1、Designation: D 4445 03Standard Test Method forFungicides for Controlling Sapstain and Mold onUnseasoned Lumber (Laboratory Method)1This standard is issued under the fixed designation D 4445; the number immediately following the designation indicates the year oforiginal adoption or, in the case of re
2、vision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This (laboratory) method is used for determining theminimum concentration of fungicide, or formul
3、ation of fungi-cides, that is effective in preventing biodeterioration by sap-stain fungi and molds in selected species of wood underoptimum laboratory conditions.NOTE 1From the results of this test, commercial treating solutionconcentrations cannot be estimated without further field tests.1.2 The r
4、equirements for test materials and procedures arediscussed in the following order:SectionSummary of Method 3Apparatus 5Reagents 6Wood 7Test Fungi 8Culture Media 9Preparation of Inoculum 10Preparation of Test Chambers 11Treatment of Samples 12Inoculation and Incubation 13Evaluation of the Test 14Repo
5、rt 151.3 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. Referen
6、ced Documents2.1 ASTM Standards:D 1193 Specification for Reagent Water23. Summary of Method3.1 Unseasoned sapwood specimens are treated either byspraying with, or by immersing in, solutions or dispersions ofa fungicide formulation prepared at five or more concentrationlevels. The specimens are expos
7、ed to sapstain fungi and molds.The toxicity of fungicides may be tested against individualfungi, in which case sterilization of the samples is necessary, oragainst several fungi by using a mixed spore suspension for theinoculation of the specimens; in the latter case, sterilization isunnecessary.3.2
8、 The intensity of surface fungal growth is estimated afterincubation and the results used to determine the chemicaltreatment concentration giving zero growth (CGo).4. Significance and Use4.1 This method is useful as a screening procedure forselecting fungicides or formulations for more rigorous fiel
9、devaluation.5. Apparatus5.1 Incubation Room (or Incubation Cabinet), maintained ata temperature of 25 6 1C, and relative humidity between 70and 80 %.5.2 Steam Sterilizer.5.3 Containers:5.3.1 Petri Dishes, with minimum size of 100 (diameter) by20 mm (height) with lid or,5.3.2 Aluminum Pans, with mini
10、mum size of 240 by 100 by20 mm (height) with aluminum foil cover.6. Reagents6.1 Purity of WaterReference to water shall be understoodto mean sterile reagent water conforming to Type IV ofSpecification D 1193.7. Wood7.1 General PropertiesThe wood species to be testedshould be locally available commer
11、cial species selected on thebasis of their susceptibility to staining fungi (pine or sprucespecies are preferred). Sapwood of the selected wood species,unseasoned (moisture content higher than 40 %), free of knots,visible decay, sapstain and mold, shall be used (Note 2). If thefungicide is to be use
12、d to protect hardwood, the inclusion ofsapwood from a hardwood species is recommended.NOTE 2If wood for the test is collected in a sawmill where logs arestored in water, it is necessary to collect lumber from at least threedifferent logs since depletion of nutrients during water storage may1This met
13、hod is under the jursidiction of ASTM Committee D07 on Wood andis the direct responsibility of Subcommittee D07.06 on Treatments for WoodProducts.Current edition approved April 10, 2003. Published June 2003. Originallyapproved in 1984. Last previous edition approved in 1991 as D 4445 91(1996)e1.2Ann
14、ual Book of ASTM Standards, Vol 11.01.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.strongly affect the growth of molds and staining fungi. Ensure that thelumber collected in a sawmill has not been treated with a sapstain andmold p
15、reventive, and if there is any doubt, at least 10 mm of surface woodmust be removed and discarded.7.2 Size of SpecimensSpecimens should be 7 by 20 mm incross section and 70 mm long.7.3 Preparation of SpecimensWithin two days of collect-ing, the samples shall be cut from the wood using a sharp sawbla
16、de. To prevent drying, the specimens shall be stored inpolyethylene bags. For storage longer than one day, tightlypacked specimens may be kept frozen (20C or lower) inpolyethylene bags for up to one year. In this case, one bagshould contain as many specimens as are used for oneexperiment.8. Test Fun
17、gi38.1 Hardwoods:8.1.1 Sapstain Fungi:8.1.1.1 Diplodia natalensis P. Evans (ATCC 34643).8.1.1.2 Ceratocystis virescens (Davidson) C. Moreau(ATCC 11066) a form of C. coerulescens found on Americanhardwoods.8.1.1.3 Aureobasidium pullulans (d. By) Arnaud. (ATCC16624).8.1.2 Mold Fungi:8.1.2.1 Trichoderm
18、 pseudokoningii Rifai (ATCC 26801).8.1.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).8.1.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521).8.2 Softwoods:8.2.1 Sapstain Fungi:8.2.1.1 Diplodia natalensis P. Evans (ATCC 34643).8.2.1.2 Ceratocystis pilifera (Fr.) C. Moreau (ATCC15457).8.2.1.3 Aureobasi
19、dium pullulans (d By) Arnaud (ATCC16624).8.2.2 Mold Fungi:8.2.2.1 Trichoderma pseudokoningii (Rifai (ATCC 26801).8.2.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).8.2.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521).8.3 General ConsiderationIn addition to the above fungi,others that are known to c
20、ause discoloration on wood speciesused in test may be included, for example, Cytospora sp.(Pine); Phialophora sp.; Graphium sp.; Ceratocystis sp.; Al-ternaria sp.; Penicillium sp.; Aspergillis sp.; Trichoderma sp.9. Culture Media9.1 Malt Agar SubstrateFor both stock culture tube andpetri dish cultur
21、es of the test fungi, use a nutrient mediumconsisting of 2 % malt extract and 2 % agar. Sterilize themedium at 121C, 15 psi (0.1 MPa) for 20 min.10. Preparation for Inoculum10.1 If the toxicity of a fungicide is being tested againstindividual fungi, maintain aseptic conditions when preparingthe spor
22、e suspension; if the general effectiveness of a fungicideis being tested using a mixed spore supension, aseptic condi-tions are unnecessary. Most laboratory experiments require arelatively small volume (about 100 mL) of inoculum that maybe prepared using only the stock test tube cultures; preparelar
23、ger volumes of inoculum from cultures grown on petridishes.NOTE 3Before using any stock test tube culture, reinoculate newtubes for future use.10.2 For the preparation of a spore suspension, add 5 mL ofsterile water to each culture tube or 10 mL to petri dishes, andrub the surface of the malt agar c
24、ulture with a blunt glass rodto loosen the spores. After collecting the spores and combiningthem with other similarly collected spores, if desired, adjust thewater volume to that required. Although it is a good practice toprepare fresh spore suspensions just before use, they may bekept, even without
25、 refrigeration, for 2 to 3 weeks.10.3 For nonsporulating cultures, obtain a mycelial suspen-sion for use by aseptically scraping the surface mycelium offand blending it with sterile water.10.4 To evaluate a fungicide use at least six test fungi (threesapstain and three mold) individually, as well as
26、 one mixedspore suspension of selected fungi.11. Preparation of Test Chambers11.1 To maintain high humidity in the petri dishes duringthe test period, place eight to ten layers of absorbent paper onthe bottom of each dish. Wet the papers with water until freewater appears, and press out any air bubb
27、les trapped under andbetween the paper disks (thoroughly if the dishes are to besterilized). Place a U-shaped glass rod (3 mm in diameter) ontop of the papers and sterilize the petri dishes if required (Fig.1).11.2 Aluminum ContainersTo maintain high humidity inthe containers, treat as with the petr
28、i dishes. Instead of aU-shaped glass rod however, place two (2) straight rods (3 mmin diameter by 200 mm long) on top of the papers. Sterilize ifrequired.12. Treatment of Specimens12.1 SpecimensIf the wood samples were stored frozen,allow them to thaw in the polyethylene bags. Because of thevariatio
29、n in the susceptibility of wood to fungi, distribute anequal number of specimens from each log, into each treatmentper fungus. If specimens were taken from lumber where logidentity is not available, select the specimens randomly fortesting. Autoclave the specimens before treatment at 121C, 15psi (0.
30、1 MPa), for 20 min.12.2 Number of SpecimensUse a minimum of ten speci-mens per concentration of a fungicide for each fungus tested.Also, use a minimum of ten untreated control specimens foreach fungus tested.12.3 Preparation of Treating SolutionEvaluate each fun-gicide using at least five concentrat
31、ions. Select the lowestconcentration of a fungicide or formulation to be below theexpected effective strength and each of the following concen-trations shall be twice the previous concentration. Start thepreparation of the set of concentrations of each fungicide bypreparing the highest concentration
32、 in an amount equal to twice3The following numbers refer to standard strains of test fungi maintained in theAmerican Type Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852.D4445032the volume required for treatment of the samples. Then dilutehalf of this preparation with an equal volume of
33、 water to obtainthe next preparation. Therefore, a serial set of concentrations isprepared by continuing the dilutions in this way.12.4 Treating ProcedureCarry out the treatment in a600-mL beaker (Fig. 2). Place two unused test pieces edgewiseon the bottom of the beaker, and the specimens, four or f
34、ive ina layer, also on edge, crosswise on the previous layers untilthey reach the top, but not extending above the rim of thebeaker. Holding down the specimens with a finger bearingdown on a watch glass, pour the prepared solution into thebeaker. After 15 s, pour the solution out, still holding thes
35、pecimens down so that they cannot move. Similarly, treatuntreated control specimens with water. After the treatment,tightly cover the beaker with a piece of plastic sheet to preventdrying, and store overnight. This allows draining of excesssolution and time for the fungicide to be deposited or fixed
36、 inthe wood before inoculation.12.5 After overnight storage, place the samples into theprepared petri dishes or aluminum pans for inoculation.13. Inoculation and Incubation13.1 Inoculation of the SpecimensStir the spore suspen-sion frequently during inoculation. Perform inoculation using atransfer p
37、ipet fitted with a rubber bulb; streak about 0.25 mL ofspore suspension along the length of one flat side of eachspecimen in the culture vessels. Application may also beaccomplished by spraying. Allow a small amount of the sporesuspension to run down on at least one of the crosscut ends.13.2 Place t
38、he petri dishes in polyethylene bags to preventdrying and incubate at 25C in an incubator preferably in thedark. Incubation time is between 2 and 4 weeks. Rewet thepaper pads with sterile water as necessary during the incuba-tion period to maintain a “damp condition.”13.3 Incubate the aluminum pans
39、at 25C for a period ofbetween 2 and 4 weeks.14. Evaluation of the Test14.1 After 2 or 4 weeks, or both, estimate the growth offungi visually and score using a scale of 0 to 5, the 5 beingmaximum intensity (Table 1). Base the estimate on the inten-sity of growth and discoloration, and not only on the
40、 surfacearea covered by the fungi, since the latter may be correlatedonly to the distribution of the original inoculum and notnecessarily to the subsequent growth activity of the fungi.FIG. 1 Arrangement of Treated Wood Specimens Within the PetriDishes Before IncubationFIG. 2 Arrangement of Test Mat
41、erial for Treatment in a 600-mLBeakerTABLE 1 Fungicide Scoring After IncubationAFungicideConcentrationin TreatingSolutionC.f. T.p. MTotal Scoresfor Stain andMoldPercent ofStain andMold(Based onControl)A 0 (control) 5.0 4.7 5.0 14.7 0.011 2.2 4.3 5.0 11.5 780.022 1.5 3.3 4.7 9.5 670.045 0.9 1.6 2.0 4
42、.5 310.09 0.0 0.5 0.9 10.0 100.18 0.0 0.1 0.1 0.1 0NaTCP 0 (control) 5.0 4.3 5.0 14.3 0.12 5.0 3.0 5.0 13.0 910.24 5.0 1.5 5.0 11.5 800.48 3.8 0.2 2.2 6.2 430.96 2.5 0.5 1.5 4.5 311.9 0.1 0.0 0.0 0.1 0AScoring assessed after three weeks incubation, for two fungicides, “A” andsodium tetrachlorophenat
43、e (NaTCP) at five concentrations, using Cephaloascusfragrans (C.f.), Trichoderma pseudokoningii (T.p.) and a mixture (M) containing thespores of two Penicillium sp., Aspergillus niger and Ceratocystis pilifera. Eachscore is an average of eight samples.D444503314.2 Determine the effective concentrati
44、on, or concentrationfor zero growth (CGo), as follows:14.2.1 At each concentration, average the scores given foreach fungus or for the mixed fungi, or both.14.2.2 If the toxicity was tested with individual fungi orwith more than one mixture of fungi, sum the average scoresfor each concentration (as
45、shown under “Total” in Table 1).14.2.3 Express fungal growth for each concentration as apercentage of the fungal growth in the controls (for example, inTable 1 at a concentration of 0.011 % for fungicide “A”),percent of total 511.514.73 100 5 7814.2.4 Plot the “percentage of total(s)” against the lo
46、garithmof treating solution concentration and draw the best-fit, straightline to these points (Fig. 3).14.2.5 The concentration where the line crosses the axis oftreating solution concentration is the estimated CGo. Forexample, the line for Fungicide A crosses the X-axis atapproximately 0.88. The an
47、ti-log of 0.88 is 0.13, so theestimated CGo is 0.13 %.14.3 If no growth is observed on untreated controls, discardall results from the test and repeat the test.14.4 For the final evaluation, compare the results with theresults from a similar test using a commercial sapstain andmold preventive, the e
48、ffectiveness of which is well known(Table 1).15. Report15.1 Report the following information:15.1.1 Species of wood,15.1.2 Details of fungicide composition,15.1.3 Fungi used (culture numbers),15.1.4 Results and calculations presented in Table 1 and Fig.3, and15.1.5 Estimated chemical concentration f
49、or zero growth(CGo).16. Precision and Bias16.1 This test method is dependent upon the physiologicalaction of living organisms. Therefore, the results may not berepeatable or reproducible. While the relative efficacy betweenexperimental levels within each individual test group is obtain-able, repeatablility and reproducibility cannot be applied tomake any inference of relative performance between differenttest groups.17. Keywords17.1 fungicides; lumber; mold; sapstain; test methodASTM International takes no position respecting the validity of any patent rig
