HomeMy WebLinkAbout12 - Newport Bay Viral TestingITEM 12
TO: Members of the Newport Beach City Council
FROM: Dave Kiff, Deputy City Manager
SUBJECT: Contract Approval - Viral Testing in Storm Drains and Tributaries to
Newport Bay (Phase II)
RECOMMENDED Authorize the City Manager to execute a contract with the Southern California
ACTION: Coastal Water Research Project (SCCWRP) for human enteric virus testing (Phase
II) in storm drains and tributaries to Newport Bay.
BACKGROUND: At least nine locations in Newport Bay routinely 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)
Bayshore Beach
Santa Ana/ Delhi Channel
— San Diego Creek at Jamboree
— Big Canyon Wash
— Back Bay Drain /East Side of Newport Dunes
The Orange County Health Care Agency's Environmental Health Division
(HCA) tests these nine areas and about 25 more at least one time a week. If the
areas exceed AB 411 standards for total coliform, fecal coliform, 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.
Beach Closure Beach Posting
KEEP OUT
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SEWAGE CONTAMINATED
WATER
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WARNING!
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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 (a single
bird dropping can have 2.4 million fecal coliforms), pet waste, garden fertilizer,
or other decomposing organic matter.
Sourcing the bacteria is an expensive challenge. Some scientists - like Dr.
Mansour Samadpour with the University of Washington - have proposed getting
a genetic fingerprint of the bacteria and attempting to match it with bacteria from
animals, humans or birds. Others question this method, arguing that the data
resulting from Dr. Samadpour s methods has no statistical significance.
The "holy grail" of water quality testing may be tests which identify the presence
or absence of human enteric viruses in a water sample. But such testing
involves both young science and high prices.
With this in mind, the City sought and received a grant from the California State
Water Resources Control Board in the FY 1999 -2000 State Budget for $175,000 to
conduct a source identification study in the Newport area. Working with a
Study Advisory Team that includes staff from the California Regional Water
Quality Control Board (Santa Ana Region), local health officials at OC HCA, and
local water quality expert Dr. Jack Skinner, we developed a three - phased Scope
of Work for our Study.
The Phases are as follows
• Phase I - Preliminary Virus, Coliphage, and Bacteria Testing of Newport
BU Storm Drains and Tributaries. This phase tested nine sites for viruses,
f- specific coliphage, and the three indicator bacteria once a week for three
weeks, including:
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
The results from Phase I of the Study 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.
• Phase II -- Re- Verification of Positive and Negative Results from Areas
Identified within Phase I. This Phase re- examined and re- tested (with
greater frequency) sites at:
43A Street Beach (Channel Place Park)
— Santa Ana Delhi Channel (several locations)
— San Diego Creek (near Campus and near Michelson)
Newport Dunes North
This Phase may also include a study to attempt to determine the extent, if
any, of vessel waste discharges into the Harbor and of the contribution, if
any, of fecal coliform and viruses by swimmers (known to health experts
as "shedding ") in popular swimming locations in the Bay. The vessel
waste and swimmer contribution sections of Phase II will likely occur in
Summer 2001.
• Phase III — Rapid Testing of Hot - Spots. As initially proposed, Phase III
would look at instances where the OC HCA has identified a spike in
bacteria numbers and will immediately attempt to test the same waters for
the presence or absence of viruses. The Study Advisory Team may modify
Phase IIrs tasks as the results from Phase II become known.
Two contracts will initially draw from the $175,00 0 State Grant. Since the Grant
is administered through the Regional Board with the City serving as the local
project manager, the City administers both contracts. We identified the Grant
monies in the Fiscal Year 2000 -01 Capital Improvement Program (Item #250-
7251, Page 133).
Our contractor for the viral testing services within Phases I and II of the Study
(approximately $30,000 in Phase I and $50,000 in Phase II) is the Southern
California Coastal Water Research Project ( SCCWRP), with Dr. Rachel Noble of
SCCWRP acting as the lead scientist. The Study Team recommended and
selected SCCWRP because it is a local public agency and because it is one of only
a handful of entities nationwide who are capable of performing testing for
human enteric viruses in ambient waters.
Our contractors for the coliphage portion of the Study (approximately $12,000 for
both Phases I and II) are the Regents of the University of California. Dr. Sunny
Jiang of UCI is the lead for the coliphage portion of the Study.
In September 2000, your City Council approved the contracts associated with
Phase I. This Agenda Item authorizes the City Manager to approve the Phase II
contract in the amount of $50,000 with SCCWRP. As noted, the revenues
associated with the contract are budgeted via the State Grant in the CIP.
ATTACHMENTS: Attachment A - Contract with SCCWRP
PROFESSIONAL SERVICES AGREEMENT
THIS AGREEMENT, entered into this 13th day of December, 2000, by and
between CITY OF NEWPORT BEACH, a Municipal Corporation (hereinafter referred to
as "City "), and the Southern California Coastal Water Research Project or
SCCWRP (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 project management and
staff services upon the terms and conditions contained in this Agreement.
C. The principal member of Consultant is, for purpose of this Project, Dr.
Rachel Noble.
D. City has reviewed the previous experience and evaluated the expertise of
Consultant and desires to contract with Consultant under the terms of
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 1St day of September, 2000,
and shall terminate on the 31St day of December, 2000, 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 scheduled billing rates, attached as Exhibit "B" and incorporated
herein by reference. No rate 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 Fifty
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thousand dollars ($50,000). The City Manager shall have the authority to authorized
work beyond this amount, not to exceed an additional $5,000.
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
personnel required to perform the services required by this Agreement, and that it will
perform all services in a manner commensurate with the community 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.
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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.
6. 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 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 City's designated
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 Project.
7. PROJECT MANAGER
Consultant shall assign the Project to a Project Manager, who shall coordinate all
phases of the Project. This Project Manager shall be available to City at all reasonable
times during the Project term. Consultant has designated Dr. Rachel Noble to be its
Project Manager. Consultant shall not bill any personnel to the 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 hourly billing rate. Consultant
shall not remove or reassign any personnel designated in this Section or assign any
new or replacement person to the Project without the prior written consent of City. City's
approval shall not be unreasonably withheld with respect to removal or assignment of
non -key personnel.
Consultant, at the sole discretion of City, shall remove from the Project any of its
personnel assigned to the performance of services upon written request of City.
Consultant warrants that it will continuously furnish the necessary personnel to
complete the Project on a timely basis as contemplated by this Agreement.
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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, and the assessment of damages against Consultant for delay.
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 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 Project proceeds in a manner consistent with City goals and policies.
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
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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. This indemnity shall apply
even in the event of negligence of City, or its employees, or other contractors, excepting
only the sole negligence or willful misconduct of City, its officers or employees, and shall
include attorneys' fees and all other costs incurred in defending any such claim.
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.
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 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 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,
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.
The sale, assignment, transfer or other disposition of any of the issued and
outstanding capital stock of Consultant, or of the interest of any general partner or joint
venturer or syndicate member or cotenant if Consultant is a partnership or joint- venture
or syndicate or cotenancy, which shall result in changing the control of Consultant, shall
be construed as an assignment of this Agreement. Control means fifty percent (50 %) or
more of the voting power, or twenty-five percent (25 %) or more of the assets of the
corporation, partnership or joint-venture.
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 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, Deputy City Manager, shall be considered the Project Administrator and shall have
the authority act for City under this Agreement. The Deputy City Manager or 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. ERRORS AND OMISSIONS
In the event of errors or omissions that are due to the negligence or professional
inexperience of Consultant which result in expense to City greater than would have
resulted if there were not errors or omissions in the work accomplished by Consultant,
the additional expense shall be borne by Consultant. Nothing in this paragraph is
intended to limit City's rights under any other sections of this Agreement.
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21. CITY'S RIGHT TO EMPLOY OTHER CONSULTANTS
City reserves the right to employ other consultants in connection with the Project.
22. 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
hold harmless City for any and all claims for damages resulting from
Consultant's violation of this Section.
23. SUBCONSULTANT AND ASSIGNMENT
A. 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.
24. 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. 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:
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Attention: Dr. Rachel Noble
Southern California Coastal Water Research Project
7171 Fenwick Lane
Westminster, California 92683 -5218
714/372- 9203(phone)
714/894 -9699 (fax)
25. TERMINATION
In the event either part 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
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 shall have the option, at its sole discretion and without cause, of terminating
this Agreement by giving seven (7) days' prior written notice to Consultant 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.
26. COMPLIANCES
Consultant shall comply with all laws, state or federal and all ordinances, rules
and regulations enacted or issued by City.
27. 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.
28. 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.
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29. OPINION OF COST
Any opinion of the construction cost prepared by Consultant represents his
judgment as a design professional and is supplied for the general guidance of City.
Since Consultant has no control over the cost of labor and material, or over competitive
bidding or market conditions, Consultant does not guarantee the accuracy of such
opinions as compared to contractor bids or actual cost to City.
30. PATENT INDEMNITY
The Consultant shall indemnify City, its agents, officers, representatives and
employees against liability, including costs, for infringement of any United States' letters
patent, trademark, or copyright infringement, including costs, contained in Consultant's
drawings and specifications provided under this Agreement.
IN WITNESS WHEREOF, the Parties have caused this Agreement to be
executed on the day and year first written above.
APPROVED AS TO FORM:
By:
Robert Burnham
City Attorney
for the City of Newport Beach
ATTEST:
By:
LaVonne Harkless
City Clerk
CITY OF NEWPORT BEACH
A Municipal Corporation
M
Homer L. Bludau
City Manager
for the City of Newport Beach
CONSULTANT
Stephen B. Weisberg, Ph.D.
Executive Director
SCCWRP
i1i
EXHIBIT A
Phase Two Sampling Plan
for
Viral Detection of Tributaries and Storm Drains
Discharging into Newport Bay
The presence of enteroviruses will be determined by the method of reverse transcriptase-
polymerase chain reaction (RT -PCR) at the laboratory of Dr. Jed Fuhrman at USC. This
method will provide a presence /absence result, and can be used to provide semi - quantative
results if used with a series of sample dilutions. Quantitative RT -PCR will also be conducted on
the same samples when new instrumentation at USC has been properly calibrated
(approximately 3 months time). Samples of known human waste and animal waste will be
tested in the laboratory as positive and negative controls for use with quantitative RT PCR.
Sampling Sites. Samples will be collected directly from each tributary and storm drain at the
safest accessible location upstream of the point of discharge to the Bay or at the mouth of the
discharge. Of the nine sites originally sampled in Phase I, six sites have been chosen as either
priority sites for continued sampling or as problem areas by the Newport Bay Watershed
Committee based on Phase UI bacterial indicator, coliphage, and enterovirus data.
Sampling and Concentration Procedures. Sampling for Phase II will begin September 13th,
2000. Sites previously demonstrating negative results for enterovirus detection (Back Bay
Drain, 43rd St., San Diego Creek at Michelson, and San Diego Creek at Campus) will be
sampled a total of 8 more times, providing a total number of samples between Phase I
and Phase II that will provide statistically rigorous results (i.e. if all samples are negative, the
sample size will be large enough to ensure that statistical confidence in the result). Samples will
be collected from the Santa Ana Delhi sites at both Mesa and University a total of 4 more times.
These areas were identified as problem areas in Phase 1, and further sampling will reveal if
these problems are persistent. Samples will be collected as many times over the following
weeks as logistically possible with the aim of completing the sampling before a measurable and
heavy rain (0.5 "). If a small rainfall event occurs that is only a one or a few tens of an inch, the
sampling will be suspended for a few days.
Samples will be collected at the same time of day at each respective site to ensure collection of
samples through tidal changes. However, the day of the week that samples are taken will be
changed in order to capture samples that are not "day" dependent. For detection of
enteroviruses by RT -PCR, large volumes of seawater (20 -40L) will be retrieved from each of six
sample sites using an acid - rinsed (5% HCI acid), and triple sample- rinsed bucket. The samples
will be put on ice, and transported immediately to the laboratory at USC. Samples will be
dispensed into a 40 -liter stainless steel pressure vessel and pressure filtered (at < 18 kPa)
through a glass fiber filter (Whatman, nominal pore size 1.2 um). The filter will be frozen
immediately at —80 C. When possible, the filtrate will be retained in a carboy, and while on ice
will be concentrated to a final volume of 40 -150 ml with the use of a spiral cartridge filtration
system (Amicon, Inc., 30 kDa molecular weight cutoff, SY130). The filtrate will be further
concentrated with the use of Centriprep -30 concentration to a final volume of approximately 1
ml. Some filtrates are full of suspended materials, etc. that can damage the spiral cartridge. In
these cases, the recovery of viruses on the glass fiber filter is extremely, and the concentration
of the filtrate becomes unnecessary.
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Table 1:
Sampling
Sites
San Diego
Creek @
Campus 8
Santa Ana Delhi @
University 4
Santa Ana Delhi @
Mesa 4
Back Bay
Drain 8
43rd St.
8
San Diego
Creek @
Michelson 8
Reverse Transcriptase Polymerase Chain Reaction (RT -PCR)
Detection of enteroviruses by RT -PCR will use modified pan - enterovirus "universal" primers
originally designed by DeLeon at al., (1990), which were designated as EV -L and EV -R. The
upstream primer sequence is 5'- CCTCCGGCCCCTGAATG -3', and the downstream primer
sequence is 5'- ACCGGATGGCCAATCCAA -3'. This primer set can amplify at least 25
enteroviral types, and the relatively short length of the PCR amplicon ensures efficient
amplification. Perkin Elmer's RNA PCR Core kits will be used for the RT -PCR. Before the RT-
PCR step, the RNA on one - quarter of the Glass fiber filter will be extracted using a Qiagen
Rneasy viral RNA extraction kit. Afterwards, a 2 ul subsample of the extract will be added to 14
ul of RT Reaction mixture. Each sample will be heated at 99 ° C for 5 min and cooled at 4 ° C
before the addition of 1 ul of RT (50 U /ul) and 1 ul of Rnase inhibitor (20 U /ul). The RT step will
be carried out by the following steps: 25 ° C for 10 mins, 42 ° C for 30 mins, 99 ° C for 5 mins,
and 4 ° C for 10 mins. The finished RT mixture will be a total volume of 20 ul, and will be mixed
with the PCR reaction mixture (80 ul) as suggested by the manufacturer, for a total PCR
reaction volume of 100 ul. The target templates are amplified DNA fragments of 196 by after 40
cycles. Visualization of amplified DNA was by staining a 1.5 % agarose gel with ethidium
bromide and illumination with UV light. 100 by lane markers will be used for size comparison.
Sensitivity assays will be performed by spiking ocean samples with known amounts of poliovirus
and subsequent amplification with pan - enterovirus primers. This allowed us to determine the
sensitivity of each assay, which our preliminary results suggested varied depending on the
presence of potentially interfering substances in seawater.
Negative and positive controls will be performed for each RT -PCR run. For the negative
controls, 2 ul of deionized, RNase free H2O will be added to the PCR reactions rather than RNA
extract. The positive controls will be RNA extracts or concentrated seawater samples spiked
with 2 ul of Poliovirus Type 1 LsC at 105 to 10 "2 PFUM 00 ul of high titer stock. Positive controls
from the Perkin Elmer GeneAmp RNA PCR Core kit were run with each set of reactions to
ensure the activity of all components in the PCR Kit
Data Analyses and Deliverables
R. Noble will perform data analyses based on the results of the enterovirus, bacteriophage and
standard bacterial testing performed at USC, UCI and OCHCA laboratories, respectively. Upon
completion of the data analyses, a brief report will be written to outline the findings, present the
results, and to provide suggestions for future actions. This report will be presented to the
Newport Bay Study committee.
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EXHIBIT B
B u d g et/S u b co n s u l to n is
Budget
The following table summarizes the costs for the analyses of the enterovirus samples.
Enterovirus sample analysis = $ 1,000 per sample
Sample Analysis Cost/sample X number of samples
$1000X 40 = $40,000
Data Analysis and Report writing 10.000
Total $ 50,000
Subconsultants
Consultant will be retaining Ms. Alison Davis at their expense to assist Dr. Rachel Noble
on this project.
As noted in Professional Services Agreement, services billed shall not exceed $50,000.
All services shall be provided in accordance with Sampling Plan in Exhibit A.
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