Biocide efficacy testing

These tests are set up with the test fuel over a simulated water bottom. The ratio of fuel to the water bottom, mentioned in the literature, ranges anywhere from 1 1 to 1,000 1. The industry standard for the simulated water bottom is Bushnell Haas Mineral Salts Enrichment Medium (Bushnell and Haas, 1941). 

The Department of the Army has a Performance Specification for Stabilizer Additive for Diesel Fuel, US MIL Specs 53021 A, which describes testing for the performance of fuel biocides. The fuel/water ratio used in this test is currently 1/100 but 1/1,000 is also recommended. This test consists of a single inoculation with hydrocarbonutilizing microorganisms. Metal coupons are added to the water bottom to check the ability of the microorganisms to cause corrosion in the system. 

ASTM E1259-01 Standard Test Method for Evaluation of Antimicrobials in Liquid Fuels Boiling Below 390°C is a multiple challenge test method. The fuel to water ratio decreases over time from 1/400 to 1/200 to a final ratio of 1/100 after eight weeks of the evaluation. 

SABS 1434-1987, the South Africa Standard Specification for Biocides , was reaffirmed in 2000. This method covers efficacy testing of three types of organic biocides (differentiated by their physical state - liquid, paste or solid) that are soluble in fuels and soluble in or miscible with water. These biocides are intended for use in hydrocarbon liquid fuels. 

Two valuable resources of information on fuel biodeterioration are ASTM D6469-99 (ASTM, 2001) and U.K. Institute of Petroleum (Institute of Petroleum, 1996) guidelines. 

---

NOTE It is also recommended that a biocide efficacy test be performed annually on the cooling system water. This checks the effectiveness of a range of biocides and it enables the most cost-effective program to be in place. It provides a response to possible changing circumstances, such as the development of any resistant species. 

Biocide efficacy tests Fossil fuel process water testing Hygiene monitoring in production areas Microbial contamination testing of textiles... 

Since bacteria are capable of forming biofilms on most surfaces, future tests should be focused on biofilm quantification. In assaying biocide efficacy, tests should be conducted based on biofilm populations rather than on liquid culture efficacy (Gu et al. 1998c, 2000c). Planktonic cells are not representative of conditions on surfaces of materials. 

To date no guidelines are available for the efficacy testing of active substances, however, for biocidal products. Paragraph 52 of Annex VI states that testing should be carried out according to Community guidelines if these are available and applicable. Where appropriate other methods can be used e.g. 

In fact, the lack of appropriate efficacy methods is internationally recognised by the authorities and there is an OECD initiative to (i) improve knowledge on what methods are available for efficacy testing of the different biocidal product types, their validity and any problems they may have and (ii) identify gaps where new methods are needed. 

It is important to note that antimicrobial and biofilm resistance are two different characteristics though some materials show both properties at the same time. Antimicrobial materials do not automatically prevent biofilm formation and vice versa. Antimicrobial surfaces could kill bacteria on contact but if dead bacteria cell debris blocks the active biocidal surface, biofilm formation could eventually occur. For example, quaternary anunonium polymers can effectively kill bacteria but when the surface is fouled with dead bacteria debris, biofilm formation is inevitable [188]. Materials with antibiofilm properties will repel the bacterial adhesion very effectively but may not kill the bacteria when they do colonize the surface. PEG surfaces are well known to repel bacteria adhesion. However, PEG surfaces show little antimicrobial activity. Quantitative antibiofilm efficacy tests can be divided into two categories static (minimum biofilm eradication concentration assay, MBEC) and dynamic (flow cell assay). In addition, SEM is a semiquantitative assay, which is discussed in Section 2.5. 

Carrier tests with dried cells on surfaces are not suitable for the evaluation of biocide efficacy towards biofilms. In general, microbial cells dried on carriers are less susceptible to biocides compared to planktonic cells however, established biofilms grown on surfaces usually display enhanced resistance compared to organisms simply dried on carriers (e.g., Samrakandi et al., 1994 Ntsama.Essomba et al., 1997). A possible reason may be physiological changes which are associated with biofilm formation, and can result in enhanced resistance to biocides. These processes are not involved in carrier tests. 

Only in few publications is the action of biocides described for aqueous systems with undefined biofilms with mixed composition (Exner et al., 1987 Mathieu et al., 1990 Goroncy-Bermes and Gerresheim, 1996 Morin, 2000 Holtmann and Sell, 2001 Walker et al., 2001). The advantage of efficacy testing on natural biofilms lies in the greater relevance to the practical situation. 

After defined exposure times, the test substrata can be removed from the system and treated with biocide, or they remain in the system and are analysed at the end of the experiment to determine biocide efficacy. Other, new variations of the CSTR can be suitable as test systems. These include the Calgary Biofilm Device, which utilises shaken microtitre plates (Ceri et al., 1999 Ceri et al., 2001), the flow-cell (Stoodley et al., 2001), the artificial biofilm system (Harkonen et al., (1999), the colony biofihn system (Anderl et al., 2000) and the drip flow reactor (Xu et al., 1998). 

A number of methods are available for evaluating the microbial resistance of polymer dispersions and biocides added to them but there are, as yet, no recognised international standards. Nevertheless, the work of the International Biodeterioration Research Group (IBRG) in this field has lead to the production of a draft method (IBRG, 2001), which, it is believed, can be used as an efficacy test for biocides within the scope of the European Biocidal Products Directive and for predicting the in-use performance of polymer dispersion biocides. 

Figure 9 Biocidal efficacy of formulations with and without added bleach systems simulated industrial washing conditions. ECE is a standard test detergent.

In fact, there is a recognition that a range of different test data may be required to support label claims. An applicant for authorisation of a biocidal product can confirm their efficacy claim with the provision of data from simple laboratory studies, laboratory simulated-use tests, pilot system studies or field studies according to either standard and/or non-standard test methods. 

By allowing this level of flexibility the directive recognises that the laboratory-based Standard Test as a universal tool for predicting accurately and consistently the relative efficacies of all biocidal products within a particular application area is not feasible. It also acknowledges the fact that the available Community standards may not always be best fitted to support the Directives requirements i.e. the support of a label claim. 

Factors are discussed which need to be taken into account when evaluating and designing new biocides for use in plastics formulations. In addition to establishing compatibility with the polymer matrix, the antiniicrobial efficacy has to be tested. The selection of a suitable test method is discussed, together with advantages and disadvantages of traditional test methods. An alternative method based on a modified version of the Film Adherence Method for the evaluation of biocidal performance is presented, which is claimed to be more representative of the end use conditions. 

In other circumstances performing only a test on Total ATP with microbial extractant can give results that might be misinterpreted. The typical mode of action of many biocides leads to the rupturing of microbes. Cellular ATP is released into the environment and the high ATP reading may be incorrectly interpreted as indicating poor biocide efficiency. In these situations it is beneficial to use two tests, one with microbial extractant (Total ATP) and one without (Free ATP) and thereby obtain a better assessment of the treatment efficacy. 

There are a number of test methods available to demonstrate the efficacy of biocides and to test directly whether they might present health hazards [44]. The EU s Biocidal Products Directive plays a significant role, both in selecting/recommending and restricting... 

The agar difihision test is used to qualitatively assess the efficacy of textiles treated with diffijsible biocides. Samples are plamd in the centre of nutrient agar plates which have been inoculated widi the test bacteria. The samples are incubated at 37°C for 18-24 hours. The evaluation of this test is based on the level of growth both under and around the sanqrie. (Fig. 2) The zone of inhibition around the test material is measured and any growdi present underneath the sample is scored. 

In many advanced countries there are legal restrictions on the claims that can be made about antimicrobials, because they are not all completely effective in killing all microorganisms. So expressions like self-sterilising , hygienic and germ-ifee are associated with specific levels of performance and not automatically acceptable just because a biocide is used. As a result, there has been considerable interest in the development of appropriate test methods for measuring antimicrobial efficacy. 

---