ASTM E875-15

Designation: E875 − 15Standard Practice forEvaluation of Fungal Control Agents as Preservatives forAqueous-Based Products Used in the Paper Industry1This standard is issued under the fixed designation E875; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (´) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This laboratory practice is used to determine the efficacyof a fungal control agent to prevent spoilage of in-processaqueous-based products used in the paper industry.1.2 For information on bacterial control agents, see TestMethod E723.1.3 It is the responsibility of the investigator to determinewhether good laboratory practices (GLP) are required and tofollow them when appropriate (see 40 CFR 160).1.4 A knowledge of microbiological techniques is requiredfor these procedures.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE723 Practice for Evaluation of Antimicrobials as Preserva-tives for Aqueous-Based Products Used in the PaperIndustry (Bacterial Spoilage)E1839 Test Method for Efficacy of Slimicides for the PaperIndustry—Bacterial and Fungal Slime2.2 Federal Standard:40 CFR 160 Good Laboratory Practice Standards33. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 fungal control agent, n—an agent that either kills orprevents growth of fungi and either kills or prevents thegermination of fungal spores. This term is applied to chemicalbiocidal or biostatic agents.3.1.2 preservative, n—chemical agent used to prevent mi-crobial spoilage of products due to microbial growth.4. Summary of Practice4.1 Aqueous material to be preserved is inoculated with anappropriate fungal innoculum followed by addition of a con-centration of fungal control agent that will kill the fungi orprevent their growth for a desired period of time, or both. Inaddition, the agent will also prevent fungal spore germination.Fungal growth is determined by visible signs of deterioration inthe test sample, and by obtaining fungal numbers and compar-ing them to a sample without any fungal control agent. Theproper level of fungal control agent is one that prevents productdeterioration and reduces and keeps the organisms to anacceptable level in the test material, as determined by the testeror user.5. Significance and Use5.1 This practice should be used to determine if a fungalcontrol agent is effective to preserve pigment suspensions, dyesolutions, pulp slurries, starch solutions, polymers, sizingagents, latex emulsions, and other specific aqueous-basedmaterials used in the paper industry. Separate evaluationsshould be made on a representative type for each specific classof product to be preserved.NOTE 1—Control of bacterial spoilage of similar products can beevaluated by Test Method E723.NOTE 2—Slimicides for control of fungal or bacterial slime can beevaluated by Test Method E1839.1This practice is under the jurisdiction of ASTM Committee E35 on Pesticides,Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved May 1, 2015. Published July 2015. Originally approvedin 1982. Last previous edition approved in 2010 as E875 – 10. DOI: 10.1520/E0875–15.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at service@astm.org. For Annual Book of ASTMStandards volume information, refer to the standard’s Document Summary page onthe ASTM website.3Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://www.access.gpo.gov.Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16. Apparatus6.1 Two Balances— One should be sensitive to 0.1 g at aload of 200 g with a platform to accommodate bottles beingused in the test. The second balance (analytical) should besensitive to 0.1 mg and used for weighing test chemicals.6.2 Clean Sample Containers, Containers (120 mL) withscrew- cap lids are ideal for test aliquots. Other suitablecontainers include milk dilution bottle, 4 oz glass bottles, orsterile sampling bags.6.3 Flaming Equipment—An alcohol lamp, bunsen burner,or electric device may be used to flame inoculating needles andother equipment.6.4 Incubators—Incubators that control the temperature ofthe test 6 2°C. Temperatures for test should be temperature atwhich the product will be stored.6.5 Petri Dishes, 100 by 15-mm, plastic or borosilicateglass, sterile.6.6 pH Measurement—Any pH meter is suitable to stan-dardize the pH of the culture medium or to determine pH ofsamples. Nonbleeding test strips may be used for determiningpH of test aliquots.6.7 Pipets—1.0-mL graduated in 0.01 mL and 10-mL gradu-ated in 0.1 mL. Serological pipets should not be used for highlyviscous materials. Automatic pipettors may be used.6.8 Pipetting Aid—rubber bulb or other device to eliminatemouth pipetting.6.9 Sterilizers—pressurized steam sterilizer (121°C at 15psi) or hot air oven capable of reaching 180 6 2°C for 2 60.2h.6.10 Swabs—Sterile swabs (cotton or other appropriatefabric type) for aiding in removal of fungal spores from slants.6.11 Sterile Funnel—Funnel with sterile glass wool forfiltration of spore suspension.6.12 Sterile Glass Beads—Glass beads (3-5 mm).6.13 Tubes—Tubes for preparation of slanted media.6.14 Milk Dilution Bottles, (100 mL).7. Reagent and Materials7.1 Purity of Water—Unless otherwise indicated, water shallbe understood to mean distilled water or water of equal purity,as defined in Specification D1193, Type 3.7.2 Freshly prepared test solutions of the fungal controlagent shall be used in all tests. Some preservatives can beadded with a micropipet.7.3 Test Materials—Freshly prepared pigment slurries,adhesives, dye rosin, polymer, sizing solutions, and otherclasses of aqueous-based materials to be preserved should beused as the substrate.7.4 Culture Medium—Dehydrated Sabouraud’s Agar (malt-ose or dextrose) is recommended for fungi. A more selectivemedium may be used provided it is used in addition toSabouraud. Results should indicate the data obtained with eachmedium.7.4.1 Spore Suspending Medium and Container—Milk dilu-tion bottles containing 100 mL Butterfield Buffer4with solidglass beads, for preparing sterile spore suspensions.7.4.2 Culture Media, slants of the selected agar.8. Test Organisms8.1 The test organisms selected may vary with the purposeof the test. If evaluating the basic effectiveness of a fungalcontrol agent, the use of standard fungal cultures is recom-mended (see 8.2). If attempting to qualify a fungal controlagent for a particularly difficult, or highly specific preservationapplication, the use of fungal spoiled product or selected fungalorganisms isolated from the problem system, or similarsystems, may be appropriate (see 8.3 and 8.4).8.2 Standard fungal cultures suitable for this procedureinclude the following:8.2.1 Aspergillus niger: ATCC 6275.8.2.2 Penicillium pinophalum: ATCC 9644.8.2.3 Trichoderma virens: ATCC 9645.8.2.4 Candida albicans: ATCC 10231.8.2.5 Saccharomyces cerevisiae: ATCC 4111.8.3 To verify that a spoiled sample contains fungalorganisms, the spoiled sample should be streaked onto plates ofSabouraud Maltose Agar (or other media selected). Whenfungal contamination is verified, if sample is large enough, itmay be used directly as the inoculum (see 10.1). If sample istoo small for use as inoculum, add the spoiled sample to alarger sample of unprotected material and incubate for 7 to 14days at an appropriate temperature (based on use conditions)until spoiled, then proceed with testing (see 10.1).8.4 If specific or combinations of fungal isolates fromspoiled material are to be used, proceed with testing as forstandard fungal organisms. See 9.1.9. Inoculum Preparation9.1 Standard Fungi and Fungal Isolates—Organisms shouldbe grown as slant cultures on the culture medium selected.Grow for 7 to 14 days at 25 to 30°C, until cultures sporulate.Add 10 mL of sterile Butterfields buffer to the slants. Gentlyremove the spores from the surface of the agar by rubbing witha sterile swab. Add the resulting suspension to a bottlecontaining glass beads and buffer. Cap the bottle tightly, andthen shake vigorously to liberate spores from fruiting bodiesand to break up spore clumps. Filter the resulting suspensionthrough sterile glass wool in a sterile funnel into a sterilecontainer to remove mycelial fragments. This filtrate is thespore suspension to be used as an inoculum for test samples.10. Procedure10.1 To provide a uniform inoculated substrate, the inocu-lum of spoilage organisms or fungal spore suspension shouldbe added to the entire quantity of the test substrate at one time,mixed thoroughly, then divided into test aliquots.4Butterfields Buffered Phosphate Diluent, Official Methods of Analysis of theAssociation of Official Analytical Chemists, K. Helrich, 15th ed., 1990, p. 429.E875 − 15210.1.1 Streak-out the test substrate (aqueous-based prod-ucts) before and after fungal inoculation to and determine thefungal contamination. Incubate plates at 25 to 30°C for sevendays or until the control plates show sufficient growth forrating. Some test materials may not support growth of fungi.These materials most likely do not require a preservative toprevent fungal growth. If the test material only supports slowgrowth of the fungi in question, then incubation periods mustbe sufficiently long to allow those fungi to grow.10.1.2 Evaluate the level of growth with either a growth/no-growth rating, or a rating scale (such as 0 to 4, with 0 beingno-growth and 4 being heavy-growth).10.2 Disperse 50-g aliquots, or any other suitable quantity,of the inoculated test substrate aseptically into the containersselected. Set up controls (no biocide added) in duplicate.10.2.1 Aseptically add the stock solution of the test fungalcontrol agent to each bottle to give the desired concentration inparts per million (mg/mL) or percent. Shake vigorously using20 complete cycles in a vertical motion or other method toensure complete mixing. Stock solution of the fungal controlagent should be of such strength that the volume of the solutionadded is no more than 1 % of the total volume of sample ineach bottle. Do not add a fungal control agent to the untreatedcontrols. Include a minimum of five concentrations of thefungal control agent in each test. The test concentration rangeshould include at least one concentration that is ineffective andone that is effective. Record the initial pH of all samples andother physical characteristics such as color, odor, and viscosity.10.2.2 Incubate all samples at 25 to 30°C, (or other tem-perature that the product would encounter either in use orstorage situations), with the bottles loosely capped. At weeklyor other suitable time intervals, mix each sample thoroughlyand immediately streak on Sabouraud’s Agar according tostandard streaking techniques and incubate at 25-30°C forseven days. In addition, observe each sample bottle for theevidence of spoilage such as visual growth of fungi, or changesin color, odor, and pH. Record the growth of the fungi for thestreaked samples using rating system in 10.2.1.10.2.3 Rechallenge all treated samples showing no visiblegrowth with 1 mL of inoculum obtained from one of theduplicate fungal controls samples (no biocide added). Incubatefor at least one more week.10.2.4 Base success for each preservation experiment uponthe expected time of usage of the material to be preserved.Continue the test for at least the average length of time that thespecific type of product would need protection, generally sixweeks, unless the material under test is preserved for a shorterperiod of time, for example, one week. Record the nature andextent of spoilage based upon the criteria used (10.1.2 and10.2.1).11. Results11.1 At each sampling time and at the end of the test, recordall results from observations of the sample bottles and thestreak plates. Visual deterioration or other signs of degradationin the bottles, such as changes in pH color, odor, and loss ofviscosity, should also be used to judge the degree of preserva-tion obtained. The study is not valid unless fungal spoilage isevident in the control, based on visual fungal growth, obviouschanges in physical characteristics, or other reliable criteria.12. Precision and Bias12.1 A precision and bias statement cannot be made for thispractice.13. Keywords13.1 aqueous-based products; fungi; fungal control agent;fungal spores; paper; preservativeASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. 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