Designation: G 160 03

Standard Practice for

Evaluating Microbial Susceptibility of Nonmetallic Materials By Laboratory Soil Burial1

1. Scope 1.1 This practice is limited to the method of conducting an evaluation of a nonmetallic materials microbiological susceptibility when in contact with the natural environment of the soil. This practice is intended for use on solid material test specimens that are no larger than approximately 2 cm (34 in.) thick and 100 cm2 (20 in.2) or on lm forming materials such as coatings which may be tested in the form of lms at least 50 by 50 mm (2 by 2 in.) in size. This practice may be applied to articles that do not spend the majority of their service life in soil. 1.2 A wide variety of properties may be affected by microbial attack depending on material or item characteristics. Standard methods (where available) should be used for each different property to be evaluated. This practice does not attempt to enumerate all of the possible properties of interest nor specify the most appropriate test for those properties. Test methods must, however, be appropriate to the material being tested. 1.3 It is recommended that this practice be combined with appropriate environmental exposures (for example, sunlight simulating weathering devices, the hydrolytic effects of extended aqueous contact, or extraneous nutrients) or fabrication into articles (for example, adhesive bonding of seams) which may promote microbiological susceptibility during the service life of the material. 1.4 The values given in parentheses are provided for information purposes only. 2. Signicance and Use 2.1 These results may be used to compare the susceptibility of materials when exposed to this test procedure.
1 This practice is under the jurisdiction of Committee G3 on Weathering and Durability and is the direct responsibility of Subcommittee G03.04 on Biological Deterioration. Current edition approved Dec. 10, 2003. Published January 2004. Originally approved in 1998. Last previous edition approved in 1998 as G 16098.

2.2 Microbiological susceptibility may be reected by a number of changes including staining, weight loss, or reduction in tensile or exural strength. 2.3 This practice may be considered an inoculation with a mixed culture of fungi and bacteria. 3. Soil 3.1 Composition Soil shall be composed of equal parts of fertile topsoil (soil with a high clay content should not be used), well-rotted and shredded horse manure, and coarse sand (10 to 40 mesh). 3.2 MixingThe soil composition of 3.1 should be prepared by simple mixing and sifting through 14-in. mesh screen. 3.3 AgingThe mixture is aged for three months and resifted twice at four-week intervals during the three months. After three months, a viability control of untreated cotton cloth, 400 to 475 g/m2 (12 to 14 oz/yd2), buried in the soil shall have a tensile strength loss of at least 50 % after ve days.
NOTE 1The soil mixture may be used for sequential tests as long as the cotton cloth control degrades within the specied time period.

3.4 pHThe soil shall have a pH between 6.5 to 7.5, checked periodically, and maintained by the addition of ground limestone to raise the pH or owers of sulfur to lower the pH. The soil pH may be taken by dispersing 1 weight part soil in 20 parts of water, shaking or stirring, then allowing the mix to settle for 1 h. The pH is measured with indicator paper, electrodes, or by titration. 3.5 MoistureThe soil shall be maintained at between 20 and 30 % moisture, based on the dry weight of the soil. (The percent moisture is calculated by weighing approximately 50 mL of a representative portion and taking the portion to constant weight by placing the soil in an oven at a temperature of 101 to 106C.) Water lost during use as a result of evaporation shall be replaced without deforming the soil bed. If the surrounding atmosphere is maintained at 85 to 95 % relative humidity, this loss is negligible, however, the moisture level should be periodically measured.

Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.

G 160 03
4. Apparatus 4.1 Soil Container The container shall be any material of suitable mechanical strength and chemical/microbial resistance and, if porous, shall be lined with impermeable material. It shall be of any size that is convenient to handle and having a depth of at least 12.7 cm (5 in.). 4.2 External EnvironmentAn apparatus capable of maintaining a temperature of 30 6 2C (86 6 3.6F) and a relative humidity to 85 to 95 %, into which the assembled container is inserted, is necessary. An incubator or controlled tropical chamber is adequate. 5. Test Specimens 5.1 Completely fabricated parts or sections cut from fabricated parts may be used as test specimens. The simplest specimen may be a 50- by 50-mm (2- by 2-in.) piece, a 50-mm (2-in.) diameter piece, or a piece (rod or tubing) at least 76 mm (3 in.) long cut from the material to be tested. 5.2 Film-forming materials such as coatings may be tested in the form of lms at least 50 by 50 mm (2 by 2 in.) in size. Such lms may be prepared by casting on glass and stripping after cure or by impregnating (completely covering) lter paper or ignited glass fabric. 5.3 For visual evaluation, a minimum of three test specimens shall be used. 5.4 In devising a test program intended to reveal quantitative changes occurring during and after exposure, an adequate number of specimens should be evaluated to establish a valid value for the original property. For example, if ve replicate test specimens are required to establish a tensile strength of a lm material, at least that number of test specimens shall be removed and tested for each exposure period. It is to be expected that values of physical or mechanical properties at various stages of fungal attack will be variable. The ASTM Manual 72 may be used as a guide. 6. Viability 6.1 The viability must be recommended as in 3.3 concurrent with the test specimen and exposed consistent with the test specimen duration and number of test specimens. 7. Replicates 7.1 A minimum of four replicates are recommended. 8. Duration 8.1 The exposure period for soil burial, unless otherwise specied, shall be for a minimum of 60 days.
NOTE 2The test specimens cannot be removed for the soil bed, once they have been buried, until the exposure period has been completed. Disturbing the soil bed in such a manner as removing the test specimens may affect the growth of soil microbes and thus cause inconsistent results. A separate set of specimens must be used for each exposure interval (for example, a set of replicates for 30, 60, and 90 days as needed).

9. Calculation and Interpretation of Results 9.1 VisualAt the end of the exposure period, the specimens shall be removed from the soil bed and conditioned according to methods appropriate to the material being tested. For visual evaluation, the material is rinsed under a stream of tap water while gently rubbing between ngers to remove soil and air-drying at 20C (68F) for minimum amount of time. Alternatively, the material may be gently vacuumed or very gently air-brushed. 9.2 Microbial staining shall be evaluated as follows:
Observed Growth or Stain None Trace (less than 10 % coverage) Light (10 to 30 % coverage) Moderate (30 to 60 % coverage) Heavy (60 % to complete coverage) Rating 0 1 2 3 4

9.3 Property Changes Physical and mechanical changes such as tensile strength, exibility, weight loss, or other tests, may be performed as described in appropriate ASTM or other test methods. Tests shall be conducted on unexposed and exposed specimens for the purpose of comparison in determining the extent of microbial degradation of the test material. 9.4 Calculate the change in property for each replicate specimen using one of the following equations:
Ce, i 5X e, i 2 Xo Ce, i 5 Xe, i 2 Xf (1) (2)

where: Xe, i = measured property of each exposed specimen, X o = mean of property from initial measurements on unexposed specimens, and Xf = mean of property from measurements on le specimens. 9.5 Use the following equation to determine the mean change in property:
C5

( Ce, i i51
n

(3)

where: n = number or exposed specimens. 9.6 Use the following equation to determine the standard deviation of the change in property:

Sc 5

n
~Ci 2C ! n21
2

(4)

10. Report 10.1 Report the following information: age of bed, specimen size, number of replicates, performance of viability control, visual staining in accordance with 9.1 and 9.2 or property change in accordance with 9.3, duration of burial, and any specic measurements requested. 10.2 Satisfactory or unsatisfactory performance of a material is dependent on the applicable standard for that material or methods agreed upon between the investigators.
2

2 Manual on Presentation of Data and Control Chart Analysis, 6th ed., Manual 7, American Society for Testing and Materials, 1990.

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11. Precision and Bias 11.1 A precision and bias statement cannot be made at this time. 12. Keywords 12.1 biodegradeable; biological deterioration; defacement; disgurement; fungal resistance; fungi; laboratory soil culture;
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microbial susceptibility; microbiological deterioration; mildew; mixed microbial innoculum; mold growth; soil environment