ASTM E645-18. Standard Practice for Evaluation of Microbicides Used in Cooling Water Systems.

Test accredited by ENAC (Spanish National Accreditation Entity).

This standard describes a procedure to evaluate the effectiveness of microbicides (bactericides, fungicides and/or algaecides), which are used to control microbial growth in water cooling systems. For the evaluation according to this guideline, cooling water and/or sediments/biofilms obtained from reality are used. The addition of sediments/biofilms addresses the need to include the main sources of microorganisms in cooling water systems. However, even with this addition, laboratory results may not be fully predictive of microbicidal efficacy in the real use. This is because the conditions that can affect the effectiveness of microbicides in the real use are difficult to simulate in the laboratory. These conditions that affect the effectiveness of microbicides include the rate of purge, the addition of replenishment water, water hardness, hydrocarbon leaks, pH, amount of sediment present, dissolved solids, presence of microbes inside the biolayers (biofilms), and the presence of other sediments (salts, iron minerals, organic substances, etc.) on surfaces. The main difficulty of this method lies in the difficulty to list all the microbes present in the water due to its great diversity and different requirements for its cultivation, which implies using different culture media to achieve an adequate recovery.

The ASTM E645 is intended for the evaluation of microbiocides using synthetic or real cooling water. There are three possibilities for testing 1) evaluating the biocidal activity against microorganisms present in the actual cooling water, 2) to test the biocidal activity against microorganisms present in microbiological deposits (biofilms) from operating cooling systems, or 3) to assess the biocidal against microorganism strains known to contaminate cooling water systems, adding them to cooling water used only as a substrate (previously sterilized) or synthetic cooling water.

In the test, microbicides are evaluated against water or sediment microbes, under conditions that simulate a cooling water system. For this, the microbicides, in the concentration/s chosen by the manufacturer, are faced with cooling water or sediment suspension. The contact time must be chosen by the manufacturer, but the standard indicates that a time of 3 ± 1 hour and 24 ± 0.25 hours must be included. The contact temperature is recommended to be ± 5ºC the temperature in the drain, when the water/sediment sample was taken to perform the test. When test is carried out using synthetic cooling water and known microorganisms are added as inoculum, the client must choose the test temperature attempting to simulate actual conditions of use. According to the ASTM E645 standard, the volume of the microbiocide stock solution added should not exceed 1% of the total volume. After the chosen contact time, the number of microbes (aerobic bacteria, anaerobic bacteria, fungi or algae) is determined and compared with the values ​​at the beginning (basal) of the test not exposed to the product.

The manufacturer must decide the conditions in which he wishes the evaluation to be carried out (water or sediments), real or simulated cooling water, the concentration used of the product, the contact time/s, and the test temperature, the one measured in the refrigeration system from which the matrix was taken for the test (water or sediment) or other indicated by the client, if synthetic colling water is used.

Results are expressed according to the logarithmic reduction of each group of microorganisms (aerobic bacteria, anaerobic bacteria, fungi and algae) obtained for the concentration of biocide tested in relation to the initial count of the control sample that has not been exposed to the product, considering that a reduction of 1 log equals 90% death; 2 log equals 99%; 3 log is 99.9% and 4 log is 99.99%.

The minimum bactericidal concentration to consider the product effective will depend on the manufacturer's requirements or the requirements for its registration. In most cases 90% death (1 log reduction), is the minimum level considered acceptable to show effectiveness of a microbicide.

When performing the test with synthetic cooling water, adding known microorganism strains, the ASTM E645 Standard does not state the microorganisms that should be tested for each activity. In IVAMI, as an internal procedure we have stated that:

-   For bactericidal activity: A test with a pool of S. aureus and P.aeruginosa inoculum, and an independent test for Legionella pneumophila.

-   For fungicidal activity: A test with a pool of C.albicans and A.brasiliensis inoculum.

-   For algicidal activity: An independent test with Chlorella vulgaris.

Nevertheless, you can choose any microorganism (bacteria, fungi or algae) you are interested in since the Standard does not specific any mandatory microorganism.