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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be removed permanently or temporarily withdrawn.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If immediately corrected, flag is not withdrawn.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fully and flag is withdrawn until problem is corrected and noted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Flag is withdrawn until all problems are corrected and homepage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 ?