appendix E10

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Table E.10

Summary of the analysis requirements by determinand for microbiological parameters in EU directives and international agreements for surface waters

Determinand Analytical methods and comments Directive/International Agreement
1. DIRECTIVES
Total coliforms1 Multiple tube (MPN), Confirmation of positive tubes Bathing Water (76/160/EEC)
Total coliforms2 MPN (multiple tube) or membrane filtration, with an appropriate medium, such as Tergitollactose agar, Endo agar or 0.4% Teepol broth. Confirmation. Incubation at 37°C for total coliforms, 44°C for faecal coliforms. Surface Water (75/440/EEC) and Sampling and Analysis (79/869/EEC)

Drinking Water (80/778/EEC)

Faecal coliforms1 Membrane filtration (Tergitol lactose agar, Endo agar, 0.4% Teepol broth) Confirmation.

Media are given as examples. An 'appropriate medium' should be used. Teepol broth is obsolete, Endo agar inhibitory to organisms exposed to seawater and sunlight. All are recommended in ISO 9308-1 (under revision).

Bathing Water (76/160/EEC)
Faecal coliforms MPN (multiple tube) or membrane filtration, with an appropriate medium, such as Tergitollactose agar, Endo agar or 0.4% Teepol broth. Confirmation. Incubation at 37°C for total coliforms, 44°C for faecal coliforms. Surface Water (75/440/EEC) and Sampling and Analysis (79/869/EEC)

Drinking Water (80/778/EEC)

Faecal coliforms3 Multiple tube (at least three tubes in three successive dilutions) Confirmation. Incubation at 44°C Shellfish Waters (79/923/EEC)
Escherichia coli 4 Incubation at 44°C. Multiple tube MPN or membrane filtration, using an appropriate medium (examples unchanged from Directive (76/160/EEC.) Confirmation Proposed Bathing Water COM(94) 36
Faecal streptococci5 Membrane filtration (or direct plating) or multiple-tube (MPN) method, using appropriate medium, such as sodium azide Surface Water (75/440/EEC) and Sampling and Analysis (79/869/EEC)
Faecal streptococci6 MPN or membrane filtration - Litsky method (Litsky, Mallman and Fifield (1953) - now obsolete, poor efficiency) Bathing Water (76/160/EEC)
Faecal streptococci4 Litsky method, incubation at 37°C; MPN or membrane filtration. Unchanged from 76/160/EEC. Proposed Bathing Water COM(94) 36
Faecal streptococci7 Sodium azide method (Litsky), MPN Drinking Water (80/778/EEC)
Salmonella Bare details Surface Water (75/440/EEC) and Sampling and Analysis (79/869/EEC)8

Bathing Water (76/160/EEC)9

Drinking Water (80/778/EEC)10

Salmonella Not specified Shellfish hygiene (91/492/EEC)
Enteroviruses Bare details. Bathing Water (76/160/EEC)11

Proposed Bathing Water COM(94) 364

Drinking Water (80/778/EEC)12

Table E.10 continued

Determinand Analytical methods and comments Directive/International Agreement
Escherichia coli 13 Test of mollusc flesh and intervalvular liquid using multiple tube MPN (at least five tubes in three successive dilutions) or any other test of equivalent accuracy. Shellfish Hygiene (91/492/EEC)
Bacteriophages14 No details Proposed Bathing Water COM(94) 36
Faecal bacteriophages15 Guélin's process Drinking Water (80/778/EEC)
Sulphite - reducing Clostridia16 Spore count after heating sample to 80°C. Colony count in glucose, sulphite iron medium, membrane filtration or differential reinforced clostridial medium (DRCM), with confirmation. Drinking Water (80/778/EEC)
Total bacteria17 Nutritive agar; incubation at 37° for 48 hours and at 22°C for 72 hours Drinking Water (80/778/EEC)
Pathogenic staphylococci18 Membrane filtration; specific medium, e.g. Chapman's agar, test for pathogenic characters. Drinking Water (80/778/EEC)
2. INTERNATIONAL AGREEMENTS
Total coliforms Not specified Drava Water economy 1954

Water economy 1956

Frontier Waters -Czech Republic 1970

Frontier Waters -Slovak Republic 1970

Bucharest Danube 1985

Regensburg Danube 1987

Co-operation with Bulgaria 1991

Total coliforms Endo-agar 37oC, 24h and 48h count (red colonies only) Elbe 1990
Faecal coliforms Not specified Water economy 1956

Frontier Waters -Czech Republic 1970

Frontier Waters -Slovak Republic 1970

Bucharest Danube 1985

Regensburg Danube 1987

Co-operation with Bulgaria 1991

Faecal coliforms Endo-agar 43oC, 24h count (red colonies only) Elbe 1990
Escherichia coli Not specified Drava Water economy 1954
Enterococcus Not specified Drava Water economy 1954
Faecal streptococci Not specified Water economy 1956

Frontier Waters -Slovak Republic 1970

Regensburg Danube 1987

Salmonella Not specified Water economy 1956

Frontier Waters -Czech Republic 1970

Frontier Waters -Slovak Republic 1970

Regensburg Danube 1987

Thermophilic bacteria Not specified Drava Water economy 1954
Pathogens Not specified Co-operation with Bulgaria 1991
Micro-organisms - biomass Acridine Orange direct count (AODC)

Shaking and addition of formalin and refrigerate

Baltic Sea 1974/92

Table E.10 continued

Determinand Analytical methods and comments Directive/International Agreement
Micro-organisms - number of colony Pour plate method

Shaking, dilution with sterile water if necessary 30-300 and inoculation into three sterile Petri dishes and incubation

Baltic Sea 1974/92
Micro-organisms - production Tritiated Thymidine Incorporation

Incubation with TT, filtration, solubilisation with ethyl acetate

Baltic Sea 1974/92
Micro-organisms - total number Acridine Orange direct count (AODC)

Shaking and addition of formalin and refrigerate

Baltic Sea 1974/92
Bacterial Plate count Not specified Bucharest Danube 1985

Water economy 1956

Bacterial Plate count 22oC Frontier waters -Czech Republic 1970

Frontier waters -Slovak Republic 1970

Bacterial Plate count 37oC Frontier waters -Czech Republic 1970

Frontier waters -Slovak Republic 1970

Bacterioplankton - biomass Not specified Protection Lake Constance 1960
Total bacteria 22oC Water economy 1956
Total bacteria Not specified Water economy 1956

Notes:

1 ISO 9308-1 permits all three methods to be used. UK now uses membrane filtration with 0.1% sodium lauryl sulphate broth; incubation at 30°C for 14 hours for faecal coliforms. Faecal coliform isolates are confirmed, giving a count of Escherichia coli. This procedure gives the best recovery (highest counts) of various media tested and is standardised in NRA microbiological methods manual (National Rivers Authority (1992)).

2 Methods are given in ISO 9308-2, which is being revised. Teepol broth is obsolete, Endo agar inhibitory to damaged coliforms. UK uses long-established procedures in The Microbiology of Water 1994 - Part 1 - Drinking Water, HMSO, London. UK always confirms for presence of Escherichia coli.

3 Examination of the Shellfish flesh and intervalvular liquid. Guideline value of 300 per 10 ome must be observed in shellfish harvested for direct human consumption in the waters in which they are reared. This requirement is now superseded by the microbiological requirements of the Shellfish Hygiene Directive (91/492/EEC)

4 Examples of analytical methods unchanged; the same comments apply.

5 Standard methods of ISO 7899/1 and /2, now under revision. Favoured medium is Slanetz and Bartley's (1957) m-Enterococcus agar with membrane filtration. UK procedure given in The Microbiology of Water 1994 - Part 1 - Drinking Water. HMSO, London.

6 The most used medium is Slanetz and Bartley's (1957) m-Enterococcus agar with membrane filtration. Incubation either at 37°C for 48 hours or at 37°C for 48 hours or at 37°C for 4 hours and 44°C for 44 hours. Latter procedure is very specific for enterococci and is used by UK. Former procedure is less specific for faecal streptococci and gives higher counts. Neither method, nor Litsky method is given in ISO 7899/2, which is being revised.

7 Litsky, Mallman and Fifield (1953) method is of low efficiency and obsolete. Generally replaced by membrane filtration, using Slanetz and Bartley's (1957) m-Enterococcus agar. See Note 2 for Bathing Water Directive.

8 A difficult test. UK guideline document. (Note 2) gives procedures. Draft International Standard, ISO/DIS 6340

9 Salmonella method used in UK is specified in NRA's manual. A difficult test. Salmonellae are ubiquitous in surface water and can originate from birds, waterfowl and animals, hence not specific indicator of faecal pollution. To be examined if bathing water has deteriorated or if it is suspected that they may be present

10 Not an analysis for routine use, but when waterborne Salmonellosis is suspected. UK guideline (see note 1) has procedures. Draft International Standard ISO/DIS 6340.

11 Enteroviruses are detected with difficulty, using an expensive and poorly efficient method. They are not the viruses thought to be responsible for gastro-enteritis in bathers, hence are yet another indicator of faecal contamination. To be examined if bathing water has deteriorated or if it is suspected that they may be present

12 Not a routine test.

13 The UK shellfish industry has long used a draft standard procedure (West and Coleman 1986); a multiple tube method with minerals modified glutamate medium, with confirmation of E.col}. Impedimetric methods are under development as a possible European alternative.

14 Awaits a decision on parameter level, to be inserted at a later date when information is available. Parameter could be either somatic coliphages or more likely 'male-specific' coliphages (F+ RNA coliphages), for which a draft international standard now exists ISO/DIS 10705-1. It is intended to replace the Enterovirus parameter.

15 Guélin's method obsolete. Not a routine test. Standard for F+ RNA coliphages in water now being developed as ISO/DIS 10705-1; methods for somatic coliphages in draft stages ISO/CD 10705-2 and 3. A faecal indicator parameter, not a pathogen.

16 ISO 6461-1 and 6461-2 give standard methods for multiple tube and membrane filtration methods. Suggested by WHO as a test for water treatment efficiency, since chlorine resistant (Guidelines for Drinking-Water Quality - Vol. 1 Recommendations, WHO, Geneva).

17 ISO 6222 gives procedures, but is being revised within CEN/TC230 and ISO/TC 147. UK uses incubation at 37° for 24 hours.

18 Not a routine test. Staphylococcal infections are not waterborne. Not an appropriate parameter.

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