ASTM D4445-10(2015)
(Test Method)Standard Test Method for Fungicides for Controlling Sapstain and Mold on Unseasoned Lumber (Laboratory Method)
Standard Test Method for Fungicides for Controlling Sapstain and Mold on Unseasoned Lumber (Laboratory Method)
SIGNIFICANCE AND USE
5.1 This test method is useful as a screening procedure for selecting fungicides or formulations for more rigorous field evaluation.
SCOPE
1.1 This (laboratory) test method is used for determining the minimum concentration of fungicide, or formulation of fungicides, that is effective in preventing biodeterioration by sapstain fungi and molds in selected species of wood under optimum laboratory conditions.
Note 1: From the results of this test, commercial treating solution concentrations cannot be estimated without further field tests.
1.2 The requirements for test materials and procedures are discussed in the following order:
Section
Summary of Test Method
4
Apparatus
6
Reagents
7
Wood
8
Test Fungi
9
Culture Media
10
Preparation of Inoculum
11
Preparation of Test Chambers
12
Treatment of Samples
13
Inoculation and Incubation
14
Evaluation of the Test
15
Report
16
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
General Information
Relations
Buy Standard
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D4445 − 10 (Reapproved 2015)
Standard Test Method for
Fungicides for Controlling Sapstain and Mold on
Unseasoned Lumber (Laboratory Method)
This standard is issued under the fixed designation D4445; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope D9Terminology Relating to Wood and Wood-Based Prod-
ucts
1.1 This(laboratory)testmethodisusedfordeterminingthe
D1165Nomenclature of Commercial Hardwoods and Soft-
minimum concentration of fungicide, or formulation of
woods
fungicides, that is effective in preventing biodeterioration by
D1193Specification for Reagent Water
sapstain fungi and molds in selected species of wood under
optimum laboratory conditions.
3. Terminology
NOTE 1—From the results of this test, commercial treating solution
3.1 Definitions—For definitions of terms used in this test
concentrations cannot be estimated without further field tests.
method, refer to Terminologies D9 and D1165.
1.2 The requirements for test materials and procedures are
discussed in the following order:
4. Summary of Test Method
Section
4.1 Unseasoned sapwood specimens are treated either by
Summary of Test Method 4
spraying with, or by immersing in, solutions or dispersions of
Apparatus 6
Reagents 7 a fungicide formulation prepared at five or more concentration
Wood 8
levels.Thespecimensareexposedtosapstainfungiandmolds.
Test Fungi 9
Options for testing the toxicity of fungicides include testing
Culture Media 10
Preparation of Inoculum 11 against individual fungi or against several fungi by using a
Preparation of Test Chambers 12
mixed spore suspension for the inoculation of the specimens.
Treatment of Samples 13
Inoculation and Incubation 14
4.2 Theintensityofsurfacefungalgrowthisestimatedafter
Evaluation of the Test 15
incubation and the results used to determine the minimum
Report 16
chemical treatment concentration giving zero growth (CGo).
1.3 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
5. Significance and Use
standard.
5.1 This test method is useful as a screening procedure for
1.4 This standard does not purport to address all of the
selecting fungicides or formulations for more rigorous field
safety concerns, if any, associated with its use. It is the
evaluation.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
6. Apparatus
bility of regulatory limitations prior to use.
6.1 Incubation Room (or Incubation Cabinet),maintainedat
a temperature of 25 6 1°C, and relative humidity between 70
2. Referenced Documents
and 80%.
2.1 ASTM Standards:
6.2 Steam Sterilizer.
6.3 Containers:
6.3.1 Sterile Petri Dishes, with minimum size of 140
This test method is under the jurisdiction ofASTM Committee D07 on Wood
(diameter) by 20 mm (height) with lid or,
and is the direct responsibility of Subcommittee D07.06 on Treatments for Wood
Products.
6.3.2 Aluminum Pans,withminimumsizeof240by100by
Current edition approved Nov. 1, 2015. Published December 2015. Originally
20 mm (height) with aluminum foil cover.
approved in 1984. Last previous edition approved in 2010 as D4445–10. DOI:
10.1520/D4445-10R15.
6.4 Spacers:
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
6.4.1 U-Shaped Glass Rod, with 3 mm diameter or,
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
6.4.2 Polyethylene Mesh, cut to cover the bottom of the
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. selected container(s).
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4445 − 10 (2015)
7. Reagents 9.2.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).
9.2.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521).
7.1 Purity of Water—Referencetowatershallbeunderstood
to mean sterile reagent water conforming to Type IV of 9.3 General Consideration—In addition to the above fungi,
Specification D1193. others that are known to cause discoloration on wood species
used in test include, for example, Cytospora sp. (Pine);
8. Wood
Phialophora sp.; Graphium sp.; Ceratocystis sp.; Alternaria
sp.; Penicillium sp.; Aspergillis sp.; Trichoderma sp.
8.1 GeneralProperties—Thewoodspeciestobetestedshall
be selected on the basis of their susceptibility to staining fungi
10. Culture Media
(pine or spruce species are preferred). Sapwood of the selected
10.1 Agar Substrate—For both stock culture tube and petri
wood species, unseasoned (moisture content higher than
dish cultures of the test fungi, use a nutrient medium: that is,
40%),freeofknots,visibledecay,sapstain,andmold,shallbe
malt extract agar (MEA, 2% malt extract plus 2% agar),
used (Note 2). If the fungicide is to be used to protect
potato dextrose agar (PDA, 0.4% potato starch, 2% dextrose
hardwood, the inclusion of sapwood from a hardwood species
plus 2% agar), or similar commercial mixtures of MEA or
is recommended.
PDA prepared in accordance with manufacturer instructions.
NOTE 2—If wood for the test is collected in a sawmill where logs are
PDA stimulates sporulation in some sapstain fungi (for
stored in water, it is necessary to collect lumber from at least three
example, Aureobasidium pullulans). Sterilize the medium at
different logs since depletion of nutrients during water storage may
strongly affect the growth of molds and staining fungi. Ensure that the 121°C, 0.1 MPa, for 20 min.
lumber collected in a sawmill has not been treated with a sapstain and
moldpreventive,andifthereisanydoubt,atleast10mmofsurfacewood 11. Preparation for Inoculum
must be removed and discarded.
11.1 If the toxicity of a fungicide is being tested against
8.2 Size of Specimens—Specimens shall be 7 by 20 mm in
individual fungi, maintain aseptic conditions when preparing
cross section and 70 mm long.
thesporesuspension;ifthegeneraleffectivenessofafungicide
is being tested using a mixed spore supension, aseptic condi-
8.3 Preparation of Specimens—Within two days of
tions are unnecessary. For laboratory experiments requiring a
collecting, the samples shall be cut from the wood using a
relatively small volume (about 100 mL) of inoculum, prepara-
sharp saw blade. To prevent drying, the specimens shall be
tion using only the stock test tube cultures is an option. For
stored in polyethylene bags. For storage longer than one day,
larger volumes of inoculum, prepare from cultures grown on
butlessthanoneyear,tightlypackedspecimensshallbefrozen
petri dishes.
(–20°C or lower) in polyethylene bags . For these longer
storage cases, the contents of one bag shall be limited to as
NOTE3—Beforeusinganystocktesttubeculture,reinoculatenewtubes
many specimens as are used for a single experiment.
for future use.
11.2 For the preparation of a spore suspension, add 5 mLof
9. Test Fungi
sterile water to each culture tube or 10 mLto petri dishes, and
9.1 Hardwoods:
rub the surface of the MEAor PDAculture with a blunt glass
9.1.1 Sapstain Fungi:
rod to loosen the spores. After collecting the spores and
9.1.1.1 Diplodia natalensis P. Evans (ATCC 34643).
combining them with other similarly collected spores, if
9.1.1.2 Ceratocystis virescens (Davidson) C. Moreau
desired,adjustthewatervolumetothatrequired.Althoughitis
(ATCC 11066) a form of C. coerulescens found on American
a good practice to prepare fresh spore suspensions just before
hardwoods.
use, their storage for up to one week with refrigeration is
9.1.1.3 Aureobasidium pullulans (d. By) Arnaud. (ATCC
permissible.
16624).
11.3 For nonsporulating cultures, obtain a mycelial suspen-
9.1.2 Mold Fungi:
sion for use by aseptically scraping the surface mycelium off
9.1.2.1 Trichoderma pseudokoningii Rifai (ATCC 26801).
and blending it with sterile water.
9.1.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).
9.1.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521). 11.4 Toevaluateafungicideuseatleastsixtestfungi(three
sapstain and three mold) individually, as well as one mixed
9.2 Softwoods:
spore suspension of selected fungi.
9.2.1 Sapstain Fungi:
9.2.1.1 Diplodia natalensis P. Evans (ATCC 34643).
12. Preparation of Test Chambers
9.2.1.2 Ceratocystis pilifera (Fr.) C. Moreau (ATCC
12.1 To maintain high humidity in the petri dishes during
15457).
the test period, place eight to ten layers of absorbent paper on
9.2.1.3 Aureobasidium pullulans (d By) Arnaud (ATCC
the bottom of each dish. Wet the papers with water until free
16624).
waterappears,andpressoutanyairbubblestrappedunderand
9.2.2 Mold Fungi:
between the paper disks. Place a U-shaped glass rod (3 mm in
9.2.2.1 Trichoderma pseudokoningii (Rifai (ATCC 26801).
diameter) (Fig. 1) or polyethylene mesh spacer (Fig.
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D4445 − 10 D4445 − 10 (Reapproved 2015)
Standard Test Method for
Fungicides for Controlling Sapstain and Mold on
Unseasoned Lumber (Laboratory Method)
This standard is issued under the fixed designation D4445; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This (laboratory) test method is used for determining the minimum concentration of fungicide, or formulation of fungicides,
that is effective in preventing biodeterioration by sapstain fungi and molds in selected species of wood under optimum laboratory
conditions.
NOTE 1—From the results of this test, commercial treating solution concentrations cannot be estimated without further field tests.
1.2 The requirements for test materials and procedures are discussed in the following order:
Section
Summary of Test Method 4
Apparatus 6
Reagents 7
Wood 8
Test Fungi 9
Culture Media 10
Preparation of Inoculum 11
Preparation of Test Chambers 12
Treatment of Samples 13
Inoculation and Incubation 14
Evaluation of the Test 15
Report 16
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2.1 ASTM Standards:
D9 Terminology Relating to Wood and Wood-Based Products
D1165 Nomenclature of Commercial Hardwoods and Softwoods
D1193 Specification for Reagent Water
3. Terminology
3.1 Definitions—For definitions of terms used in this test method, refer to Terminologies D9 and D1165.
4. Summary of Test Method
4.1 Unseasoned sapwood specimens are treated either by spraying with, or by immersing in, solutions or dispersions of a
fungicide formulation prepared at five or more concentration levels. The specimens are exposed to sapstain fungi and molds.
Options for testing the toxicity of fungicides include testing against individual fungi or against several fungi by using a mixed spore
suspension for the inoculation of the specimens.
This test method is under the jurisdiction of ASTM Committee D07 on Wood and is the direct responsibility of Subcommittee D07.06 on Treatments for Wood Products.
Current edition approved March 1, 2010Nov. 1, 2015. Published March 2010December 2015. Originally approved in 1984. Last previous edition approved in 20092010
as D4445 – 09a.D4445 – 10. DOI: 10.1520/D4445-10.10.1520/D4445-10R15.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4445 − 10 (2015)
4.2 The intensity of surface fungal growth is estimated after incubation and the results used to determine the minimum chemical
treatment concentration giving zero growth (CGo).
5. Significance and Use
5.1 This test method is useful as a screening procedure for selecting fungicides or formulations for more rigorous field
evaluation.
6. Apparatus
6.1 Incubation Room (or Incubation Cabinet), maintained at a temperature of 25 6 1°C, and relative humidity between 70 and
80 %.
6.2 Steam Sterilizer.
6.3 Containers:
6.3.1 Sterile Petri Dishes, with minimum size of 140 (diameter) by 20 mm (height) with lid or,
6.3.2 Aluminum Pans, with minimum size of 240 by 100 by 20 mm (height) with aluminum foil cover.
6.4 Spacers:
6.4.1 U-Shaped Glass Rod, with 3 mm diameter or,
6.4.2 Polyethylene Mesh, cut to cover the bottom of the selected container(s).
7. Reagents
7.1 Purity of Water—Reference to water shall be understood to mean sterile reagent water conforming to Type IV of
Specification D1193.
8. Wood
8.1 General Properties—The wood species to be tested shall be selected on the basis of their susceptibility to staining fungi
(pine or spruce species 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 the fungicide is to be used to protect hardwood, the inclusion
of sapwood from a hardwood species is recommended.
NOTE 2—If wood for the test is collected in a sawmill where logs are stored in water, it is necessary to collect lumber from at least three different logs
since depletion of nutrients during water storage may strongly affect the growth of molds and staining fungi. Ensure that the lumber collected in a sawmill
has not been treated with a sapstain and mold preventive, and if there is any doubt, at least 10 mm of surface wood must be removed and discarded.
8.2 Size of Specimens—Specimens shall be 7 by 20 mm in cross section and 70 mm long.
8.3 Preparation of Specimens—Within two days of collecting, the samples shall be cut from the wood using a sharp saw blade.
To prevent drying, the specimens shall be stored in polyethylene bags. For storage longer than one day, but less than one year,
tightly packed specimens shall be frozen (–20°C or lower) in polyethylene bags . For these longer storage cases, the contents of
one bag shall be limited to as many specimens as are used for a single experiment.
9. Test Fungi
9.1 Hardwoods:
9.1.1 Sapstain Fungi:
9.1.1.1 Diplodia natalensis P. Evans (ATCC 34643).
9.1.1.2 Ceratocystis virescens (Davidson) C. Moreau (ATCC 11066) a form of C. coerulescens found on American hardwoods.
9.1.1.3 Aureobasidium pullulans (d. By) Arnaud. (ATCC 16624).
9.1.2 Mold Fungi:
9.1.2.1 Trichoderma pseudokoningii Rifai (ATCC 26801).
9.1.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).
9.1.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521).
9.2 Softwoods:
9.2.1 Sapstain Fungi:
9.2.1.1 Diplodia natalensis P. Evans (ATCC 34643).
9.2.1.2 Ceratocystis pilifera (Fr.) C. Moreau (ATCC 15457).
9.2.1.3 Aureobasidium pullulans (d By) Arnaud (ATCC 16624).
9.2.2 Mold Fungi:
9.2.2.1 Trichoderma pseudokoningii (Rifai (ATCC 26801).
9.2.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).
The following numbers refer to standard strains of test fungi maintained in the American Type Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, www.atcc.org.
D4445 − 10 (2015)
9.2.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521).
9.3 General Consideration—In addition to the above fungi, others that are known to cause discoloration on wood species used
in test include, for example, Cytospora sp. (Pine); Phialophora sp.; Graphium sp.; Ceratocystis sp.; Alternaria sp.; Penicillium sp.;
Aspergillis sp.; Trichoderma sp.
10. Culture Media
10.1 Agar Substrate—For both stock culture tube and petri dish cultures of the test fungi, use a nutrient medium: that is, malt
extract agar (MEA, 2 % malt extract plus 2 % agar), potato dextrose agar (PDA, 0.4 % potato starch, 2 % dextrose plus 2 % agar),
or similar commercial mixtures of MEA or PDA prepared in accordance with manufacturer instructions. PDA stimulates
sporulation in some sapstain fungi (for example, Aureobasidium pullulans). Sterilize the medium at 121°C, 0.1 MPa, for 20 min.
11. Preparation for Inoculum
11.1 If the toxicity of a fungicide is being tested against individual fungi, maintain aseptic conditions when preparing the spore
suspension; if the general effectiveness of a fungicide is being tested using a mixed spore supension, aseptic conditions are
unnecessary. For laboratory experiments requiring a relatively small volume (about 100 mL) of inoculum, preparation using only
the stock test tube cultures is an option. For larger volumes of inoculum, prepare from cultures grown on petri dishes.
NOTE 3—Before using any stock test tube culture, reinoculate new tubes for future use.
11.2 For the preparation of a spore suspension, add 5 mL of sterile water to each culture tube or 10 mL to petri dishes, and rub
the surface of the MEA or PDA culture with a blunt glass rod to loosen the spores. After collecting the spores and combining them
with other similarly collected spores, if desired, adjust the water volume to that required. Although it is a good practice to prepare
fresh spore suspensions just before use, their storage for up to one week with refrigeration is permissible.
11.3 For nonsporulating cultures, obtain a mycelial suspension for use by aseptically scraping the surface mycelium off and
blending it with sterile water.
11.4 To evaluate a fungicide use at least six test fungi (three sapstain and three mold) individually, as well as one mixed spore
suspension of selected fungi.
12. Preparatio
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.