acquire against City by virtue of the payment of any loss under such insurance. 14. PROHIBITION AGAINST TRANSFERS Consultant shall not assign, sublease, hypothecate or transfer this Agreement or any for the services to be performed under this Agreement, directly or indirectly, by operation of law or otherwise without prior written consent of City. Any attempt to do so without consent of City shall be null and void. 15. OWNERSHIP OF DOCUMENTS Each and every report, draft, work product, map, record and other document reproduced, prepared or caused to be prepared by Consultant pursuant to or in connection with this Agreement and delivered to City shall be the exclusive property of City. Documents, including drawings and specifications, prepared by Consultant pursuant to this Agreement are not intended or represented to be suitable for reuse by City or others on any other project. Any use of completed documents for other projects and any use of incomplete documents without specific written authorization from Consultant will be at City's sole risk and without liability to Consultant. Further, any and all liability arising out of changes made to Consultant's deliverables under this Agreement by City or persons other than Consultant is waived against Consultant and City assumes full responsibility for such changes unless City has given Consultant prior notice and has received from Consultant written consent for such changes. Consultant shall, at such time and in such form as City may require, furnish reports concerning the status of services required under this Agreement. 16. CONFIDENTIALITY Because the City deems the Scope of Services outlined in Attachment A to be of benefit to a wide variety of constituencies, the City and its Consultant apply no specific confidentiality limitations on any information which results from the services in this Agreement. Both parties reserve the right to retain any information as confidential if agreed to by both parties. 17. ADMINISTRATION This Agreement will be administered by the City Manager's Department. Dave Kiff, Assistant City Manager, shall be considered the Project Administrator and shall have the authority to act for City under this Agreement. The Assistant City Manager or 0 0 his authorized representative shall represent City in all matters pertaining to the services to be rendered pursuant to this Agreement. 18. RECORDS Consultant shall keep records and invoices in connection with the work to be performed under this Agreement. Consultant shall maintain complete and accurate records with respect to the costs incurred under this Agreement. All such records shall be clearly identifiable. Consultant shall allow a representative of City to examine, audit and make transcripts or copies of such records during normal business hours. Consultant shall allow inspection of all work, data, documents, proceedings and activities related to the Agreement for a period of three (3) years from the date of final payment under this Agreement. 19. WITHHOLDINGS City may withhold payment of any disputed sums until satisfaction of the dispute with respect to such payment. Such withholding shall not be deemed to constitute a failure to pay according to the terms of this Agreement. Consultant shall not discontinue work as a result of such withholding. Consultant shall have an immediate right to appeal to the City Manager or his designee with respect to such disputed sums. Consultant shall be entitled to receive interest on any withheld sums at the rate of seven percent (7 %) per annum from the date of withholding of any amounts found to have been improperly withheld. 20. CITY'S RIGHT TO EMPLOY OTHER CONSULTANTS City reserves the right to employ other consultants in connection with the Research Project. 21. CONFLICTS OF INTEREST A. The Consultant or its employees may be subject to the provisions of the California Political Reform Act of 1974 (the "Act "), which (1) requires such persons to disclose financial interest that may foreseeably be materially affected by the work performed under this Agreement, and (2) prohibits such persons from making, or participating in making decisions that will foreseeably financially affect such interest. B. If subject to the Act, Consultant shall conform to all requirements of the Act. Failure to do so constitutes a material breach and is grounds for termination of this Agreement by City. Consultant shall indemnify and K 0 0 hold harmless City for any and all claims for damages resulting from Consultant's violation of this Section. 22. SUBCONSULTANT AND ASSIGNMENT Except as specifically authorized under this Agreement, the services included in this Agreement shall not be assigned, transferred, contracted or subcontracted without prior written approval of City. 23. NOTICES All notices, demands, requests or approvals to be given under this Agreement shall be given in writing and conclusively shall be deemed served when delivered personally or on the third business day after the deposit thereof in the United States mail, postage prepaid, first class mail, addressed as hereinafter provided. All notices, demands, requests or approvals from Consultant to City shall be addressed to City at: City of Newport Beach City Manager's Office 3300 Newport Boulevard P. 0. Box 1768 Newport Beach, CA, 92658 -8915 949/644 -3002 (phone) 949/644 -3020 (fax) All notices, demands, requests or approvals from City to Consultant shall be addressed to Consultant at: Attention: Doug Bickham Contracts Officer Sponsored Projects Administration University of California, Irvine 160 Administration Building Irvine, CA 92697- 1875 (949) 824- 2644 (phone) (949) 824- 2094 (fax) 24. TERMINATION In the event either party hereto fails or refuses to perform any of the provisions hereof at the time and in the manner required hereunder, that party shall be deemed in 9 0 0 default in the performance of this Agreement. If such default is not cured within a period of two (2) days, or if more than two (2) days are reasonably required to cure the default and the defaulting party fails to give adequate assurance of due performance within two (2) days after receipt of written notice of default, specifying the nature of such default and the steps necessary to cure such default, the nondefaulting party may terminate the Agreement forthwith by giving to the defaulting party written notice thereof. City or Consultant shall have the option, at its sole discretion and without cause, of terminating this Agreement by giving seven (7) days' prior written notice to the other party as provided herein. Upon termination of this Agreement, City shall pay to the Consultant that portion of compensation specified in this Agreement that is earned and unpaid prior to the effective date of termination. 25. COMPLIANCES Consultant shall comply with all laws, state or federal and all ordinances, rules and regulations enacted or issued by City. 26. WAIVER A waiver by either party of any breach, of any term, covenant or condition contained herein shall not be deemed to be a waiver of any subsequent breach of the same or any other term, covenant or condition contained herein, whether of the same or a different character. 27. INTEGRATED CONTRACT This Agreement represents the full and complete understanding of every kind or nature whatsoever between the Parties hereto, and all preliminary negotiations and agreements of whatsoever kind or nature are merged herein. No verbal agreement or implied covenant shall be held to vary the provisions herein. Any modification of this Agreement will be effective only by written execution signed by both City and Consultant. IN WITNESS WHEREOF, the Parties have caused this Agreement to be executed on the day and year first written above. 10 APPROVED AS TO FORM: By: Robert Burnham City Attorney for the City of Newport Beach ATTEST: LaVonne Harkless City Clerk F CITY OF NEWPORT BEACH A Municipal Corporation la Garold B. Adams Mayor for the City of Newport Beach CONSULTANT By: The Regents of the University of California 11 0 0 EXHIBIT A Scope of Work and Study Description Determining the Relationship Between Fecal Indicators and Human Pathogenic Viruses in Newport Bay Watershed - What do Indicators Indicate? Phase Background and Objectives: The microbial quality of water is a prime public health concern in today's world. It is estimated that the risk of becoming ill as a result of microbial contamination of food and water is 100,000 times greater than the risk from pesticide contamination (WHO 1993). To protect public health, the World Health Organization and the U.S. Environmental Protection Agency have established microbial pollution indicator standards and recommended routine monitoring of water for fecal bacterial indicators (Dufour, 1984, U.S. EPA 1986,1994, WHO 1993). Coastal recreational water quality standards in California, and throughout most of the world, are based on the concentration of total coliform, fecal coliform, and enterococcus groups of indicator bacteria (Bartram and Rees 2000). However, the adequacy of current water quality standards to indicate the presence or absence of human pathogens is still questionable. For example, human viruses are more resistant to sewage treatment processes and environmental conditions than bacterial indicators and therefore may pose a substantial threat. It is now recognized that the absence, or a low concentration, of indicator organisms in water may not adequately reflect the absence of human viruses. In our previous study of southern California coastal waters, we found over 30% of coastal waters tested contained human viruses, and the presence of these viruses did not correlate with an elevated level of bacterial indicators (Jiang et al. 2000). Waterborne infectious viruses are suspected to be more important than current knowledge suggests because viral diseases are hard to identify by current diagnostic techniques. While the causative agent of nearly 50% of all acute gastrointestinal illnesses cannot be identified (Center for Disease Control, 1988, 1990, 1991, 1993) most of these illnesses are suspected to be caused by viruses. Therefore, viral quality of recreational coastal water is of particular importance and a rising public health concern. Newport Bay, California is an important estuarine system in Southern California. Tourists worldwide visit this area for boating, kayaking, sailing, swimming and other water recreational activities. To protect public health and the beneficial use of this valuable resource, the State Water Quality Control Board requires strategies to be developed and stepwise implemented to improve Newport Bay water quality to meet recreational standards (REC -1) by the year 2014. With the goal of understanding the relationship between fecal indicator bacterial concentrations and human pathogenic viral loads in this watershed, the specific objectives of this study are the following: 12 0 0 1. To determine the presence and distribution of human enteric viruses in various locations in Newport Bay and its tributaries using polymerase chain reaction (PCR) analysis; 2. To determine the abundance and distribution of F- specific coliphage in order to evaluate its adequacy as an alternative indicator of human viruses in the Newport Bay watershed. 3. To investigate the infectivity of human viruses present in the Watershed by combining PCR with tissue culture analysis. 4. To statistically determine the relationship between human viruses and fecal indicators in the Watershed. Project Approach: Results from the phase I & II Newport Bay water quality investigation conducted during the summer of 2000 indicated that the occurrence and distribution of human enteroviruses did not correlate with the abundance of total coliform, fecal coliform and enterococcus. However, only a few locations around the Bay were sampled repetitively to produce a statistically valid analysis of the relationship between human viruses and indicators. This proposed study will expand sampling locations and frequencies for human viral testing, guided by a desired statistical sensitivity and power. In addition, two more viruses will also be included in our detection effort because the presence of viral contamination in the watershed may shift with the human disease load and the populations harboring the disease (either symptomatic or asympotomatic) in a local community. Additional viruses that will be tested in this research are adenoviruses and hepatitis A viruses. Adenoviruses are widespread in coastal waters of Southern California according to a recent study (Jiang et al. 2000). Seasonal outbreaks of hepatitis A virus in Southern California are also documented by the Los Angeles Health Care Agency. The addition of these two viruses to the testing panel will increase the sensitivity and resolution of detection of human viral pathogens in the Newport Bay watershed. Sampling Plans This project will be conducted over a two -year period. The first year, the phase I part of the study, will primarily focus on a large -scale sampling effort from multiple locations in Newport Bay. The primary sampling locations will include 5 of the 6 sites collected in the previous year (Figure 1). The Backbay drain location will not be sampled this year due to the divergence of the flow. An additional location at Newport Dunes swimming lagoon will be added as the 6t' site due to high usage of this location by bathers. Samples will be collected from each location on a weekly basis starting on August 15th for three consecutive weeks. Sampling times on each day will be determined based on the tidal chart to ensure all samples are collected during the ebb - tide period. This design will maximize the possibility of capturing urban runoff without being diluted with intruding seawater during the flood -tide period. The sampling frequencies at each site will be determined by statistical tests to achieve the desired resolution. 13 0 0 Ten to twenty liter water samples will be collected from each site and split for detection of fecal bacterial indicators, human viruses and coliphage. Table 1. shows the proposed sampling schedule and parameters that will be determined at each location. Simultaneously, the temperature and salinity of the water will also be measured in situ to provide background information on basic environmental conditions. All data collected will be logged into a database on the same day of the sample collection. Table 1. Baywide Bacti[Virus Sampling Location: Six locations in Bay and tributaries — similar sites as sampled in Summer /Fall 2000 Discussion: This effort would attempt to keep continuity with 2000's enteric virus /phage /bacti studies in that found the presence of virus particles in one (SA Delhi Channel) of the six sites tested. Dr. Sunny Jiang will lest for phage plus viruses (using RT -PCR) and then save the concentrates for infectivity analysis to be completed later. OCHCA will take a split sample and test for the three bacteriological indicators. 43rd Street Sampling Sites: SA1Delhi - SA/Delhi - Univ Mesa SD Creek — Campus SD Creek — Michelson Dunes East August 15, 22, August 15, 22, August 15, 22, August 15, 22, August 15, 22, August 15, 22, Dales: and 29 and 29 and 29 and 29 and 29 and 29 Time of Day: Morning Morning Morning Morning Morning Morning Liter) Liter) Liter) Liter) Liter) er Types of Samples Taken: 1 (3< Liter) 1 (3� Dter) 1 (34 Liter) 1 (34 Liter) 1 (34 Liter) 1 (3-4 Liter) Testing for: FC/rC FC/rC FCfTC FC/TC FC TC FCrrC Ent Ent Ent Ent Ent Enteric Virus Entedc Virus Enteric Virus Enteric Virus Enteric Virus Infectivity Infectivity Infectivity Infectivity Infectivity EEnlenc Concentrate Concentrate Concentrate Concentrate Concentrate Coliphage Coliphage Coliphage Coliphage Coliphage Bacti Tests /Day: 78 Viral Tests /Day: 6 Coliphage Tests /Day: 6 Viral Infect Conc/Day. 6 14 14 E Mein Upper Island The Nartov Middle Ial•w Northstar Bea 0 Sant Ana- elhi Channel \. J mbaree Road like B -:: M .... .......... '.Marsh :' :.:::. . i n i ne na I Baan Unit 11 Bern ughn's Launch ig Canyon Wash 1 Sh.Unaker bland' ?� y Dr. Pipe un s M,E,W,N �Ne.port ^.a. x Ave. bland ark Ave. • - Harbor Patrol Beach Corona Del Mar Drain N St. Beacl Rocky Pt. Beach Figure 1:Map of Newport Bay watershed, California. • indicates OCHCA Sampling Stations and 4indicates sampling locations of this study. Detection of Human Adeno -, Entero- and Hepatitis A Viruses A minimum of ten liters of water from each site will be concentrated using an ultrafiltration system as previously described (Jiang et al. 2000). The final concentrates will be split for determination of coliphage by plaque assay and human viruses by PCR. For human virus detection, the concentrates will either be used immediately for viral assay or frozen after concentration for analysis until the completion of the intensive sampling effort. 15 Oe ass• Merino 0.v" Short., (Planned) ' (, Ar s Drain 3r PCH Bridge nine It \ La enoa shore Be 38th 3 St. b Island $• \ s. Yac lub 19th t each S t.-Beac Balboa Pawns a th L Be' Ne.port Pier a., Island a arbor Sap Island Alvarado Bav Isle I sae+.i B•Ibos Pier Pacific Ocean 0 Sant Ana- elhi Channel \. J mbaree Road like B -:: M .... .......... '.Marsh :' :.:::. . i n i ne na I Baan Unit 11 Bern ughn's Launch ig Canyon Wash 1 Sh.Unaker bland' ?� y Dr. Pipe un s M,E,W,N �Ne.port ^.a. x Ave. bland ark Ave. • - Harbor Patrol Beach Corona Del Mar Drain N St. Beacl Rocky Pt. Beach Figure 1:Map of Newport Bay watershed, California. • indicates OCHCA Sampling Stations and 4indicates sampling locations of this study. Detection of Human Adeno -, Entero- and Hepatitis A Viruses A minimum of ten liters of water from each site will be concentrated using an ultrafiltration system as previously described (Jiang et al. 2000). The final concentrates will be split for determination of coliphage by plaque assay and human viruses by PCR. For human virus detection, the concentrates will either be used immediately for viral assay or frozen after concentration for analysis until the completion of the intensive sampling effort. 15 0 0 Viral nucleic acid extraction and purification will follow the method originally developed by Boom et al. (1990) with minor modifications. This method uses guanidinium thiocyanate (GuSCN) with Triton X -100 to lyse viral particles and silica beads to absorb nucleic acid, allowing effective purification of DNA and RNA at the same time. In brief, 50 Id of viral concentrate are lysed by 900 ld of GuSCN lysis buffer at room temperature for 10 min. Then 40 µl of silica particles are added and nucleic acids absorbed at room temperature for 10 min with gentle shaking. Silica beads are pelleted, washed and dried. The nucleic acid will then eluted from the beads using 50 µl TE buffer at a temperature of 56 °C. The purified nucleic acid will be used for detection of human viruses by PCR or RT -PCR as described below. This method has been shown to be effective at purification of viral nucleic acid from seawater concentrates and sewage effluent. I do not expect a great deal of difficulty in purification of viral nucleic acids from Newport Bay waters. Primers specific for human enterovirus, hepatitis A virus and adenovirus will be used for amplification of the respective viral genomes (Table 1). The procedure for RT- PCR of enterovirus and hepatitis A virus will follow the protocol developed by Tsai et al. (1993) with a modification of the total reaction volume. Amplification products will be further confirmed by probing with internal oligonucleotide probes in a dot - blotting format. Probing will also increase the detection limit of the PCR amplification product. For adenovirus detection, a nested PCR protocol will be used as previously described by Pina et al. (1998). This procedure has been successfully used in my laboratory for detection of viruses in coastal waters. Table 1. PCR primers and probes for human virus detection Target viruses Primer and probe sequences Amplicon and target Pan- Upstream, 5'- CCTCCGGCCCTGAATG -3' 197 -bp highly enteroviru Downstream, 5'- ACCGGATGGCCAATC -3' conserved 5' s Probe, 5'- TACTTTGGGTGTCCGTGTTTC -3' untranslated region. Hepatitis Upstream, 5'- CAGCACATCAGAAAGGTGAG -3' 192 -bp VP 1 A virus Downstream, 5'- CTCCAGAATCATCTCCAAC -3' and VP 2 Probe, 5'- TGCTCCTCTTTATCATGCTATG -3' capside protein interphase. Adenoviru First upstream, 5'- 301 -bp Hexon s GCCGCAGTGGTCTTACATGCACATC -3' First downstream, 5'- CAGCACGCCGCGGATGTCAAAGT-3' 143 -bp Hexon Nested upstream,5'- GCCACCGAGACGTACTTCAGCCTG-3' Nested downstream 5'- TTGTACGAGTACGCGGTATCCTCGCGGTC-3' 16 0 0 Quantification of F- specific Coliphage by Plaque Assay The presence of F- specific coliphage will be quantified in each sample by a top agar overlay method using either freshly concentrated or unconcentrated water samples. One milliliter and 0.1 ml of sample are mixed with one ml of exponential E. coli HS (pFamp)R host in 1% soft agar, then overlayed over bottom nutrient agar containing 15 µg /ml of each ampicillin and streptomycin to prevent background growth of indigenous bacteria. E. coli HS (pFamp)R contains a plasmid coding for both ampicillin and streptomycin resistance and is a specific host for F- specific RNA coliphage. Plaques will be enumerated after 12 hours of incubation at 37 °C. Quantification of F- specific Coliphage by Culture enrichment In addition to direct plaque assay using concentrated viral samples, 1 -liter water samples from each site will be enriched with 50 ml sterile 11x TSB medium, 10 ml late log phase E. coli host and a final concentration of 15 mg /L of ampicillin and streptomycin. The enrichment method is expected to encourage the recovery of injured phage. Since this method was used in our previous study, enrichment will also be performed this year to provide consistency in sampling method. Five -tube enrichment will be used to determine the most probable number (MPN) of the phage. For MPN determination, five 100 -ml water samples from each site were amended with 5 ml sterile 11 x TSB medium, 1 ml E. coli host and antibiotics. Negative controls will contain five tubes of 100 -ml sterile DI water amended with nutrient medium, E. coli host and antibiotic as in regular sample assay. The enrichment cultures will be incubated at 37 °C for 24 hours before spot testing for the presence of F- specific coliphage. For replicate spot testing, 1 ml of log phase E. coli host will be mixed with top agar and overlaid onto TSB agar plates containing antibiotics to form an even bacterial lawn. Two microliters of each enrichment culture was spotted onto the freshly prepared bacterial lawn. Plates are incubated at 37 °C for 8 to16 hours. Clearing spots are scored and the MPN calculated using Thomas' formula (Clesceri et al. 1998). Determine the Infectivity of Human Viruses by Integrated Cell Culture PCR For samples that test positive for human viruses by PCR assay, the frozen concentrate will be analyzed to determine the infectivity of these viruses by integrated cell culture PCR as described by Ballaster et al. (2000). This portion of the study will be conducted during the second year of the project once initial PCR screening have been completed. Due to the intensity of labor and time requirements for integrated cell culture PCR, analyses will focus on samples that are initially PCR positive for human viruses. A random selection of PCR negative samples will also be tested to ensure the correlation between the PCR results and infectivity analysis. The integrated cell culture PCR assay will be conducted by subcontracting with the Metropolitan Water District of southern California. Final results from these analyses will be compared with the results on presence of human viruses by direct PCR, standard fecal indicators, coliphage and basic environmental parameters. 17 0 0 Statistical Analysis to Determine the Relationship Between Human Viruses and Fecal Indicators Statistical analysis of the entire data set will be performed during the second project year when all tests for all samples are completed. Both linear regression analysis and multiple regression analysis will be conducted using the data set. The concentration of fecal indicators, human viruses and coliphage may be natural log transformed to achieve normal distribution. Human viruses will be used as the dependent variable and all other measured factors will be used as independent variables in the linear regression analysis. The relationship between independent variables will be examined by analysis of variance (ANOVA) to ruleout of the possibility of covariance. Other statistical analysis may also be used if considered necessary when data becomes available. All statistical analyses will be performed using the SAS or SPSS software packages (SPSS Inc. Chicago, IL). W] a • EXHIBIT B RESEARCH PROJECT BUDGET Budget Period: 8115101 — 08/14/02 TOTAL A. PERSONNEL SERVICES - SALARIES AND WAGES Name: Position Title: Salary /hrly rate % or hrs S. Jiang Prin Investigator $5711 (summer) 0.5 $2,856 TBN Lab Asst III 2535x1.5mo; 2586xa.5mo 50% 6 mo. $7,720 Total Salaries And Wages $ 10,576 Staff Fringe Benefits: Sunny Jiang PI 12.7% fib x $2856 sal $363 TBN Lab Asst III 23% fb x $7720 sal 1.776 Total Fringe Benefits $ 2,139 Total Personnel Cost $12,715 B. OPERATING EXPENSES Supplies: sample bottles, plastic petridishes, pippets, flasks, and microbiological media Sample Preservation Expenses per sample for coliphage analysis @ $30x18 samples $540 Expenses per sample for enteric virus analysis @ $600x18 samples $10,800 Total Operating Expenses $11,340 Total Direct Costs $24,055 C. UNIVERSITY ADMINISTRATIVE OVERHEAD @ 50.4% $12,124 D. TOTAL COSTS 36 179 Breakdown of Costs per Sample: Direct Indirect Coliphage $ 100 $ 50 Human Viruses $ 1,236 $ 623 (Include entero -, hepatitus A- and adenovirus) 19 r_1 L F- SPECIFIC COLIPHAGE ANALYSIS AT UC IRVINE P.I. - S. JIANG CITY OF NEWPORT BEACH, CA BUDGET JUSTIFICATION PERSONNEL Principal Investigator: Sunny Jiang, Ph.D. will be in charge of the study design, data analysis for F- specific coliphage results. She will also train the research assistant for conducting the research, assist with sample collection, and sample analysis. Dr, Jiang will take compensation in the form of summer salary calculated at approximately 42% of one summer salary month for Phase I and Phase II of the study. Laborato[y Research Assistant: Ms. Waxia Fu will be in charge of conducting the experiments and collection of the samples for F- specific coliphage analysis using the multi -tube enrichment method. She will conduct experiment for plaque assay and spot test to confirm the presence of coliphage. She will devote 5.5 hours per sample at the rate of $11.40 /hour. Fringe Benefits: Benefits for academic and staff personnel have been calculated at the University - established benefit rate of 9.2% for summer for academic personnel and 5.2% for casual staff. OPERATING EXPENSES Materials and supplies for this project are estimated to cost $30 per sample. These supplies include sample bottles, plastic petridishes, pippets, flasks, and microbiological media. Estimation is based on historical costs and listed catalog pricing. ADMINISTRATIVE OVERHEAD University Overhead (Facilities and Administrative costs) were estimated in accordance with UCI's current indirect cost rate agreement. UCI's indirect cost rate agreement was approved by DHHS, the Federal Cognizant Audit Agency for UCI on 4/10/97 and remains valid through June 30, 2001. 20