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

Summary and conclusions

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 of 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.

Based on information stated in the Standard (EN ISO 9308-1:2000) about interference of background growth and on 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 is of very good microbiological quality with low heterotrophic counts. In Denmark however there are quite many private water supplies, e.g. wells with higher levels of background flora than normally found in 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 too.

The Danish equivalency study was based on Niemela, Lee & Frickers publication (2003) and one earlier Danish study from 2000/2001. Two limited practical equivalency studies were undertaken on spiked respectively natural water samples 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
demonstrate the effect of Colilert as a quantitative method as this method is approved for qualitative testing (P/A) of Danish drinking water (annex A).

The equivalency studies were performed on the EN ISO 9308-1:2000 (designated “EU reference method” in this report) and included in total five other internationally recognized methods: Lauryl Sulphate Agar (LSA), Membrane Lactose Glucuronide Agar (MLGA), Chromogenic agar, Chromocult, Colilert and the Danish national reference method until now: MPN in MacConkey broth (DS 2255:2001).

The final conclusion on the data from the Danish equivalency studies with spiked as well as natural drinking water samples are summarized as follows:

Colilert was shown to be equivalent to the EU reference method for the detection of coliform bacteria and E. coli in spiked samples with a low background flora.

Furthermore the same study with spiked samples confirmed well-known problems by using the reference method for detection of coliform bacteria and E. coli in waters with high heterotrophic counts due to overgrowth of the membrane filters. Colilert showed good recoveries in the same samples.

The study demonstrated that the problem with overgrown filters was also seen for three other membrane filtration methods: LSA37, Chromogenic agar and Chromocult.

MLGA showed relatively good recoveries in spiked samples, but the method was excluded from the further studies due to technical reasons.

The Danish reference method until now (DS 2255:2001) was included in testing of natural drinking water samples where it was found not to be able to detect coliform bacteria in any of 15 samples where one or more of three other methods (EU Reference method, Colilert and Chromogenic agar) detected coliform bacteria.

The Danish EPA has therefore decided to implement the EU Reference method EN ISO 9308-1:2000 with Colilert as an alternative quantitative method for the examination of coliform bacteria and E. coli in Danish drinking water.