Standard Practice for Evaluating Water-Miscible Metalworking Fluid Bioresistance and Antimicrobial Pesticide Performance. Internal procedure based on ASTM E2275-24 Standard.
Test accredited in our laboratory by ENAC (Spanish National Accreditation Entity), for microorganism indicated by the standard and others that it could be required.
This test method is aimed for evaluating the relative intrinsic bioresistance of water-miscible metalworking fluids bioresistance, the antimicrobial biocides performance, or both, defining bioresistance as the capacity to withstand biological attacks. This method is applicable to antimicrobial agents that are incorporated into the concentrated metalworking fluids or its final in-use dilution.
In this test the relative bioresistance is determined by inoculating the metalworking fluids with a biological inoculum that can be known (characterized) including one or more known biological cultures, or unknown (uncharacterized), when a metalworking fluid with an unidentified biological contamination (microorganisms) or an isolated unidentified microorganism of the altered metalworking fluid, is used. The bioresistance of the fluid is defined in terms of resistance to the increase of biomass, to the increase in the recovery of viable cells, and to changes in chemical properties, changes in physical properties, or some combination of them.
To perform the test, the metalworking fluids are dispensed into test containers that simulate the conditions of use (microcosm), containing or not the antimicrobial biocide, into which the biological inoculum is introduced, with known microorganisms (characterized) or unknown (not characterized). After the inoculation, the microcosms are aerated to simulate the recirculation conditions in the refrigeration systems. In addition, metal chips can be added to simulate the accumulation of chips that usually occurs in the cooling systems. After the inoculation, samples are periodically extracted from the microcosm and the parameters of interest are evaluated (macroscopic aspects, biomass, colony forming units -UFC-, chemical or physical properties).
Depending on the objectives of the assay, the test ranges from 5 days to 3 months. The shorter analysis periods are used to evaluate the microbiocidal speed of the biocide and the initial bioresistance of the metalworking formulation. The longer periods are used to evaluate the bioresistance of the metalworking fluids formulations to repeated biological inoculum. For tests lasting more than a week, extractions of 10 to 80% of the fluid are carried out weekly, replacing it with new fluid before inoculation.
The tests, according to their objectives, can be carried out according to 3 possible test methods:
1. Comparative evaluation of the bioresistance of metallurgical fluid formulations
This test determines the bioresistance of two or more formulations of metalworking fluids through 5 periodic evaluations: before 30 minutes after introducing the inoculum; at 2 days; at 5 days; after the weekend non-aeration system; and 5 days after the new aeration.
2. Evaluation of microbiocidal performance to know the speed of action
This test determines how quickly a microbiocidal treatment reduces the counts of viable microorganisms. In this case the biocide is added to a previously inoculated fluid and analyzed 6 times: at 10 minutes before adding the antimicrobial biocide, at 4 hours, 8 hours, 24 hours, 72 hours and 5 days after inoculation.
3. Evaluation of microbiocidal performance, to know the effectiveness of conservation
This test is used to determine the effectiveness of a microbiocide or a combination of them, to protect a metalworking fluid against biodeterioration. It can be used to determine the product and the dose of use. In this test, the sample must be tested for at least 6 weeks or until the microbiocide loses effectiveness. Besides the microbiological characteristics, the chemical and physical characteristics should be evaluated. The chemical and physical characteristics are not realized in this laboratory. We only analyze gross observations and microbiological tests).
Evaluation of the bioresistance and performance evaluation
These evaluation tests depend on the specific objectives desired for each sample and include the evaluation of macroscopic aspects (appearance of flocs, discoloration, separation in phases, etc.), chemical changes (alkalinity, pH, concentration of dissolved oxygen, ...), physical analysis (dispersion stability, corrosivity and foaming tendency) and microbiological.
The bioresistance determines the relative capacity of the test fluid to prevent the proliferation of the inoculated microorganisms, retain their original chemical and physical properties, or a combination of several parameters. In IVAMI we carried out these tests evaluating the gross observations and the microbiological parameters through the determination of the count in CFUs of the inoculated microorganisms, known or unknown, and macroscopic examination (detection of floc formation, discoloration and phase formation).
The ASTM E2275 standard is designed to give relative values rather than absolute values for fluid bioresistance or microbiocide performance. Different fluids may have different stabilities to large numbers of microorganisms. Therefore, the results obtained in test formulations should be compared to changes in both negative (uninoculated) and positive (inoculated untreated) controls.
According to this standard, when this method is used to evaluate microbiocide performance, it is prudent to always evaluate at least two antimicrobial treatments. Preferably one treatment should serve as a positive control. Furthermore, if the primary intent is to evaluate the general efficacy of the microbiocide being tested, then it should be tested in various types of formulations. On the other hand, if the primary aim is to protect a particular formulation, then a microbiocide-free version of that formulation should be used as the control.
The client must indicate in the submission form the end-use dilution to be tested, the method and the variables that he wishes to analyze. In addition, it should be specified the microorganisms against which the bioresistance must be analyzed. IVAMI offers the possibility of inoculation with all the microorganisms indicated in the standard: Aeromonas hydrophila, Candida albicans, Desulfovibrio desulfuricans, Escherichia coli, Flavobacterium ferrugineum, Fusarium oxysporum, Klebsiella pneumoniae, Mycobacterium immunogenum, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas oleovorans and Saccharomyces cerevisiae. If it is desired to employ a contaminated metalworking fluid, or an undefined culture isolated from a metalworking fluid, the applicant should provide it.