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 1
OBJECTIVES 2 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
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. 3
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of compliance If beach is not in compliance with the criteria, the flag may be removed permanently or temporarily withdrawn. 4
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 If immediately corrected, flag is not withdrawn.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 If it cannot be corrected immediately, given 10 days to comply fully and flag is withdrawn until problem is corrected and noted on the homepage. 5
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 Flag is withdrawn until all problems are corrected and homepage is updated accordingly.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. 6
Blue Flag Beach Criteria Flag is withdrawn immediately and for the rest of the season. Site is registered as withdrawn on the Blue Flag homepage. Beach information clearly indicates that Blue flag status has been withdrawn and posted manually or electronically.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. 7
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. 8
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. 9
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. 10
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. 11
Blue Flag Beach Criteria Comply with the requirements for the microbiological parameter for E.coli and enterococci. 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 Accepted percentile: Requires 95 th percentile compliance of the limit values. 12
Calculation of the 95 th 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 13
Guidelines Values for Microbial Quality of Recreational Waters 95 th percentile value of enterococci/100 ml 40 Estimated risk per exposure < 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 14
Examples of Guidelines/ Standards (per 100 ml) for Bathing Water Country/Region/ Organization WHO Water Quality Guidelines/Standards for Bathing waters: All values/100 ml MW: Enterococci </= 40 (GM) E.coli not established Mexico Enterococci 200/100 ml (2010) United States European Guidelines MW: Enterococci GM <35, single sample - 104 FW: E. coli GM - 126, single sample 235 or enterococci GM - 33, single sample - 61 Based on excellent water quality MW: enterococci -100, E.coli 250 FW: enterococci - 200, E.coli 500 Notes Based on 95 percentile GM based on >5 samples/month. Higher values in place for moderate, lightly used or infrequently used waters Based on 95 percentile Higher values for good & sufficient quality 15
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. 16
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 17
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 18
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 19
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 20
21 Defined Substrate Technology: Enterolert, Colilert & Colilert-18
22 Enterolert Positive Reaction
ONPG Positive Reaction Colilert &Colilert-18 23
MUG Positive Reaction Colilert & Colilert-18 24
25 Use of Enterolert & Colilert, Colilert-18 with Quanti-Tray for Recreational Waters
26 Separation of Snap Packs
27 Dispensing of Reagent into Sample
Quanti-Tray Demonstration Pour mixture into a Quanti-Tray 28
Quanti-Tray Demonstration cont. Seal Quanti-Trays for Incubation 29
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 30
Enterolert Demonstration Count fluorescent wells and refer to MPN table 31
32 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 35
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 36
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 37
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 38
Mucho Gracias Questions? 39