Environmental Project No. 1155 – Coliform bacteria and E. coli in drinking water. Comparison of EU reference method with alternative methods

Coliform bacteria and E. coli in drinking water. Comparison of EU reference method with alternative methods

Background

Coliform bacteria and E. coli are two important parameters for control of the quality of drinking water. The Council Directive on the Quality of Water intended for Human consumption (98/83/EF) specifies in Annex III, part 1 the method for analysis of these parameters: EN ISO 9308-1:2000. This method is based upon the use of Lactose TTC agar with sodium heptadecylsulphate (Tergitol 7), also called the TTC-Tergitol method.

The Directive states that Member States may use alternative methods, providing the provisions of Article 7, part 5 are met. The Article states that:

5a). Member States shall comply with the specifications for the analyses of parameters set out in Annex III of the Directive.

5b). Methods other than those specified in Annex III, Part 1, may be used providing it can be demonstrated that the results obtained are at least as reliable as those produced by the methods specified. Member States which have recourse to alternative methods shall provide the Commission with all relevant information concerning such methods and their equivalence.

The large European study with the Council Directive reference method for coliform bacteria and E. coli in drinking water has shown that the EU reference method fails to detect a significant proportion of coliforms and E. coli in drinking water (Niemela, Lee & Fricker, 2003). Especially water with high heterotrophic counts may cause problems with competing flora in drinking water. These types of water, e.g. water from wells or contaminated mainwater, are precisely the types of water in which coliform bacteria and E. coli are looked for and often should be found. It should be noted that in the EU study E. coli were analysed by direct incubation at 44°C which is not in compliance with the EN ISO standard. Nevertheless this does not change the conclusions regarding coliform bacteria at 37°C. It might affect the results for E. coli due to on one hand lower findings of competing flora leading to higher counts of E. coli but on the other hand stressed E. coli due to the combination of maximum temperature at the same time as growing on a selective agar (“hurdle-effect”).

EN ISO 9308-1:2000 states specifically in the scope that: “The Standard Test has a low selectivity, allowing the detection of injured bacteria. Due to the low selectivity, background growth can interfere with the reliable enumeration of coliform bacteria and E. coli, for example in some drinking waters, like shallow well waters, that have not been disinfected and yield a high background growth. This part of EN ISO 9308 is therefore especially suitable for disinfected water and other drinking waters of low bacterial numbers”. Later in the scope it is concluded that the method is “… applicable to other kinds of water provided that suspended matter or background flora does not interfere with filtration, culture and counting”.

Based on this information stated in the Standard and on the studies done on comparison of Colilert™ with the EU reference method for the analysis of drinking water for coliform bacteria including E. coli (Niemela, Lee & Fricker, 2003) Denmark decided not to conduct a fully equivalency study but instead verify the existing knowledge by testing Danish drinking water of different microbiological quality in a limited equivalency study. It was also decided that it should be aimed to validate and approve a method that could be used for water with as well as without background flora. Most of the Danish drinking water from public water supplies has a very good microbiological quality with a low content of heterotrophic microorganisms. In Denmark however there are many small private water supplies e.g wells with higher levels of background flora than normally found in the public water supplies and this background flora may affect the detection of coliform bacteria. Furthermore will the good quality water normally contain very high levels of background flora when it is contaminated with coliform bacteria including E. coli and the method used shall therefore be valid for this purpose as well.

In the end of 2003 an equivalency study was conducted in one laboratory with the specific purpose to:

document that the already known problems with using EN ISO 9308-1:2000 for drinking water with high heterotrophic counts also were valid for Danish drinking water
compare other well-known methods to EN ISO 9308-1:2000 (without deviations from the Standard)
support a Danish equivalency study from 2000/2001 (chapter 2)
demonstrate the effect of Colilert as a quantitative method as Colilert is approved for qualitative testing (P/A) of Danish drinking water in case of testing contaminated water to find the source of contamination (annex A).

Colilert is already used as a supplementary test in several microbiological laboratories in Denmark for quantitative testing and seems to be a robust and well-known method that needs validation.

It was decided to use only one microbiological testlaboratory (Danish Environmental Protection Agency’s (EPA) Reference Laboratory) for this equivalency study as it is a requirement for other laboratories to demonstrate verification of the method before using it routinely. This meant that there was no need to use more laboratories to verify potential problems with the methods as problems found by the reference laboratory when analysing Danish drinking water will be enough to discriminate the method.

The equivalency studies were performed on the EN ISO 9308-1:2000 (designated “EU reference method” in this report) against five other internationally recognized methods: Lauryl Sulphate Agar (LSA), Membrane Lactose Glucuronide Agar (MLGA), Chromogenic agar, Chromocult and Colilert. Results from the equivalency studies are given in chapter 3 and 4.