ASTM C1904-22

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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: C1904 − 20 C1904 − 22
Standard Test Methods for
Determination of the Effects of Biogenic Acidification on
Concrete Antimicrobial Additives and/or Concrete Products
This standard is issued under the fixed designation C1904; 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 standard presents test methods for the determination of the effects of biogenic acidification on concrete products and/or
efficacy of antimicrobial products to resist microbially-induced corrosion (MIC) of concrete. In these tests, the biogenic
acidification is achieved by sulfur-oxidizing bacteria (SOB) that can convert elemental sulfur or thiosulfate to sulfuric acid without
the use of H S gas.
1.2 This standard is referenced in the guideline document for MIC of concrete products. Guide C1894 provides guidance for
microbially-induced corrosion of concrete products and an overview of where this test, and its options, can and should be used.
This document is not intended to be a guideline document for MIC of concrete products.
1.3 This standard does not cover controlled breeding chamber tests, in which H S gas is produced by bacterial activity and
acidification is the result of the conversion of this H S gas to sulfuric acid.
1.4 This standard does not cover chemical acid immersion tests, in which acidification is achieved by chemical sulfuric acid
addition, not by bacterial activity. Testing protocols for chemical acid immersion are described in Test Methods C267 and C1898.
1.5 This standard does not cover tests that assess field exposure conditions or sewage pipe, concrete tank, or concrete riser network
design.
1.6 This standard does not cover live trial tests where concrete coupons or other specimens are monitored in sewers.
1.7 The tests described in this standard should not be performed on concrete samples that have already been exposed to MIC
conditions.
1.8 This standard does not cover concrete deterioration due to chemical sulfate attack, which is caused by the reaction of sulfate
compounds that exist in wastewater with the hydration products of cement. Test methods for assessing sulfate attack are provided
by Test Methods C452 and C1012/C1012M.
1.9 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
These test methods are under the jurisdiction of ASTM Committee C13 on Concrete Pipe and is the direct responsibility of Subcommittee C13.03 on Determining the
Effects of Biogenic Sulfuric Acid on Concrete Pipe and Structures.
Current edition approved Oct. 1, 2020Sept. 1, 2022. Published October 2020October 2022. Originally approved in 1920. Last previous edition approved in 2020 as
C1904 – 20. DOI: 10.1520/C1904_C1904M-2010.1520/C1904-22.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
C1904 − 22
1.10 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.
1.11 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:
C125 Terminology Relating to Concrete and Concrete Aggregates
C150/C150M Specification for Portland Cement
C192/C192M Practice for Making and Curing Concrete Test Specimens in the Laboratory
C260/C260M Specification for Air-Entraining Admixtures for Concrete
C267 Test Methods for Chemical Resistance of Mortars, Grouts, and Monolithic Surfacings and Polymer Concretes
C452 Test Method for Potential Expansion of Portland-Cement Mortars Exposed to Sulfate
C494/C494M Specification for Chemical Admixtures for Concrete
C595/C595M Specification for Blended Hydraulic Cements
C618 Specification for Coal Fly Ash and Raw or Calcined Natural Pozzolan for Use in Concrete
C822 Terminology Relating to Concrete Pipe and Related Products
C989/C989M Specification for Slag Cement for Use in Concrete and Mortars
C1012/C1012M Test Method for Length Change of Hydraulic-Cement Mortars Exposed to a Sulfate Solution
C1017/C1017M Specification for Chemical Admixtures for Use in Producing Flowing Concrete (Withdrawn 2022)
C1157/C1157M Performance Specification for Hydraulic Cement
C1240 Specification for Silica Fume Used in Cementitious Mixtures
C1600/C1600M Specification for Rapid Hardening Hydraulic Cement
C1768/C1768M Practice for Accelerated Curing of Concrete Cylinders
C1894 Guide for Microbially Induced Corrosion of Concrete Products
C1898 Test Methods for Determining the Chemical Resistance of Concrete Products to Acid Attack
D1193 Specification for Reagent Water
2.2 Other Standards:
AASHTO TP 119-20 Standard Method of Test for Electrical Resistivity of a Concrete Cylinder Tested in a Uniaxial Resistance
Test
ATCC (American Type Culture Collection) Bacterial Culture Guide
ATCC Microbial Media Formulations
EPA 375.4 Sulfate (Turbidimetric)
ISO 20391-1 Biotechnology—Cell counting—Part 1: General guidance on cell counting methods
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this practice, refer to Terminology standards C125 and C822, and Guide C1894.
4. Significance and Use
4.1 As described in Guide C1894, the MIC of concrete is considered to be a three-stage process with the reduction in pH (Stage
I) (for example, 12.5 > pH > 9-10), the establishment of biofilms which further lowers the pH (Stage II) (for example, 9-10 > pH
> 4-6) and eventual deterioration due to biogenic acid exposure (Stage III) (for example, < ~4 pH). This standard provides standard
test methods to assess the effects of different stages of MIC on concrete products and efficacy of antimicrobial products used in
or on concrete.
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.
The last approved version of this historical standard is referenced on www.astm.org.
Available from American Association of State Highway and Transportation Officials (AASHTO), 444 N. Capitol St., NW, Suite 249, Washington, DC 20001,
http://www.transportation.org.
Available from American Type Culture Collection (ATCC) 10801 University Boulevard Manassas, VA 20110, http://www.atcc.org
Available from U.S. Government Printing Office, Superintendent of Documents, 732 N. Capitol St., NW, Washington, DC 20401-0001, http://www.access.gpo.gov.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
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4.2 The tests are performed in simulated exposure solutions containing well-controlled bacterial strains that are grown in the
laboratory. These tests do not require an environmental chamber and are intended to be performed as benchtop tests in biosafety
level 1 laboratory conditions. These tests are suitable for simulation of the Stage II and III of MIC because the pH range of the
solution can be controlled within the ranges of each stage.
4.3 This standard provides three test methods.
4.3.1 Test Method A is suitable for assessing the efficacy of antimicrobial admixtures in delaying or preventing biogenic
acidification in a nutrient-rich simulated wastewater exposure solution.
4.3.2 Test Method B is suitable for assessing the effectiveness of antimicrobial admixtures in a prescribed cementitious system
(Option B1) or assessing the performance of different cementitious systems (Option B2) in delaying or preventing microbially-
induced corrosion of concrete in the Stage II of MIC.
4.3.3 Test Method C is suitable for assessing the suitability of cementitious systems in delaying or preventing microbially-induced
corrosion of concrete in the Stage III of MIC.
4.4 The results obtained by these test methods should serve as information to be used with Guide C1894 in, but not as the sole
basis for, selection of a biologically-resistant material for a particular application. No attempt has been made to incorporate into
these test methods all the various factors that may affect the performance of a material when subjected to actual service.
5. Apparatus
5.1 Analytical Balance, accurate to at least 60.0001 g.
5.2 Controlled-Temperature Laboratory or Chamber—The laboratory or chamber shall maintain the temperature of 25 6 2°C.
5.3 Autoclave, capable of maintaining 121-123°C, to be used in sterilization and waste disposal stages (Note 1).
NOTE 1—Sterilization is important to avoid cross contamination and to dispose of waste properly. An autoclave shall be used to sterilize all media/solution
and borosilicate glass media bottles used to promote bacterial growth to prevent cross-contamination by other species. Sterilization shall be performed
prior to commencement of any bacterial inoculation or testing, and before waste disposal after the tests. Additional guidance on sterilization and waste
disposal is provided in Section 6.
5.4 Incubator—capable of maintaining temperature in the range of 23-30 6 2°C.
5.5 Orbital Shaker, capable of achieving at least 80 rpm.
5.6 Pipets and Syringes, 1 mL, 5 mL, and 10 mL.
5.7 Automatic Pipetor, capable of delivering 10 mL 6 0.05 mL liquid.
5.8 Petri Dishes, sterile 15 mm by 100 mm.
5.9 Inoculating Loop.
5.10 Borosilicate Glass Media Bottles, of sufficient capacity to prepare nutrient media and bacteria cultures in all test methods.
These are also used to perform tests using the Test Method A. The size of the bottles should be decided depending on the size of
the nutrient media and bacterial cultures to be prepared. Guidance is provided in the relevant sections.
5.11 Containers—The containers are used to immerse the paste or mortar specimens used in Methods B and C in the exposure
media. They shall be chemically compatible with sulfuric acid. Soda-lime glasses should not be used since they are prone to
decalcification under acidic conditions. Polypropylene containers are suggested. Since most of the plastic containers are not
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autoclavable, other sterilization methods (for example, 70 % ethanol solution) must be used to sterilize the container. The size of
the bottles should be decided depending on the size of the nutrient media and bacterial cultures to be prepared. Guidance is
provided in the relevant sections regarding the size of the containers depending on the number of samples to be tested.
5.12 Vacuum Mixer, Bowl, and Paddle, to vacuum unit will minimize entrapped air formation.formation and dust.
5.13 Diamond Blade Wet-Saw, to cut cylindrical paste or mortar specimens into 2.65 6 0.15 mm thick disks.
5.14 Flat Surface pH Electrode, with a precision of 60.02 pH.
5.15 pH Electrode, with a precision of 60.02 pH.
+2
5.16 Calcium Combination Ion Selective Electrode (ISE), which can detect 0.15 6 0.05 mg/L to 40,000 6 1000 mg/L Ca , and
can work in a pH range from 2.5 to 11.
5.17 Multiparameter Meter, compatible with pH electrodes and ISE.
5.18 Loading Machine, which is equipped with the-Ball-on-Three-Ball (B3B) test apparatus (Fig. 1) (1) and a loading system that
can provide the prescribed capacity and rates of loading. It shall have been verified to have an accuracy of 1.0 %, or better, within
twelve months of the time prior to use.
5.19 Digital Caliper, with a precision of 60.02 mm.
6. Sterilization and Disposal of Waste
6.1 Bacteria used in the tests covered by this standard are classified as biosafety level 1 (BSL-1) based on U.S. Public Health
Safety Guidelines (2). Laboratory personnel conducting the testing must have proper training to perform standard microbiological
procedures. Personal protective equipment (PPE) should be worn during testing to prevent contamination as required by BSL-1
criteria.
6.2 Sterilize all apparatus and media prior to perform any bacterial inoculation to prevent cross-contamination.
6.2.1 For autoclavable apparatus and liquid media (for example, borosilicate glass, nutrient media, water), perform sterilization
by autoclaving at 121-123°C for a minimum of 15 minutes.
FIG. 1 Schematic Sketch of a B3B Test Apparatus
The boldface numbers in parentheses refer to a list of references at the end of this standard.
It is the responsibility of the testing facility to comply with biosafety regulations for their own country.
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6.2.2 For non-autoclavable materials and apparatus, (for example, polypropylene containers, paste or mortar specimens, pH
electrode, calcium ISE), use other sterilization/disinfection methods, such as rinsing with 70 % ethanol solution.
6.2.3 To prevent cross contamination of simultaneously tested cells, particularly the cells without bacterial inoculation (for
example, those used on control tests without biogenic acidification), it is recommended that separate sets of apparatus be used for
cells with and without bacterial cultures.
6.3 During testing, care must be taken to prevent contamination of the laboratory spaces, apparatus and supplies by proper
sterilization and disinfection. After completion of the tests, sterilize all apparatus and supplies coming into contact with the bacteria
media and all liquid waste, by autoclaving at 121-123°C for a minimum of 15 minutes. Sterilized waste must be disposed in
accordance with related regulations mandated by related federal, state and local agencies.
7. Nutrient Media
7.1 The nutrient media (NM) is used to promote bacterial growth in all test methods.
7.1.1 In Test Method A, the NM represents the simulated wastewater solution.
7.1.2 In Test Method B, the NM is used as the exposure solution for the paste or mortar specimens.
7.1.3 In Test Method C, the NM is inoculated with the bacterial cultures to prepare a biogenic sulfuric acid solution as described
in 8.2.2.
7.2 Prepare the NM by adding the following compounds to Specification D1193 Type 2 de-ionized water: 10 g/L Na S O , 0.25
2 2 3
g/L CaCl , 3 g/L KH PO , 3 g/L K HPO , 0.8 g/L MgCl × 6H O, 0.1 g/L (NH ) SO , and 5 mg/L FeSO where Specification
2 2 4 2 4 2 2 4 2 4 4
D1193 Type 2 de-ionized water is to be used as the solvent.
7.3 It is normal for the NM to have a cloudy appearance, as it will contain some undissolved solids. The NM should be stirred
before it is transferred to the test cell to homogenize the suspended solids in the liquid phase.
7.4 Measured pH of the NM should be 6.55 6 0.05.
7.5 The amount of prepared NM depends on the number of tests to be performed. The ratio between the NM volume and the total
3 2
surface area of the paste or mortar specimens during tests shall be 7.0 6 0.5 cm /cm . This corresponds to approximately 300 mL
of NM per paste or mortar specimen (disc) as described in Section 8 (Note 2).
NOTE 2—For seven specimens and solutions, 2500 mL of NM needs to be prepared.
7.6 Additional NM is required for the conditioning of the specimens as described in 9.3.
7.7 To avoid possible bacterial and/or fungal contamination during extended storage periods, the NM should be prepared as
needed.
8. Bacterial Cultures and Exposure Media
8.1 The methods involve the use of bacteria that can consume elemental sulfur or thiosulfate, instead of H S, to acidify
biogenically the exposure environment for concrete. These bacteria can be cultivated, preserved and reproduced as needed using
conventional microbiological techniques such as agar plates, agar slants, and glycerol stock strains. Follow standard
microbiological techniques to prepare all bacteria cultures described below.
8.2 Test Method A—This method uses Halothiobacillus Neapolitanus (ATCC 23641) and Acidithiobacillus Thiooxidans (ATCC
19703) as seed for biogenic sulfuric acid production. H. Neapolitanus is a neutrophilic sulfur-oxidizing bacteria (NSOB); and A.
Thiooxidans is an acidophilic sulfur-oxidizing bacteria (ASOB).
8.2.1 Cultivate H. Neapolitanus in the ATCC Medium 290 S6 for Thiobacilli as described by the ATCC Microbial Media
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Formulations guide. Cultivate A. Thiooxidans in the ATCC Medium 125 for Thiobacilli as described by the ATCC Microbial Media
Formulations guide. The bacterial inoculation cultures are ready for the preparation of biogenic sulfuric acid when they reach their
exponential growth rates as per by ATCC 290 S6 and ATCC 125. Record the average live colony forming units per milliliter
(cfu/mL) of both bacterial cultures by performing a viable cell count (ISO 20391-1) (Note 3).
NOTE 3—For bacterial counting, a solid growth media that is suitable for non-fastidious bacteria (for example, TSA agar) can be used.
8.2.2 In order to test the effect of bacterial population-activity, three different acidification environments with various pH levels
are created, which are designated as Severity Levels (SL) 1, 2 and 3, as described in 8.2.3, 8.2.4, and 8.2.5, respectively (Note 4).
NOTE 4—Regarding the order of testing that a lab may want to use if tests cannot be run simultaneously, if an antimicrobial product shows efficacy in
SL3, it does not need to be tested for other severity levels. More information is provided in Section 10.
8.2.3 Severity Level 1 (SL1) is described as the environment with reduced bacterial population by dilution and low bacterial
activity due to keeping them in nutrient-deficient media for an extended period. In these tests, fresh NM shall be acidified by
actively growing bacteria until the pH drops below 3. Then, the acidified media shall be maintained in static condition in closed
bottles for six weeks. During this period, the bacterial population and activity are expected to decrease due to diminishing nutrient
concentration. After six weeks of waiting period, the pH of the bacterial suspension shall be measured. The suspension shall then
be diluted using fresh NM to obtain several suspensions with specific pH values to determine the pH range within which the
antimicrobial product is effective. Soon after the preparation of these solutions, testing should start (Note 5).
NOTE 5— It is recommended to prepare five to ten diluted solutions within the pH range from 6.5 to 2 (for example, 6.25, 6.00, 5.75, 5.10, 4.10, 3.00,
and 2.00). The increased number of solutions within this range will allow more accurate determination of effective pH range of the antimicrobial product.
8.2.4 Severity Level 2 (SL2) is described as the environment with reduced bacterial population by dilution and medium bacterial
activity due to keeping them in nutrient-deficient media for a period shorter than the waiting period specific for SL1. To prepare
this environment, fresh NM shall be acidified with actively growing bacteria until the pH drops below 3. The acidified media shall
be kept in this condition for three weeks. During this period, the bacterial population and activity is expected to decrease due to
diminishing nutrient concentration. After the waiting period, the pH of the bacterial suspension shall be measured. The suspension
shall be diluted by fresh NM to obtain several bacterial suspensions with specific pH values (see Note 5). Soon after the preparation
of these solutions, testing should start.
8.2.5 Severity Level 3 (SL3) is the environment that is expected to have high bacterial population and activity. These tests shall
be performed on media acidified by highly active bacterial cultures in fresh NM. A total of 5 % v/v of the culture of H.
Neapolitanus and a total of 5 % v/v of the pure culture of A. Thiooxidans at their exponential growth rate shall be injected to fresh
NM (Note 6). The pH of the environment shall not be modified through dilution of already acidified media; the bacteria are
expected to reduce the pH naturally by biogenic acidification. The testing should start when target pH levels (see Note 5), as
decided by the tester, are reached.
NOTE 6—As an example, 5 mL of pure bacterial culture will be injected into 100 mL of fresh NM.
8.3 Test Method B—This method uses Halothiobacillus Neapolitanus (ATCC 23641) to inoculate paste or mortar specimens. H.
Neapolitanus is a neutrophilic sulfur-oxidizing bacteria (NSOB) and can be cultivated, preserved and reproduced as needed using
conventional microbiological techniques such as agar plates, agar slants, and glycerol stock strains.
8.3.1 Cultivate the bacteria in the ATCC Medium 290 S6 for Thiobacilli as described by the ATCC Microbial Media Formulations
guide. The bacterial inoculation cultures are ready for the inoculation of paste or mortar specimen when they reach their
exponential growth rate. Record the average live colony forming units per milliliter (cfu/mL) of the bacterial inoculation cultures
by performing a viable cell count (ISO 20391-1).
8.4 Test Method C—This method uses Halothiobacillus Neapolitanus (ATCC 23641) and Acidithiobacillus Thiooxidans (ATCC
19703) as seed for biogenic sulfuric acid production. H. Neapolitanus is a neutrophilic sulfur-oxidizing bacteria (NSOB); and A.
Thiooxidans is an acidophilic sulfur-oxidizing bacteria (ASOB).
8.4.1 Cultivate H. Neapolitanus in the ATCC Medium 290 S6 for Thiobacilli as described by the ATCC Microbial Media
Formulations guide. Cultivate A. Thiooxidans in the ATCC Medium 125 for Thiobacilli as described by the ATCC Microbial Media
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Formulations guide. The bacterial inoculation cultures are ready for the preparation of biogenic sulfuric acid when they reach their
exponential growth rates as per by ATCC 290 S6 and ATCC 125 (see Note 3). Record the average live colony forming units per
milliliter (cfu/mL) of both bacterial cultures by performing a viable cell count (ISO 20391-1).
8.4.2 At their estimated exponential growth rate, inoculate the NM with 5 % (v/v) H. Neapolitanus and A. Thiooxidans (50 mL
of bacteria in the growth media per 1 L of NM) to obtain biogenic sulfuric acid. Monitor pH of the inoculated media. The solution
is ready for testing when the pH = 2 6 0.05.
9. Test Specimens
9.1 Test Method A—This test method examines the effectiveness of the antimicrobial products without the cementitious matrix.
The dosage of the tested antimicrobial product could be based on manufacturer’s recommendations or decided by the tester based
on other consideration.
9.2 Test Method B—This test method is performed on paste or mortar samples and examines either the effectiveness of
cementitious materials for resisting Stage II MIC deterioration or the effectiveness of antimicrobial additives for resisting Stage
II MIC deterioration in cementitious materials. This method has two options. Option B1 is used to determine the suitability of
antimicrobial admixtures in delaying or preventing microbially-induced corrosion of cementitious systems in the Stage II of MIC.
Option B2 is used to determine the resistance of a cementitious system in delaying or preventing microbially-induced corrosion
of concrete in the Stage II of MIC.
9.2.1 In Option B1, hardened cement paste specimens will be prepared using Type I/II ordinary portland cement manufactured in
compliance with Specification C150/C150M. Mill certificate of the cement including its oxide composition, fineness, and specific
gravity shall be part of the report. The water-to-cementitious material ratio (w/cm) of the paste will be 0.42.
9.2.2 In Option B2, hardened cement paste or mortar specimens will be prepared following the required mixture proportions. The
cementitious system may contain Specification C150/C150M portland cements (non-air entrained), Specification C595/C595M
blended portland cements, or C1600/C1600M rapid hardening hydraulic cements. Additionally, supplementary cementitious
materials (SCM) may be added to non-air entrained portland cements following Specification C150/C150M. These SCM include
Specification C618 coal fly ash and raw and calcined natural pozzolans for use in concrete, Specification C989/C989M slag cement
for use in concrete and mortars, and C1240 silica fume used in cementitious mixtures. Material specification reports for all cements
and SCM shall be part of the reporting process. Specimens may also contain admixtures including those that are intended to
increase the resistance of concrete to MIC. The type, dosage and application procedure must be specified as part of the reporting
process (Specifications C260/C260M, C494/C494M, and C1017/C1017M). Available material specification reports for the
admixtures should be provided.
9.2.3 Mixing shall be performed using a vacuum mixer with a 500 mL (or greater) bowl to minimize entrapped air formation and
increase mixture consistency. The vacuum mixer bowl. If a vacuum mixer is used, it shall be operated at 400 rpm under 70 %
vacuum.
9.2.4 If applicable, add the antimicrobial admixture to the required amount of Specification D1193 Type 2 de-ionized water at the
desired dosage as specified by the product’s manufacturer. Mix for 30 seconds.
9.2.5 Add the required amount of cement (and fine aggregate for mortar specimens) and mix for 90 seconds. Scrape the inner
surfaces of the mixing bowl and the mixing blade using a spatula. Mix for an additional 90 seconds or until mixture appears
homogeneous.
9.2.6 Cast the mixed fresh paste or mortar in plastic cylinder molds with 50.8 mm diameter and 101.6 mm height. Follow casting
procedure described in Practice C192/C192M.
9.2.7 Seal-cure the specimens at 23 6 2°C for 28 days following the procedure described in AASHTO TP 119.
9.2.8 After curing, cut the specimens into 2.65 6 0.15 mm thick disks using a diamond blade wet saw. Only the disks obtained
from the middle 60 mm part shall be used for testing. Discard the cut ends of specimens.
9.2.9 Visually inspect the specimens to discard the ones with visible cracking that was caused by the sawing process, or voids,
defects, and heterogeneities that might be artifacts of poor mixing and consolidation during casting.
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