AATCC Test Method 30-2004 Antifungal Activity, Assessment on Textile Materials: Mildew and Rot Resistance of Textile Materials Developed in 1946 by AATCC Commit- tee RABI: revised 1952, 1957, 1971, 1981, 1987, 1988 (with title change), 1983, 1996; reafirmed 1970, 1974 1879, 1909, 1998; edtarily "revised and reaffirmed 1986, 2004, 1. General Purpose and Scope 1.1 The two purposes of this test method are to determine the susceptibil- ty of textile materials to mildew and rot and to evaluate the efficacy of fungicides ‘on textile materials. 2. Principle 2.1 Teal I 1 Il and IV can be used, singly or in combination, depending on the type of exposure to which the textile material will be subjected. For example, if the final product will eome in contact with sol, Test I, which simulates tis ex- ‘posure, should be used; if the finished product will never come in contact with soil or tropical conditions, a much less severe test (eg Il or ID should be used. ‘Test ILis specifically designed for cellur lose-contsining materials while Tet Il is {or all others, For all materials intended for outdoor and above ground use, Test IV should be used. The two important considerations, when evaluating textile materials in elation to fungal growth are (1) the actual deterioration of the textile product (rot) and (2) growth not neces- sarily deteriorating the product but mak- ing st unsightly (muldewy) often with an unpleasant and musty odor 22 Certain pre-exposures of textile producis may be indicated when specific endeuses are critical (see Appendix A). ‘When the end-use will be near high tem perature and the fungicide may be vola- file, a preliminary oven exposure may be desired, When the end-use will be in tropical exposures or outside with rainfall present, # leaching exposure should be performed before mildew evaluation is made, When at all possible, the textile material should be exposed to the ex- pected conditions of use prior fo perfonm- ing ths test. 3. Terminology 3.1 mildow resistance, n—in textiles, resistance 10 development of unsightly fungal growths and. accompanying. un- ‘pleasant, musty odors on textile materials exposed” 10 conditions favoring such 78 TM 30-2004 growths, 3.2 rot resistance, n—in textiles, re- sistance to deterioration of a textile mate- "ial as a result of fungal growth in oron it. ‘NOTE: Such deterioration is normally assessed by measuring loss in tensile strength. 4, Safety Precautions NOTE: These safety preceutions are for information purposes only. The pre cautions are ancillary to the testing proce- dures and are not intended tobe all inelue sive. Iti the user’s responsibility to use Safe and proper techniques in handling materials in this test method, Manufac turers MUST be consulted for specific 1.5-40.2 in.) with the long dimen- sion paralel to the warp and unraveling 02.54 0.1 em width (1.04 0.04 in}, or, in the case of fabeio with less than 20 threads per 25 em (1.0 in. to a predeter- ‘mined numberof threads to give a speci- ‘men 2.5-+ 1.0 em in width (1.00.4). ‘A sample cutter can also be used (see 24.3). The numberof specimens will vary ‘according tothe numberof variables, The ‘suggesied number of specimens is five fo each treatment, contol end reference 17. Test Procedure 7.1 Viability control: Expose untreated cotton cloth 271 yim? (8 aziyd?) in the sil bed for seven days during the test pe- ‘od to verify fumgal activity. The soil bed Shall be considered as satisfactory if the viability control fabric loses 90% break- ‘ng strength after seven days exposure "12 Soil Bed: Place the air-ry test soil (see 24.4) in trys, boxes or suitable con- tainers to a dapth of 13.0 = 1.0 em (5.1 = (04 in) and bring to optimum moisture ‘content by gradual addition of water ec- ‘companied by mixing to avotd pudding. ‘After allowing itto stand for 24h, sieve it ‘through 6.4 mm (0.25 in.) mesh sereen. Maintain uniform moisture content by ‘covering the soil container with a suitable Tid. The moisture content ofthe soil dur- ing the test period shall be maintained be- tween 25 + 5% (based on dry weight). If the surounding air is maintained a higher than 83 = 39% relative humidity, ‘AATCC Technical Manual’2010 Copyright © 2009 American Associton of Textile Chemist and Coorsthe loss of moisture is negligible "73 Incubation: Bury the specimens horizontally on 10.0 = 1.0 om G9 = 0.4 in.) of soil, spaced at least 2.5 em (1.0 in.) apart and then cover with 2.5: 05 em (1.0 = 0.2 in) of est soil Incuba- tion periods can vary from 2-16 weeks, depending on severity of service require ‘ments, and other factors of importance to the interested parties. Maintain the tem perature at 28 + 1°C (82 + 2°F) during the test period. Evaluation and Report 8.1 Swength loss determination: Re- move specimens, gently wash with water, diy at room temperature for 22 +4 h and then condition in en atmosphere of 64 = 2% humidity and a temperature of 24 © 43°C (76. 6°F) for 24 h. Determine break ing strength by the method outlined ASTM D 5035, Standard Test Method for Breaking Force and Elongation of Textile Fabrics (Strip Fore), using 25 x75 mm (1 x3 in) jaw fies. The gage length is de- termined a6 25% ofthe specimen length. Testing can be perfommed every two ‘weeks or as specified by the end-user 82 Report: Report the length of expo- sure to the soil bed, percent retained breaking strength when compared to the unexposed textile, any pre-exposure of specimens before burying and pereent e- tained breaking strength of untreated specimen andor viability cont. Test ‘Agar Pate, Chaetomivm Globosum 9.1 This procedure is used for evaluat- ing rot resistance of cellulose-contaning textile materials that will not come in contact with soil It may also be used for determining uniformity of fungicide ‘weament 10, Test Specimen 10.1 Proceed as in Section 6, if strength loss isto be determined. IFonly a visual examination is performed, a mini mum of five samples is required. How- fever, any number can be tested depending fon end-users request. Cut 38 0.5 em (15 + 0.2 in) diameter dises from both lncated and untreated samples, 1 Test Procedure 11.1 Organism: Chaetomium giobo- sum. American Type Culture Collection No. 6205 (see 24.5) 112 Culture medium (see 244 ‘mineral salts agar should have the follow= AATCC Technical Manvll2010 Dipotasium hydrogen phosphate, KQIPO nnn 208 Magnesium sul, ‘MgSO, - 7H:0. 02g Ferous sulfite, F280," 71,0. . semen 0B Distlied water vt 1000 mL 11,3 Inoculum: Place agar solution in any desired container such as test tube, French square botle, Erlenmeyer ask, ot petri dish. Sterilize in an autoclave at {03 kPa (15 psi) and 121°C @S0°F) for 15 min and cool in a position which affords ‘maximum inoculation surface. After the agar has hardened, under aseptic condi- tions, place on its surface a disc of filler paper previously sterilized by dry beatin an oven at 71 = 3°C (160 + 5°F) for Ih. Streak the ltr paper with spores of Cha- ‘elomium globosum by use ofa sterile nee- Gle, Incubate at 28 = 1°C (82 + 2°F) for ‘approximately 10-14 days to. produce abundant growth. Remove the fille paper from the container and add it to 50 + 2 aL of sterile distilled water containing & few glass beads and shake vigorously to bring the spores into suspension. Use this suspension for inoculum in 11.5. 11.4 Culture chember: Melt mineral salts agar of the composition specified in 112 in an autoclave and distribute into any convenient coniainer. Sterile under conditions given in 113 and leave undis- ‘urbed until the agar hardens 11.5 Inoculation: Proawet the speci- ‘mens (but do not rub or squeeze) in water containing 0.05% of a nonionic wetting agent (see 24.7) and place in contact with the hardened agar medium in each con- tainer under aseptic conditions. Distribute 110-£0.1 mL ofthe inoculum evenly over the 15.0 10%4.0+05 om (6.00.4 x 1.5 + 02 in) specimens by means of a sterile pipette-Use 0.2.4 0.01 mL ofinoe- ‘lum for the 3.80.5 em (1.5 =0.2 in) ses. Set up a control specimen, celli- lose filler paper or untreated control, in & similar way by using 1.00.1 of 0.24 (0.01 mL of sterile water. 12, Evaluation and Report 1211 Surengih loss evaluation: Proceed as per 8.1 and report the change in break- ing strength as compared to the sample before exposure or the control if available. 12.2 Visual assessment: Report the ex- tent of fungal growth on the dis, using a microscope (SOX) where necessary, in ‘accordance withthe following scheme: Observed Growth ‘No growth Microscopic growth (visible only une der the misoscope) ‘Macroscopic growth (visible to the 2) Test tl ‘Agar Plate, Aspergis Nigor 13. Score 13.1 Certain fungi, of which Aspergil- dus niger sone, can grow on textile prod ots without causing measurable break. ing strength loss within a laboratory ‘experimental time frame. Nonetheless, their growth may produce undesimble and unsightly effects. This procedure is sed to evaluate textile specimens where ‘growth of these fungi is important 14, Test Specimens Cut duplicate 3.8 + 0.5 em (1.5 + 02 in.) diameter discs fFom both treated ‘and untreated samples. Other shapes and sizes can be used provided any antici. pated size of the grow-ffee zone is taken into consideration. 15, Tost Procedure 15.1 Organism: Aspergillus niger, “American Type Cultire Collection No. (6275 (see 245) 15.2 Culture medium: Proceed as per 11.2. Other suituble media are (Dox) Agar and Sabouraud Dextrose Agar (sce 248). 15,3 Inoculum: Add serapings from tipe (7-14 days) fruiting culture of ds- ‘pergilus niger grown on the medium de- ‘scribed in 112 containing 3.0+0.1% glu ‘cox, to a sterile Erlenmeyer flask ‘containing 50-4 1 mL of sterile water and fe few glass beads. Shake the flask thor- ‘oughly to bring the spores into suspen- sion, Use this suspension as the inoculum, 15.4 Inoculation: If the test medium ‘contains glucose, distribute evenly 1.0 + (0.1 ml of the inoculum over the Surface ‘of the agur. Pre-wet the dises (but do not ub or squeeze) in water containing 0.05% of a nonionie wetting agent (see 24.7) and place on the agar surface. Dis tribute evenly over each dise 02 © 0.01 a of the inoculum by means ofa sterile pipette. IF the test medium does not con- {ain glucose, a negative contol fabric is required to ensure inoculum Viability. In- ‘cubate all specimens at @ temperature of 28 4 1°C (82 + 2°F) for 14 days when ‘mineral salts ager is used and for seven, days when 3% glucose is added to the ‘mineral salts agar 16, Evaluation and Report 16.1 At the end of the incubation pe- riod report the percentage of surface area Of the dises covered with the growth of Aspergillus niger, using 2 microscope (SOX) where necessary, in accordance ‘withthe following scheme: TM 30-2004 7 (Copsigh © 2009 American Association of Tote Chomists nd ColoresObserved Growth 'No growth (if present, report the size of the growtl-free zone in mm) Microscopie growth (visible only un- er the microscope) “Macroscopic growth (visible 10 the eye) Test V Humidity Jer, Mixed Spore Suspension 17, Seope 17.1 This test method is designed to determine the fingistaic effectiveness of treatments intended to control mildew and non-pathogenic fungal growth on ar- ticles or surfaces composed of textile ma- terials intended for outdoor and above {ground use and which are usually water- 17.2 For this test method visual assess- ‘ment. is used, Additionally, breaking strength may be determined by method as peril. 16, Princip 18.1 Treated and untreated, nuttient- saturated strips of fabric are sprayed with | mixed spore suspension of miléew- ‘causing organisms and incubated at 90.4 2% relative humidity. Mildew growth on ‘weated and unvested stuips is rated at ‘weekly intervals for up to four weeks. 19. Apparatus 19.1 Glassware: $00 mL. French square {ars or equivalent with fitting serew caps. Caps are modified by center drilling and inserting en appropriate size stainless Steel or brass bolt to which =. hook (formed from a 6.5+ 0.5 em length [2.6 + 0.2 in.] of #22 nickel-chromium wire or other non-comosive wire) i attached, 19.2 Plastic paper clips or nylon thread to suspend the specimens ffom the screw caps of the French jars. 193 Atomizer, DeVilbise #152. (or equivalent operated al 69 KPa (10 psi). 19.4 Counting chamber suitable for de- termining spore concentrations; c, hemocytometer. 20. Test Specimens 20.1 The specimens are prepared by cxt- sing 2.5 +0.5emx7.5=05 em(1.0+02 % 3.0 02 in) strips from sample weigh ing 170.0 + 340 gim* (5.0 + 10 ou), For heavier fabrics use strips 20+ 0.3 em 2.0205 em (08+02%0.8-+02in). 20.2 Use at least four specimens of ‘cach treated and untreated fabric. ‘203 Untreated fabric stps, identical in all other respects to the treated speci- ‘mens uncer test, are required to establish the test validity, If untreated fabrics are 78 TM 30-2004 ‘ot available, use @ control cloth with the {lowing rejuirements: Cotton: American type, good mid- dling Wap: 18.5 tex 2 886% 25748 Well: 30 tex 2 630 x 28748 Weave: Plain 34 endsiom and 17 picksiem Mass/unit area: 230.0 pim* (6.8 aziyd") Finish: Sooured only 21, Test Procedures 21.1 Organisms: DULL Aspergillus niger, American ‘Type Culture Collection No. 6275. 21.12 Penicillin varians, American ‘Type Culture Collection No. 10509. DLL Trichoderma viride, American ‘Type Culture Collection No. 28020 (see 245). 21.2 Culture medium: 21.2.1 Maintain stock culture of each of test ube slants of potato dextrose agar for A. niger and P.varians, and malt ex- tract agar for Z. viride (see 24.6 and 24.8 for media). 21.222 Incubate new stock culture 7-10 ays at 25:4 1°C (77+ 2°F), then store at 2-10°C G6-50°F) 21.3 Preparation of conidial suspen- 21.3.1 Conidial suspensions of fungal organisms are prepared by adding 10 mL of a sterile 0.5% saline solution contain: ing 0.05% of @ non-fungicidal wetting agent (see 24,7) to a 7-10 day agar cul> tre 21.3.2 Serape the surface of the culture gently with a platinum or nichrome wire fo liberate the spores. Asitate the liquid Slightly to disperse the spores without de- ‘aching mycelial fragments, and gently ecant the mold suspension into a flask containing a few glass beads. 21.33 Shake the dispersion vigorously to break up any clumps of spores and then filter through @ thin layer of sterile cotton oF glass wool. Conidial suspen sions may be stored at 64°C (43 = 7"F) for up to four weeks. 21.34 Inoculum for tet should be ad- justed using a hemocytometer or a Peiroff-Hausser bacteria counter to con- tain five million conidia per mL on day of use by appropriate dilution of stock sus- pension with saline solution. 21.4 Preparation of test specimens: 214.1 To ensure Tuxuriant growth, both the test and control strips mast be Siturted with a sterilized glycerol nut ent solution of the following. compost tion: 97.6% distilled water, 2.0% gly rol, 0.1% KHPO, 0.1% NH.NOs, (0.05% MgSOy-7H,0, 0.1% yeast extract and 0.05% of a nonionic wetting agent (Gee 247). Adjust the pH t0 63+ 0.1, Sulicient nutrient solution should be pre= pared to saturate all the specimens used nasingletest 21.4.2 Soak cach stip in nutrient for toe minutes oF unt saturated. Squceze ‘excess liquid and allow fabric stps to at Ary before proceeding with application of test fons. 21.5 Premix equal volumes of well agli conidial suspensions of . niger, T viride and P.vartans, Evenly distib- ‘ute 1.0-+0.1 ml. ofthe above suspension ‘onto both sides ofeach specimen either by spraying or by means ofa pipette ‘21.6 Suspend fabri sis using plastic paper clips or aylon thread from the caps of individual jars containing 90-+3 rl of ‘water each. Hook position must be ade jase so that the bottom ends of aiached ‘tnps are all at uniform height above the water level. The caps are tightened, then backed off one-ighth tum to allow {for some ventilation, ‘1.7 incubate at 28 + 1°C (82 + 2°F) for id days (or non-coated cellulosic textiles) or 28 days (for noncellulosic or coated calulosi textiles). 22. Evaluation and Report 22.1 A record ofthe pereent of surftce ‘rea covered with fungal growth for each stip is made at weekly intervals, or until heavy growth occurs on each sample rep- licate. Using a microscope (50X) where necessary, assess each specimen in aecor- dance with the scheme given in 12.2. 22.2 After seven days each control ‘snip must show macroscopic growth. If not the cave repeat the fst since test conditions were not valid, 22.3 Any adverse effect of incubation on the fabri; e., color changes, flexibil- ity, water repelleney, should be qualita- tively reported. 22.4 Strength lose determination can be cared out as per 81. 722.5 The results of this test method have tobe correlated to claims and direc- tions for use recommended for the mil- dow control product plus any other eite- ‘a agreed upon by the interested parties. 23, Precision and Bias 28.1 The precision and bis of this test ‘method are being established. If the ‘breaking strength lost is determined, dhen eft othe stement given in ASTM D 24 Notes 24.1 Publication avaible fom US. De- partment of Health & Human Servies— CDCNIN-HES Publication No. (CDC) #4 395, we site: wore pov. 242 Booklet available from Publicstions fice, ACGIH, Kemper Woods Centar, 1330 Meadow Dr, Cincinnati OH 45240; tel 313/742-2020; web site: wwe agi org. "245A JDC Precision sample cutter may be AATCC Technical Manual!2010 Copyright ©2009 American Astoiton of Towle Chomiss an Colorist‘fom Thwing-Albert lassument Co, 10960 Dutton Road, Philadelphia PA 19154; tl 215/637-0100; fax: 215/682-8370; web se: wwwihwingalber.com, Cat. #99 ‘Medel JOC25, 24 Types of sol which have been found satisfictory for this purpose inclode garden and naturlly fertile topscls, composts and ‘on-atarilegresaiouss poting sil An equal blend of good topsoil, well ted and shred ed manure, and coarse sand stould be used Sulficent to eure a high depree of microbial activity and the peseace of cellulose destroy ing organisms. The optimum moisture content ofthese is abou 308% moisture above oven ry weight 245 Chactomin sodosum ATOC 62, Aspeglia niger, ACC. 6275, Peni SR An 109 and cadena vi 1 ATCC 28020, ean be purchased fom the ‘Asctzan Type Cale Collcton, P.O, Box 154, Mananas VA 20108 108/365-2700; fx 7108768-200; web se: www ale op "46 Clue medi bing oerbel in 112 (Mine! Sls) ea be pu Etsed Sm Balimore Biolog! Lebo ‘ex, 280 Schlig Ce, Coxteyvle MD 21030. 247 Teioa™ X-100 (Rohm & Hass Co, ‘lpia PA 19108 bas een found tobe ‘AATCC Technical Manval2010 ‘good wetting agent. Suitable atematives are octyl sodium sulfosecinate or N-metiyle taurid danvatives, 24.8 These culture media can be bought e- ther from Baltimore Biological Labertones (Gee 246) of Deo Laboratories, 920 Henry St, Devos Ml 48201 7249" ASTM D 5035 can be used for yar, ‘tread, corage or tape (ee 12.1) ‘24.10 I esting is being performed for Fe ‘eal Standards, use AATCC 30-2, Other or [Bnisms canbe used: Myrohect verucara AATCC 9095, QM 450 Trichoderma SP ATCC ‘9648, QM 363; Memnanitia cchnara ATCC 11973, QM 1225, Aspens niger ATOC (6275, QM 48%; Aspergilis clarans ATCC 18214, 0M 862: Aapenii A Pre-Treatments AN, Leaching ALL The leaching should conform in principle to the following procedure: ‘Water fom a faucet is passed through a ‘tubing into leaching vessels, care being taken that specimens having different ‘amounts ofthe same treatment are in sep- ‘rite vessels, Flow is adjusted to ensure & complete change of water not less thea thee times in 24h. The delivery tubes are inserted down through the center of wire mesh eylinders in the leaching sels and held in the wire eylinders with rubber bands and leached for 24 b, The pH and temperature of the water is r= Corded and included in the report of the test results. ‘2. Volailization A2.1 Standard specimens ofthe fabric to be tested are exposed continuously to dry heat at 100-105°C @Q12-221°F) for 24 hin a well ventilated oven. 3, Weathering A3.1 Portions of the material to be tested are exposed on a series of weather- ing racks at 45° to the horizontal facing South, between April 1 and October 1, ia such @ manner as to avoid sagging’ or Mapping. Tt is recommended that such racks be set up in at least four locations within the United States; eg, Washing- ton DC; Miami FL; New Orleans LA; and suitable devert locations. TM 302004 79 Copyright ©2009 American Assocation of Tentile Chests and Colorist