ASTM D4445-10(2019)

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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Historical
• Publication Date: 30-Jun-2019
• ICS: 79.040 - Wood, sawlogs and sawn timber
• Technical Committee: D07 - Wood
• Drafting Committee: D07.06 - Treatments for Wood Products

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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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 2019)
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 2. Referenced Documents
1.1 This (laboratory) test method is used for determining the 2.1 ASTM Standards:
D9 Terminology Relating to Wood and Wood-Based Prod-
minimum concentration of fungicide, or formulation of
fungicides, that is effective in preventing biodeterioration by ucts
D1165 Nomenclature of Commercial Hardwoods and Soft-
sapstain fungi and molds in selected species of wood under
optimum laboratory conditions. woods
D1193 Specification for Reagent Water
NOTE 1—From the results of this test, commercial treating solution
concentrations cannot be estimated without further field tests.
3. Terminology
1.2 The requirements for test materials and procedures are
3.1 Definitions—For definitions of terms used in this test
discussed in the following order:
method, refer to Terminologies D9 and D1165.
Section
Summary of Test Method 4
Apparatus 6 4. Summary of Test Method
Reagents 7
4.1 Unseasoned sapwood specimens are treated either by
Wood 8
Test Fungi 9 spraying with, or by immersing in, solutions or dispersions of
Culture Media 10
a fungicide formulation prepared at five or more concentration
Preparation of Inoculum 11
levels. The specimens are exposed to sapstain fungi and molds.
Preparation of Test Chambers 12
Treatment of Samples 13
Options for testing the toxicity of fungicides include testing
Inoculation and Incubation 14
against individual fungi or against several fungi by using a
Evaluation of the Test 15
mixed spore suspension for the inoculation of the specimens.
Report 16
4.2 The intensity of surface fungal growth is estimated after
1.3 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this incubation and the results used to determine the minimum
chemical treatment concentration giving zero growth (CGo).
standard.
1.4 This standard does not purport to address all of the
5. Significance and Use
safety concerns, if any, associated with its use. It is the
5.1 This test method is useful as a screening procedure for
responsibility of the user of this standard to establish appro-
selecting fungicides or formulations for more rigorous field
priate safety, health, and environmental practices and deter-
evaluation.
mine the applicability of regulatory limitations prior to use.
1.5 This international standard was developed in accor-
6. Apparatus
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
6.1 Incubation Room (or Incubation Cabinet), maintained at
Development of International Standards, Guides and Recom-
a temperature of 25 6 1°C, and relative humidity between 70
mendations issued by the World Trade Organization Technical
and 80 %.
Barriers to Trade (TBT) Committee.
6.2 Steam Sterilizer.
6.3 Containers:
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. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved July 1, 2019. Published August 2019. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 1984. Last previous edition approved in 2015 as D4445–10(2015). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/D4445-10R19. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4445 − 10 (2019)
6.3.1 Sterile Petri Dishes, with minimum size of 140 9.2.1 Sapstain Fungi:
(diameter) by 20 mm (height) with lid or, 9.2.1.1 Diplodia natalensis P. Evans (ATCC 34643).
6.3.2 Aluminum Pans, with minimum size of 240 by 100 by 9.2.1.2 Ceratocystis pilifera (Fr.) C. Moreau (ATCC
20 mm (height) with aluminum foil cover. 15457).
9.2.1.3 Aureobasidium pullulans (d By) Arnaud (ATCC
6.4 Spacers:
16624).
6.4.1 U-Shaped Glass Rod, with 3 mm diameter or,
9.2.2 Mold Fungi:
6.4.2 Polyethylene Mesh, cut to cover the bottom of the
9.2.2.1 Trichoderma pseudokoningii (Rifai) (ATCC 26801).
selected container(s).
9.2.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).
9.2.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521).
7. Reagents
9.3 General Consideration—In addition to the above fungi,
7.1 Purity of Water—Reference to water shall be understood
others that are known to cause discoloration on wood species
to mean sterile reagent water conforming to Type IV of
used in test include, for example, Cytospora sp. (Pine);
Specification D1193.
Phialophora sp.; Graphium sp.; Ceratocystis sp.; Alternaria
8. Wood
sp.; Penicillium sp.; Aspergillis sp.; Trichoderma sp.
8.1 General Properties—The wood species to be tested shall
10. Culture Media
be selected on the basis of their susceptibility to staining fungi
10.1 Agar Substrate—For both stock culture tube and petri
(pine or spruce species are preferred). Sapwood of the selected
dish cultures of the test fungi, use a nutrient medium: that is,
wood species, unseasoned (moisture content higher than
malt extract agar (MEA, 2 % malt extract plus 2 % agar),
40 %), free of knots, visible decay, sapstain, and mold, shall be
potato dextrose agar (PDA, 0.4 % potato starch, 2 % dextrose
used (Note 2). If the fungicide is to be used to protect
plus 2 % agar), or similar commercial mixtures of MEA or
hardwood, the inclusion of sapwood from a hardwood species
PDA prepared in accordance with manufacturer instructions.
is recommended.
PDA stimulates sporulation in some sapstain fungi (for
NOTE 2—If wood for the test is collected in a sawmill where logs are
example, Aureobasidium pullulans). Sterilize the medium at
stored in water, it is necessary to collect lumber from at least three
121°C, 0.1 MPa, for 20 min.
different logs since depletion of nutrients during water storage may
strongly affect the growth of molds and staining fungi. Ensure that the
11. Preparation for Inoculum
lumber collected in a sawmill has not been treated with a sapstain and
11.1 If the toxicity of a fungicide is being tested against
mold preventive, and if there is any doubt, at least 10 mm of surface wood
must be removed and discarded.
individual fungi, maintain aseptic conditions when preparing
the spore suspension; if the general effectiveness of a fungicide
8.2 Size of Specimens—Specimens shall be 7 by 20 mm in
is being tested using a mixed spore supension, aseptic condi-
cross section and 70 mm long.
tions are unnecessary. For laboratory experiments requiring a
8.3 Preparation of Specimens—Within two days of
relatively small volume (about 100 mL) of inoculum, prepara-
collecting, the samples shall be cut from the wood using a
tion using only the stock test tube cultures is an option. For
sharp saw blade. To prevent drying, the specimens shall be
larger volumes of inoculum, prepare from cultures grown on
stored in polyethylene bags. For storage longer than one day,
petri dishes.
but less than one year, tightly packed specimens shall be frozen
NOTE 3—Before using any stock test tube culture, reinoculate new tubes
(–20°C or lower) in polyethylene bags . For these longer
for future use.
storage cases, the contents of one bag shall be limited to as
11.2 For the preparation of a spore suspension, add 5 mL of
many specimens as are used for a single experiment.
sterile water to each culture tube or 10 mL to petri dishes, and
9. Test Fungi
rub the surface of the MEA or PDA culture with a blunt glass
rod to loosen the spores. After collecting the spores and
9.1 Hardwoods:
combining them with other similarly collected spores, if
9.1.1 Sapstain Fungi:
desired, adjust the water volume to that required. Although it is
9.1.1.1 Diplodia natalensis P. Evans (ATCC 34643).
a good practice to prepare fresh spore suspensions just before
9.1.1.2 Davidsoniella virescens (Davison) de Beer et al, a
use, their storage for up to one week with refrigeration is
form of C. coerulescens found on American hardwoods.
permissible.
9.1.1.3 Aureobasidium pullulans (d. By) Arnaud. (ATCC
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).
11.4 To evaluate a fungicide use at least six test fungi (three
9.1.2.3 Gliocladium roseum (Link) Bainier (ATCC 10521).
sapstain and three mold) individually, as well as one mixed
9.2 Softwoods:
spore suspension of selected fungi.
12. Preparation of Test Chambers
The following numbers refer to standard strains of test fungi maintained in the
12.1 To maintain high humidity in the petri dishes during
American Type Collection (ATCC), P.O. Box 1549,
...

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 (Reapproved 2015) D4445 − 10 (Reapproved 2019)
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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.5 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
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.
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 Nov. 1, 2015July 1, 2019. Published December 2015August 2019. Originally approved in 1984. Last previous edition approved in 20102015
as D4445 – 10.D4445–10(2015). DOI: 10.1520/D4445-10R15.10.1520/D4445-10R19.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. 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 (2019)
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.
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 CeratocystisDavidsoniella virescens (Davidson) C. Moreau (ATCC 11066)(Davison) de Beer et al, 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).
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 (2019)
9.2.1.3 Aureobasidium pullulans (d By) Arnaud (ATCC 16624).
9.2.2 Mold Fungi:
9.2.2.1 Trichoderma pseudokoningii (Rifai(Rifai) (ATCC 26801).
9.2.2.2 Cephaloascus fragrans Hanawa (ATCC 12091).
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
...