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Coliform bacteria and E. coli in drinking water. Comparison of EU reference method with alternative methods 1 International equivalency studies and approved methods
Many countries have during the latest years made a lot of studies of the equivalency between the EU reference method for drinking water (EN ISO 9308-1:2000) and other internationally recognized methods for coliform bacteria and E. coli. Due to this fact The Danish Environmental Protection Agency (EPA) decided to use these results as basic knowledge for the national Danish studies. This chapter gives an overview over international equivalency studies and approved methods. This overview does not aim to give detailed information on the studies but to highlight the conclusions used for the Danish work. 1.1 EU equivalency studyNiemela, Lee & Fricker published in 2003 a study which aimed to demonstrate the use of ISO CD 17994 2001, the Equivalency standard stating criteria for comparing two methods. They demonstrated it specifically for the comparison of Colilert and the EU reference method for detection of coliform bacteria and E. coli. They compared results from 20 laboratories in 13 European countries (including Denmark) and concluded that the Colilert detected significantly more coliform bacteria as well as E. coli than the EU reference method did. This means that the EU reference method fails to detect a significant proportion of coliform bacteria including E. coli. Furthermore Niemela, Lee & Fricker (2003) concluded that confirmation of positive wells in Colilert was not necessary. As mentioned in “Background” the EU Study by Niemela, Lee & Fricker (2003) deviated from the EU Standard by incubation of plates for E. coli directly at 44°C, which may affect part of the conclusion. The main-problem with growth of background flora will nevertheless be of increasing influence at lower temperature. As possible explanations for the better recovery of coliform bacteria with Colilert than with the EU reference method Niemela, Lee & Fricker (2003) pointed out that:
Finally they concluded that the findings of Colilert as superior to the EU reference method is limited to Colilert as a defined substrate with limited amounts of nutrients and not to all methods using b-D-galactosidase and b-D-glucoronidase as detection principle. 1.2 Equivalency studies and accepted methods in other countriesBased on experience from other European countries the status for methods either accepted or approved as alternative methods to the EU reference method in some other countries is outlined below. 1.2.1 The NetherlandsAccording to RIVM and KIWA (2001): “Comparison between NEN-EN-ISO 9308-1 and an alternative method for the enumeration of coliform bacteria and Escherichia coli” it has been demonstrated that LSA performs equally, or sometimes even better (the recovery is the same or higher) than TTC-Tergitol for the enumeration of coliform bacteria and E. coli. The study demonstrated thus that detection and enumeration of coliform bacteria and E. coli with LSA (and confirmation in accordance with the EU reference method) is at least as reliable as with the EU reference method. The data have been submitted to the Commission. 1.2.2 UKThe UK has compared Membrane Lauryl Sulphate broth (MLSB) to TTC-Tergitol. UK has undertaken sufficient equivalency testing for approving MLSB as an alternative method for coliform bacteria and E. coli and the data have been submitted to the Commision. 1.2.3 GermanyIn Germany the Federal Environmental Agency’s drinking water Commission has approved Colilert-18/Quanti-Tray as an alternative method to the EU reference method without having undertaken a specific equivalency study. 1.2.4 Colilert International ApprovalsColilert is approved or accepted in 24 countries for official drinking water analyses (amongst these 24 countries four EU Member States: Germany, Hungary, Czech and Italy and two non-EU-Member States: Iceland and Norway have approved the method according to the new drinking water directive, whilst UK and Ireland have approved the method according to the old directive). 1.3 Methods for the Danish equivalency studyAs mentioned above the Danish equivalency study intended to verify results from other European countries when used for Danish drinking water. The methods were chosen on basis of:
MLSB was not included in the Danish study. The technique used for MLSB with soaking a pad in broth is well-known in UK laboratories, but not routine in Danish laboratories. The technique is not difficult but deviating from the normal flow in the laboratories. Therefore it was assessed to be time consuming in the laboratories because of changing between different techniques for membrane filtration in the daily work.
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