E24-06 STANDARD METHOD OF EVALUATING THE RESISTANCE OF WOOD PRODUCT SURFACES TO MOLD GROWTH

Transcription

1 AMERICAN WOOD-PRESERVERS ASSOCIATION STANDARD (This Standard is promulgated according to a consensus procedure and is under the jurisdiction of AWPA Committee P-6) E24-06 STANDARD METHOD OF EVALUATING THE RESISTANCE OF WOOD PRODUCT SURFACES TO MOLD GROWTH Note: AWPA Standard E24-06 was adopted in 2006 and consists of 5 pages. 1. Scope 1.1 This Standard provides an evaluation method for assessing the resistance of surfaces of wood products, treated or untreated, coated or uncoated, to the growth of molds. The main objective of the method is to determine the relative resistance of wood products to surface growth of molds in an environment favorable for growth of fungi. Resistance to mold growth is evaluated relative to reference products and unprotected sapwood of a species of pine. Pine sapwood is included as an untreated control substrate to indicate viability of molds in the test chamber and severity of growth conditions. This test method may also be used to evaluate compounds or formulations that may inhibit fungal growth and the aggregate levels for their use. Products evaluated with this method would be intended for use in interior environments where the products may be unintentionally subjected to conditions favorable to the growth of molds. This Standard is based on the ASTM Standard Test Method D , Standard Test Method for Resistance to Growth of Mold on the Surface of Interior Coatings in an Environmental Chamber. 1.2 The method is applicable to the testing of commercial or experimental wood products designed to be resistant to the growth of molds. Performance at a certain rating does not imply any specific period of time for a fungal free surface in service, however products with a better rating would likely perform better in actual end use. 1.3 The requirements for preparation of the material for testing and the test procedure appear in the following sections: Outline of Method...2 Apparatus...3 Reagents and Materials...4 Test Specimens...5 Treatment Procedure...6 Preparation of Inoculum...7 Exposure in Environment Chambers...8 Assessment of Mold Growth...9 Interpretation of Results...10 American Wood-Preservers Association 2. Outline of Method Samples of wood products are exposed in an environment chamber where temperature and relative humidity are controlled to provide ideal conditions for the growth of molds. The chamber and samples are inoculated with specified molds, and circulating air within the chamber continually subjects samples to spores for the duration of the test. The method is non-sterile, and therefore molds from the air or soil may be present and compete with the inoculated molds for colonization on surfaces of test products. Samples are removed from the chamber and evaluated for growth of molds on the sample surfaces every two weeks for eight weeks. Each sample is assigned a rating for extent and intensity of mold growth. Average results for replicate samples are calculated and compared to results obtained for reference products and untreated pine sapwood samples to evaluate the relative resistance of products to mold growth. 3. Apparatus 3.1 Wood working equipment capable of producing samples of wood products and pine sapwood samples of required dimensions, and for drilling appropriate size holes in the samples to accommodate an eyehook screw. 3.2 Latex or vinyl gloves for handling samples. 3.3 A balance capable of weighing to within 0.1 g. 3.4 Appropriate laboratory facilities for preparation and disposal of mold inoculum, including air handling systems, a laboratory blender, distilled water, flasks, pipettes, and a trigger sprayer. 3.5 A temperature controlled room to house the environment chamber capable of being maintained approximately 5 C cooler than the selected test temperature within the chamber so that there is sufficient heat loss from the cabinet that a minimum of 95% relative humidity is maintained at the test temperature, and to prevent excessive condensation occurring inside the environment chamber. The room should be maintained at negative pressure with respect to all other rooms in the building and should be separately vented to the outside. All persons entering the room should wear a NIOSH N95 mask 1. 1 This standard does not purport to address all safety concerns, if any, associated with its use. It is the responsibility of the user of the standard to establish appropriate safety and health practices and determine the applicability of regulatory limitation prior to use.

2 2 E24-06 Standard Method for Evaluating the Resistance of Wood Product Surfaces to Mold Growth A self-contained environment chamber containing water, soil, and racks for suspending test samples, with a pitched roof to prevent deposition of condensation onto samples. The environment chamber shall be temperature controlled to within ±1 C of the desired test temperature. The chamber shall be constructed of materials that do not support mold growth, and shall consist of a small cabinet, suitable for holding about 28 approximately 75 by 100-mm (3 by 4 in.) samples under test conditions. It can be constructed as follows (Fig 1): Figure 1: Components of Controlled Environment Mold Growth Chamber Tank, polypropylene or polyethylene, with an offset shoulder at the top rim is used as the chamber. 2 A pitched (90 degree angle) top with straight sides should be constructed out of acrylic plastic so moisture condensation will run down the sides and be re-circulated within the chamber instead of dripping onto the test panels. Gasket material may be required to achieve a seal where the top rests on the lip of the tank. An immersion heater 3 is installed in the bottom of the chamber by connections through the end wall. It is so placed that it is immersed when there are approximately 80 mm (3 in) of water in the bottom of the chamber. The heater is regulated by a solid-state electronic temperature controller 4 to maintain a temperature of 25.0 C ± 1.0 C in proximity to the samples, as measured by a thermocouple located amongst the samples. Other operating temperatures may be selected between 15 C and 32.5 C to simulate specific exposure situations. Generally, lower temperatures favor growth of staining fungi and higher temperatures favor growth of molds. To aid in even heat distribution, water within the tank is constantly stirred by an aquarium circulating pump 5 set at maximum flow. An acrylic tray approximately 25 mm (1 in.) smaller than the inside dimensions of the chamber and approximately 76 mm (3 in.) deep with a non-corrodible metal 6 mesh bottom shall be supported approximately 25 mm (1 in.) above the water level and centered in the chamber. One layer of fine mesh plastic or fiberglass screen should be placed over the metal mesh for holding soil. A small fan, 7 mounted perpendicular to and just above the surface of the soil bed in the tray to provide dispersion and circulation of the fungal spores to achieve continuous inoculation of the surfaces of the samples. A series of sample holding supports, suspended across the width of the chamber at a height and spacing that allows the use of samples approximately 75 by 100 mm (3 by 4 in.), situated vertically, with 75-mm (approximately 3 in.) clearance above the inoculated soil with a suitable method of fastening. Fasteners and supports shall be of materials that do not support mold growth to avoid contamination of samples or the chamber environment. Stainless steel eyehook screws may be attached to the samples for suspension from the sample holding supports. 3.7 A good quality greenhouse-grade potting soil, suitable for plant propagation, containing about 25% peat moss, with a ph of between 5.5 and 7.6, should be used. The moisture content of the soil should be at 100% of water holding capacity at the beginning of the test. Place the soil in the tray to a depth of about 8 cm (3 in.). Soil should not be compacted. 3.8 Proper relative humidity is indicated by the constant formation of condensate on the pitched-roof cover. A data logger for measuring temperature and relative humidity may be used in the test panel area to confirm test conditions. 4. Reagents and Materials 4.1 Tween 80 dispersion agent for preparation of mold inoculum. 4.2 Two-part epoxy for sealing unprotected edges of samples to simulate a mid-panel section, if appropriate. 5. Test Specimens 5.1 Wood Species 4 Ogden model ETR universal controller, or equivalent 2 Nalgene # (approximately 24 x 18 x 18 inches), or equivalent. 3 Mighty Watt immersion heater, part # mwej06a0119-n, or equivalent 5 Aqua-pump-1, variable flow, or equivalent 6 Stainless steel is well suited for this purpose 7 A 4-inch Muffin fan, model MU2A1, or equivalent

3 3 E24-06 Standard Method for Evaluating the Resistance of Wood Product Surfaces to Mold Growth 2006 Sapwood of a pine species, free of defects and fungal growth, or another softwood species equally susceptible to fungal growth, shall be used as an untreated negative control and as a substrate for testing of surface- applied treatments. The pine species used shall be reported. All test samples should be handled with latex or vinyl gloves to avoid soiling test surfaces. 5.2 Reference Products At least one reference wood treatment or in-process treated wood product shall be included in the test for comparative purposes. The product chosen should be commercially available and in current common use in the marketplace. Source material from which reference samples are prepared should be obtained from a wholesale or retail outlet. If obtained directly from a manufacturer, the material shall be from a production run. Unique labels should be applied to samples to identify replicates and source material from which the samples were cut. 5.3 Preparation of test pieces The material should be cut into a series of 75 x 100mm samples, 12.5mm thick or normal product thickness. Should cutting of a treated panel into samples compromise the integrity of a treatment or coating, a twopart epoxy sealant may be used to seal exposed edges. A minimum of two coats should be applied and allowed to thoroughly dry before entry into the environment chamber. 5.4 Replication A minimum of six replicates shall be used in each test, with at least one replicate included in each environment chamber if multiple chambers are used simultaneously for one test. Samples of each control, reference, and test product shall be retained unexposed for future comparison with exposed samples. 6. Treatment Procedure 6.1 Surface-Applied Treatments The test preservative supplied shall be accompanied by an analytical certificate. The material supplied shall be a representative sample of the product to be tested. Samples shall be stored and handled in accordance with any written requirements such as provided in a material safety data sheet. Apply the test preservative to the samples using the process specified by the manufacturer. Examples include immersion, spraying or flow coating. For products at an early stage of development it is desirable to test at least three retentions of the active ingredients ranging around the anticipated effective retention. Weigh all samples before and after treatment to determine uptake. Retentions should be expressed as micrograms per square centimeter of surface. Treat sufficient samples to provide the necessary replication plus additional samples to allow for elimination of samples with preservative retention deviating more than 15% from the mean. Substitute an appropriate alternative that falls within this range. Samples should be labeled to identify the preservative and retention. Dry treated samples in a well ventilated protected area. If samples are stacked for drying, use supporting rods of a material that does not react with the preservative and does not support growth of molds. 6.2 In-Process-Treated Wood Products Wood products should be prepared with care to ensure uniformity of substrate, treatment, or coating. Preservative retentions should be expressed as kilograms per cubic meter of wood product. If possible, reference or analytical samples should be obtained immediately adjacent to test sample locations on the same source material. Unique labels should be applied to samples to identify replicates and source material from which samples were cut. Should cutting of source material into samples compromise the integrity of a treatment or coating, a twopart epoxy sealant may be used to seal exposed edges. A minimum of two coats should be applied and allowed to thoroughly dry before entry into the environment chamber. 7. Preparation of Inoculum 7.1 Preparation of mold cultures Pure cultures of the following molds should be used to prepare inoculum for soil in the environment chamber and for application to sample surfaces: Aureobasidium pullulans (d. By.) Arnaud, ATCC 9348 Aspergillus niger v. Tiegh, ATCC 6275 Penicillium citrinum Thom, ATCC 9849 Alternaria tenuissima group (Kunze) Wiltshire Ftk 691B The ATCC numbers refer to strains maintained by the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108, USA ( The cultures are introduced onto 1.5% malt extract, 2% agar or potato dextrose agar (Difco or equivalent) 100 mm Petri plates and incubated until spores form at about C. At least three plates should be prepared for inoculation of the soil and an additional three plates should be prepared for inoculation of the sample surfaces two weeks later. Other mold species may be added to, or substituted for, the above if it is necessary to demonstrate resistance to specific molds. For certain mold species a Type II Biohazard safety cabinet may be required Inoculation of Soil in the Environment Chamber Place the soil in the tray in the chamber and add water to the tank chamber to the appropriate depth. Allow the cabinet to equilibrate at test temperature for at least 24 hours before inoculating the soil with the specified mold suspensions. Use 14 to 20 day old pure mold cultures to prepare mold suspensions using the following procedure. An inoculation suspension is prepared by adding approximately 10 ml of distilled water containing 2 drops of Tween 80 and rubbing the surface with a glass rod to dislodge spores. Decant the spore suspension and rinse the plate surface with an additional 10 ml of distilled water. Combine the spore suspension and rinsing from all the plates and filter through a coarse filtration medium like

4 4 E24-06 Standard Method for Evaluating the Resistance of Wood Product Surfaces to Mold Growth 2006 glass wool or cheese cloth. Make up to about one liter with distilled water. Distribute the inoculum evenly over the soil surface in the chamber. Allow two weeks of continuous operation for the mold to sporulate and equilibrate within the environment chamber before introducing test samples. Additional inoculum should be prepared to apply to sample surfaces immediately before placement of samples in the chamber. Inoculate all sample surfaces with a trigger or chromatography sprayer. Apply evenly and lightly to minimize surface wetting; do not apply to the point of runoff. Application should be done in an appropriate air exhaustion hood with samples arranged to allow for consistency of application. 8. Exposure in Environment Chambers Use gloves while handling samples. Samples shall be conditioned at ambient laboratory temperature and relative humidity before installed in the environment chamber. Hang the inoculated samples vertically with the bottom approximately 75mm. (3 in) above the surface of the inoculated soil and with sufficient spacing to allow free circulation of air. The samples are suspended with the long dimension vertical and parallel to each other so that the faces are perpendicular to the fan airflow. Sample spacing should be a minimum of 2 cm between faces. A minimum of 5 cm clearance should be allowed between the outermost samples and the sides of the chamber. Sample locations in the chamber should be randomized. If more than one chamber is utilized in a test, samples should be equally distributed between chambers, ensuring at least one replicate of each test group is included in each chamber. A sample location map should be recorded for each chamber in the event mold growth was to obscure a sample label. Viability of the mold growth in the cabinet can be checked by placing several malt agar or potato dextrose agar plates, 8 open and face up, at several locations on the sample supports. After 1 hour, cover plates and place in an incubator at 32.5 ± 1 C (86 ± 2 F) for 3 days. Mold growth should be medium-heavy to heavy and cover the complete surface of the agar plate. If an incubator is not available, leave the covered plates in the cabinet. Mold growth may be less extensive at cooler chamber temperatures. The chamber and soil are not sterile; therefore organisms other than those in the inoculum may be present and become established on test samples. The environment chamber is operated at 25.0 C (or desired temperature) for eight weeks. 9. Assessment of Mold Growth All persons evaluating samples should, as a minimum, wear a NIOSH N95 mask. Following 2, 4, 6 and 8 weeks exposure within the environment chamber, samples are removed, weighed (optional), and visually rated (unaided) for the extent and intensity of mold growth using the scale in Table 1. Observations of mold growth characteristics shall be recorded. It may be appropriate for some products to be assigned separate ratings for each face or for surfaces of sapwood and heartwood. Observations comparing conditions of unexposed and exposed samples shall be recorded. Table 1 Rating Description 0 No visible growth Mold covering up to 10% of surfaces providing growth is not so intense or colored as to 1 obscure the sample color over more than 5% of surfaces Mold covering between 10% and 30% of surfaces providing growth is not so intense or 2 colored as to obscure the sample color on more than 10% of surfaces Mold covering between 30% and 70% of surfaces providing growth is not so intense or 3 colored as to obscure the sample color on more than 30% of surfaces Mold on greater than 70% of surfaces providing growth is not so intense or colored as 4 to obscure the sample color over more than 70% of surfaces Mold on 100% of surfaces or with less than 100% coverage and with intense or colored 5 growth obscuring greater than 70% of the sample color If the cabinet is operating properly, untreated pine sapwood control samples should develop a minimum average mold growth rating of at least 3 within four weeks (when chamber is operated at 25 C). If this growth is not obtained, the cabinet conditions are not satisfactory, there is some interfering substance in the chamber, or inoculation was unsuccessful. The test shall be abandoned. Upon completion of the 8-week test, samples may be weighed to determine moisture uptake during exposure in the chamber. Samples may be oven-dried at 105 C to determine pre-exposure and 8-week exposure moisture contents. 10. Interpretation of Results Results should be reported giving the mean, standard deviation and range of the six (or more) replicates. Relative resistance to mold growth is determined from comparing average ratings of replicates of each panel product. Report the results at the end of the 8-week exposure giving the mean rating, standard deviation and range of the six (or more) replicate samples. Compare results to the performance of control and reference samples in the same test and any dose/efficacy effects determined. The test report shall include: 8 Prepared agar plates can be obtained form Difco Laboratories, Inc., Detroit. Mich ; Baltimore Biological Laboratories. Baltimore, Md , or from equivalent sources. The number of this AWPA Standard and the date of publication. Species of sapwood used as untreated negative control.

5 5 E24-06 Standard Method for Evaluating the Resistance of Wood Product Surfaces to Mold Growth 2006 Reference wood product used and pertinent information such as species, source, etc. The number of replicates. Operating temperature of the environment chamber. Source of soil. Species and source of mold cultures. Description of wood products tested, including manufacturing details, information on treatment chemicals and loading or coatings, and any pre-test conditioning or exposure (leaching, weathering, sterilization), if appropriate. Summary of observations of mold growth characteristics for each wood product tested. Summary of observations of unexposed and exposed sample conditions. Any deviations from the Standard and any special factors that may have influenced the results.