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Blue Flag Beaches and Recreational
Water Testing for and Enterococci
E.coli using Enterolert & E. coli
Gil Dichter
World Wide Technical Support Manager,
Water
www.idexx.com/water
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OBJECTIVES
Blue Flag Beaches
– Background
– Criteria
WHO & Country Criteria
Define Enterococci & E.coli
Method for Testing
– Enterolert & Colilert-18 for Recreational Water
Theory
How to test
Read results
US EPA approvals and requirements
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Blue Flag Beach Criteria
Blue Flag Program is for beaches and marinas
– Started in France in 1985
– In Europe since 1987
– Areas outside Europe since 2001 (1st was South Africa)
Run by the Foundation of Environmental Education (FEE)
– It is an international organization
– Non-profit
– Non-governmental
– For both fresh and marine waters
Covers beaches for cleanliness as well as water quality.
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Blue Flag Beach Criteria
Criteria for program are either imperatives or guidelines
– Imperative: Must comply in order to be awarded the Blue Flag
accreditation.
– Guideline: Preferable that they are complied with but not
mandatory.
During the Blue Flag Season, the flag must fly at the beach
– It is a symbol of the program being run at the beach and
– Also an indication of compliance
If beach is not in compliance with the criteria, the flag may
be removed permanently or temporarily withdrawn.
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Blue Flag Beach Criteria
There are several degrees of non-compliance
– Minor non-compliance:
A problem with only 1 minor consequence to health & safety of
the beach user or the environment.
If immediately corrected, flag is not withdrawn.
Only noted or registered in the controlled visit report.
If it cannot be corrected immediately, given 10 days to comply
fully and flag is withdrawn until problem is corrected and noted
on the homepage.
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Blue Flag Beach Criteria
– Minor Multiple non-compliance:
non-compliance of 2 to 3 criteria if it is a minor consequence to
health & safety of beach user or to the environment.
Given 10 days to comply fully with all criteria.
Flag is withdrawn until all problems are corrected and homepage
is updated accordingly.
– Major non-compliance:
Beach does not comply with one or several criteria.
Can have consequence to the health & safety of the beach user
or environment.
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Blue Flag Beach Criteria
Flag is withdrawn immediately and for the rest of the season.
Site is registered as “withdrawn” on the Blue Flag homepage.
Beach information clearly indicates that Blue flag status has
been withdrawn and posted manually or electronically.
Blue Flag accreditation is the authority charged with
responsibility of the beach.
– It can be a
local municipality,
private hotel,
national park or private beach operator.
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Blue Flag Beach Criteria
Blue Flag beaches are subject to announced and/or
unannounced control visits by FEE International.
First time applicant:
– Must provide evidence in the application that the beach complied
with the imperative quality criterion for Blue Flag in the season
(year) prior to application for full status.
– This will only be considered from beaches with a minimum of 20
samples taken in the previous season(s).
Bathing water quality information must be displayed on the
Blue Flag information board.
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Blue Flag Beach Criteria
– Use a table or figures with easy identifiable symbols corresponding
to the results. Okay to swim = ☻
Results be listed as soon after the testing is completed and
updated regularly.
Water Quality:
– Require beaches to achieve excellent bathing water quality.
– Quality standards are based on the most appropriate international &
national standards & legislation.
– At least one sampling site that is located where the highest number
of bathers are.
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Blue Flag Beach Criteria
– If there are potential sources of pollution (storm water outlets, rivers
or other inlets), additional samples must be taken to provide data
that these areas do not affect bathing water quality.
– Samples taken must be at a depth of 30 cm below the surface.
– No more than 30 days between samples during the season.
– A minimum of 5 samples must be taken evenly spread out for the
season.
– First sample must be taken within 30 days before the official starting
date of the Blue Flag season.
– If results indicate a possible increase in pollution levels, it is
recommended to temporarily increase the number of samples to
track any possible pollution incident.
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Blue Flag Beach Criteria
– In the event of short term pollution, 1 additional sample is taken to
confirm that the incident has ended.
– An independent, officially authorized and trained person must collect
the samples.
– An independent laboratory must carry out the analysis of the water
samples.
– A sampling calendar must be established prior to the start of the
bathing season.
– No industrial, waste water or sewage discharges should affect the
beach area.
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Blue Flag Beach Criteria
– Comply with the requirements for the microbiological parameter for
E.coli and enterococci.
Accepted percentile:
Requires 95th percentile compliance of the limit values.
Parameter Coastal &
Transitional
Waters Limit
Values
Inland Waters
Limit Values
E. coli 250/100 mL 500/100 mL
Enterococci 100/100 mL 200/100 mL
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Calculation of the 95th Percentile
Take the log10 value of all bacterial enumerations to be
evaluated. If values = 0 or <1, replace with a value of 1
Calculate the mean log10 values (µ)
Calculate the standard deviationααα (σ) of the log 10 values
The upper 95 percentile is derived from the equation
Antilog (µ +1.65 σ)
The resulting value must be within the limit value
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Guidelines Values for Microbial Quality of
Recreational Waters
95th percentile value of
enterococci/100 mL
Estimated risk per exposure
≤ 40 < 1 % GI illness
The upper 95th percentile value relates to an
average probability of <1 case of gastroenteritis in
every 100 exposures
43-200 1-5% GI illness risk
The upper 95th percentile value relates to an
average probability of <1 case of gastroenteritis in
every 20 exposures
201-500 5-10 % GI illness
The range of the 95th percentiles represents a
probability of 1 in10 to 1 in 20 of gastroenteritis
for a single exposure
> 500 < 10 % GI illness
There is a > 10 % chance of gastroenteritis per
single exposure
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Examples of Guidelines/ Standards (per 100 mL) for
Bathing Water
Country/Region/
Organization Water Quality Guidelines/Standards
for Bathing waters: All values/100
mL
Notes
WHO MW: Enterococci </= 40 (GM)
E.coli not established
Based on 95 percentile
Mexico Enterococci 200/100 mL (2010)
United States MW: Enterococci GM <35, single
sample - 104
FW: E. coli GM - 126, single sample
235 or enterococci GM - 33, single
sample - 61
GM based on >5
samples/month. Higher
values in place for
moderate, lightly used or
infrequently used
waters
European
Guidelines
Based on excellent water quality
MW: enterococci -100, E.coli 250
FW: enterococci - 200, E.coli 500
Based on 95 percentile
Higher values for good
& sufficient quality
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Enterococci
Enterococci are the key subset of fecal streptococcus
Fecal Streptococcus
1) Enterococcus spp.:
E. faecalis, E. faecium, E. gallinarum, E. avium, E. durans, E.
casseliflavus
2) Non-enterococci streptococcus
S. bovis, S. equinus
Defined as Gram+, catalase -, grows in 6.5% saline, 40% bile
salts, and at 10°C and 45°C.
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Escherichia coli
Named after the Austrian Scientist who found this bacteria-
Dr. Escherich
A genus of Gram negative bacteria of the family
Enterobacteriaceae
A type of thermotolerant coliform bacteria commonly found in
the intestines of warm blooded animals including humans
Does not occur naturally in soil and vegetation
May occur in soil and water as a result of fecal contamination
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Colilert, Colilert-18 & Enterolert
1. Standard Methods for Water & Waste Water,
APHA, AWWA, WEF
Section 9223 B Colilert & Colilert-18
Section 9230 D Fluorgenic Substrate
Enterococcus Test (Enterolert)
2. American Society of Testing Materials (ASTM)
Method Number D6503 Standard Method for
Enterococci in Water Using Enterolert
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Introduction con’t
•Microbial contaminants: bacteria such as enterococci
and E.coli are used as indicator bacteria for
pathogenic micro-organisms (bacteria, protozoa,
viruses)
•Illness can result from swimming in contaminated
water such as:
–Gastrointestinal
–Ear infections
–Sore throat
–Hepatitis
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Requirements for an Indicator
Organism Present when pathogens are present
Absent in uncontaminated waters
Present in higher numbers than pathogens in
contaminated water
Better survival in water than pathogens
Easy and Safe to analyze
Rapid results
Readily avaialble
Inexpensive
Accurate
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Defined Substrate Technology: Enterolert,
Colilert & Colilert-18
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Enterolert Positive Reaction
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ONPG Positive Reaction
Colilert &Colilert-18
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MUG Positive Reaction
Colilert & Colilert-18
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Use of Enterolert & Colilert, Colilert-18
with Quanti-Tray for Recreational
Waters
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Separation of Snap Packs
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Dispensing of Reagent into Sample
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Quanti-Tray Demonstration
Pour mixture into a
Quanti-Tray
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Quanti-Tray Demonstration
cont.
Seal Quanti-Trays for Incubation
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Incubate Samples at 41 ± 0.5ºC for Enterolert
for 24-28 Hours &35 ± 0.5ºC for 18-22 Hours
Colilert-18 & 24-28 hours for Colilert
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Enterolert™ Demonstration
Count fluorescent wells
and refer to MPN table
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E.coli- Blue Fluorescence- Quanti-Tray
under a 365nm UV Light
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USEPA Approval for Recreational
Waters
Enterolert for Marine Waters
Colilert & Colilert 18 for Fresh Waters
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Enterolert Studies
Budnick, G.E. et al. 1996. Evaluation of Enterolert for Enumeration of Enterococci in Recreational Waters. AEM. 62:3881-3884 – 138 marine water samples compared to the 48
hour m-E/EIA -MF method at 41°C
– Strong positive correlation of 0.97; no statistical difference between methods using the Student’s t Test, p(≥0.05) = 0.63
– False positive rate of 5.1% vs. 10% for MF
– False negative rate of 0.4% vs. 11.7% for MF
– Time studies indicate less time for set up, reading and recording results permitting more efficient monitoring of recreational water beaches
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Enterolert Studies con’t
Abbott, S. et al. 1998. Evaluation of Enterolert for
the Enumeration of Enterococci in the Marine
Environment. New Zealand Journal of Marine and
Freshwater Research. 32: 505-513
– Compared 343 MW samples over a 12 month period
– Correlation (r) = 0.927, Y= 10.4 + 0.90X
– No significant difference between the 2 methods by
paired t test, p(≥0.05) = 0.39
– False + rate of 2.4% and False – rate of 0.3%
– Enterolert required less time for preparation, sample
set-up incubation and reading of tests
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Use of Idexx Colilert Quanti-Tray 2000 for the
Enumeration and Detection of E.coli in Recreational
Waters – Kinzelman et al, Lake and Reservoir
management 21(1):73-77, 2005
234 samples from 5 different beaches were compared
between m-TEC and Colilert/Quanti-Tray 2000
Correlation coefficient of 0.90 indicated acceptable
agreement between methods
Colilert-18/Quanti-Tray is an acceptable alternative to the
traditional MF technique for monitoring E.coli levels at
the beaches
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Mucho Gracias
Questions ?