ASTM D4412-19(2024)

Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits

SIGNIFICANCE AND USE
5.1 Sulfate-reducing bacteria are widely distributed in marine and fresh water muds which, in consequence, frequently are laden with the hydrogen sulfide produced by these organisms during dissimilatory sulfate reduction.  
5.2 It has been reported that Desulfovibrio spp. can form as much as 10 g of sulfide per litre during active multiplication. Sulfate-reducing bacteria can cause the external or internal corrosion of water or wastewater pipelines and pipelines for petroleum and natural gas. The formation of galvanic cells by massive growth of sulfate-reducing bacteria under suitable conditions makes the corrosion much worse than just the effect of the hydrogen sulfide on the metal or concrete.
SCOPE
1.1 These test methods cover the procedure for the detection and enumeration by the most probable number (MPN) technique of sulfate-reducing bacteria in water or water-formed deposits.  
1.2 Two media preparations are provided. Medium A which is prepared with reagent grade water, and Medium B which is prepared using the water to be sampled as the water source. Medium B is offered for those special conditions where sulfate-reducing bacterial strains have adapted to atypical non-fresh water environment.  
1.3 For the isolation and enumeration of thermophilic sulfate-reducing bacteria encountered in waters associated with oil and gas production, all broths, dilution blanks, and incubations must be maintained at temperatures of at least 45 °C and preferably within 5 °C at the sample temperature.  
1.4 The sensitivity of these test methods can be increased by purging the dilution blanks and tubes of media with nitrogen immediately prior to use.  
1.5 The analyst should be aware that adequate collaborative data for precision and bias statements as required by Practice D2777 are not provided. See Section 11 for details.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 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.  
1.8 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: Published
• Publication Date: 31-Mar-2024
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaces: ASTM D4412-19 - Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits
• Effective Date: 01-Apr-2024
• Referred By: ASTM E645-18 - Standard Practice for Evaluation of Microbicides Used in Cooling Water Systems
• Effective Date: 01-Apr-2024
• Referred By: ASTM D6469-20 - Standard Guide for Microbial Contamination in Fuels and Fuel Systems
• Effective Date: 01-Apr-2024
• Referred By: ASTM D5978/D5978M-16(2024) - Standard Guide for Maintenance and Rehabilitation of Groundwater Monitoring Wells
• Effective Date: 01-Apr-2024
• Referred By: ASTM D8506-23 - Standard Guide for Microbial Contamination and Biodeterioration in Turbine Oils and Turbine Oil Systems
• Effective Date: 01-Apr-2024
• Referred By: ASTM D8243-19 - Standard Test Method for Determination of APS Reductase to Estimate Sulfate Reducing Bacterial Bioburdens in Water – Enzyme-Linked Immunosorbent Assay Method
• Effective Date: 01-Apr-2024
• Referred By: ASTM D887-13(2022) - Standard Practices for Sampling Water-Formed Deposits
• Effective Date: 01-Apr-2024
• Referred By: ASTM D4025-18 - Standard Practice for Reporting Results of Examination and Analysis of Deposits Formed from Water for Subsurface Injection
• Effective Date: 01-Apr-2024

Overview

ASTM D4412-19(2024): Standard Test Methods for Sulfate-Reducing Bacteria in Water and Water-Formed Deposits is an international testing standard published by ASTM. This standard outlines procedures for the detection and enumeration of sulfate-reducing bacteria (SRB) in water samples and water-formed deposits using the Most Probable Number (MPN) technique. SRB are widely distributed in marine and fresh water environments and can cause significant corrosion issues in pipelines and water infrastructure.

ASTM D4412-19(2024) is relevant for water quality laboratories, environmental monitoring agencies, pipeline operators, and industries concerned with microbiologically influenced corrosion (MIC).

Key Topics

• Detection and Enumeration of SRB:
  The standard details the use of the MPN technique to quantify SRB in water and deposits, enabling reliable monitoring and risk assessment.

• Media Preparation:
  Two types of media are specified:

  • Medium A: Uses reagent grade water for standard conditions.
  • Medium B: Uses the sampled environmental water, suitable for SRB adapted to unique or non-freshwater environments.

• Test Sensitivity and Environmental Control:
  Sensitivity of the test can be increased by purging dilution blanks and media tubes with nitrogen prior to use to minimize oxygen levels. Special conditions for thermophilic SRB (common in oil and gas production) involve incubations at temperatures above 45°C.

• Interference and Analytical Limitations:
  The method notes limitations in providing collaborative data for precision and bias, and underscores the variable nature of microbial populations in samples.

• Safety and Environmental Practices:
  Users are reminded that the standard does not address all potential safety concerns and are responsible for following applicable safety, health, and regulatory guidelines.

Applications

• Corrosion Monitoring:
  SRB are known contributors to external and internal corrosion of water, wastewater, petroleum, and natural gas pipelines. By measuring SRB populations, organizations can identify corrosion risks and plan mitigation strategies.

• Water Quality Management:
  Regular testing for sulfate-reducing bacteria is vital in managing the biological quality of supplied water, preventing infrastructure damage, and meeting compliance requirements.

• Industrial Process Control:
  Industries such as oil and gas, power generation, and marine operations benefit from monitoring SRB to prevent operational inefficiencies, costly repairs, and downtime due to biofilm-related corrosion.

• Environmental Assessment:
  Environmental agencies use these methods for ecosystem health assessments, especially in locations where SRB-induced hydrogen sulfide poses risks to water quality and aquatic life.

Related Standards

• ASTM D1129: Terminology Relating to Water
  Provides definitions used throughout ASTM water testing standards.

• ASTM D1193: Specification for Reagent Water
  Outlines requirements for water purity used in laboratory testing.

• ASTM D2777: Practice for Determination of Precision and Bias
  Offers guidelines for evaluating test method performance.

• ASTM D6503: Test Method for Enterococci in Water
  Specifies microbial testing procedures relevant to water quality.

• Standard Methods 9221: Multiple-Tube Fermentation Technique
  A widely used procedure for enumerating microbial populations in water.

Summary

ASTM D4412-19(2024) delivers a systematic approach to detecting and quantifying sulfate-reducing bacteria, supporting corrosion prevention, operational reliability, environmental safety, and water quality assurance. Routine application of this standard ensures that organizations can effectively manage the microbiological risks associated with water and water-formed deposits.

Keywords: sulfate-reducing bacteria, SRB, water testing, corrosion, MPN, hydrogen sulfide, water quality, ASTM D4412, environmental monitoring, industrial water analysis.

Standards Content (Sample)

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.
Designation: D4412 − 19 (Reapproved 2024)
Standard Test Methods for
Sulfate-Reducing Bacteria in Water and Water-Formed
Deposits
This standard is issued under the fixed designation D4412; 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 mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 These test methods cover the procedure for the detection
and enumeration by the most probable number (MPN) tech-
2. Referenced Documents
nique of sulfate-reducing bacteria in water or water-formed
2.1 ASTM Standards:
deposits.
D1129 Terminology Relating to Water
1.2 Two media preparations are provided. Medium A which
D1193 Specification for Reagent Water
is prepared with reagent grade water, and Medium B which is
D2777 Practice for Determination of Precision and Bias of
prepared using the water to be sampled as the water source.
Applicable Test Methods of Committee D19 on Water
Medium B is offered for those special conditions where
D6503 Test Method for Enterococci in Water Using Enter-
sulfate-reducing bacterial strains have adapted to atypical
olert
non-fresh water environment.
2.2 Other Standards:
1.3 For the isolation and enumeration of thermophilic
Standard Methods 9221 Multiple-Tube Fermentation Tech-
sulfate-reducing bacteria encountered in waters associated with
nique for Members of the Coliform Group
oil and gas production, all broths, dilution blanks, and incuba-
tions must be maintained at temperatures of at least 45 °C and
3. Terminology
preferably within 5 °C at the sample temperature.
3.1 Definitions—For definitions of terms used in this
1.4 The sensitivity of these test methods can be increased by
standard, refer to Terminology D1129.
purging the dilution blanks and tubes of media with nitrogen
3.1.1 most probable number, n—statistical method for de-
immediately prior to use.
termining bacterial density based on the Poisson distribution.
D6503
1.5 The analyst should be aware that adequate collaborative
data for precision and bias statements as required by Practice
3.2 Acronyms:
D2777 are not provided. See Section 11 for details.
3.2.1 SRB, n—sulfate-reducing bacteria
1.6 The values stated in SI units are to be regarded as
4. Summary of Test Methods
standard. No other units of measurement are included in this
standard.
4.1 Water and water deposit samples and dilutions of these
samples are dispensed into tubes of Starkey’s medium (A or B)
1.7 This standard does not purport to address all of the
following five tube MPN procedures. The tubes are sealed with
safety concerns, if any, associated with its use. It is the
liquid paraffin, and incubated at 20 °C for 21 days. Positive
responsibility of the user of this standard to establish appro-
reactions are indicated by the deposit of a black precipitate.
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
5. Significance and Use
1.8 This international standard was developed in accor-
dance with internationally recognized principles on standard- 5.1 Sulfate-reducing bacteria are widely distributed in ma-
ization established in the Decision on Principles for the rine and fresh water muds which, in consequence, frequently
Development of International Standards, Guides and Recom-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
These test methods are under the jurisdiction of ASTM Committee D19 on contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Water and are the direct responsibility of Subcommittee D19.24 on Water Micro- Standards volume information, refer to the standard’s Document Summary page on
biology. the ASTM website.
Current edition approved April 1, 2024. Published April 2024. Originally Available from Standard Methods, http://standardmethods.org.
approved in 1984. Last previous edition approved in 2019 as D4412 – 19. DOI: Starkey, R. L., “Characteristics and Cultivation of Sulfate-Reducing Bacteria,”
10.1520/D4412-19R24. Journal of the American Water Works Association, Vol 40, 1948, pp. 1291–1298.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4412 − 19 (2024)
are laden with the hydrogen sulfide produced by these organ- 7.3.4 pH of medium should be 7.2 after autoclave
isms during dissimilatory sulfate reduction. sterilization, at 121 °C for 15 min.
5.2 It has been reported that Desulfovibrio spp. can form as
7.4 Starkey’s Medium B—The medium is similar to that
much as 10 g of sulfide per litre during active multiplication. described in 7.3, 7.3.1, and 7.3.2 with the following modifica-
Sulfate-reducing bacteria can cause the external or internal
tion:
corrosion of water or wastewater pipelines and pipelines for 7.4.1 Water collected from the sample collection site is used
petroleum and natural gas. The formation of galvanic cells by
to prepare the medium outlined in 7.3. The water sample is
massive growth of sulfate-reducing bacteria under suitable filtered to remove particulates (1.2 μm membrane filter) and the
conditions makes the corrosion much worse than just the effect
pH is recorded.
of the hydrogen sulfide on the metal or concrete. 7.4.1.1 After preparing the Medium B following 7.3.1,
7.3.2, and 7.3.3, and prior to dispensing, check and adjust pH,
6. Apparatus and Materials
if necessary to that of the original water used, then filter
sterilize the medium by passage through 0.2 μm filter and
6.1 Anaerobic Incubator, 20 °C, if available, or conven-
asceptically dispense into presterilized tubes.
tional 20 °C incubator.
7.5 Hydrogen Sulfide Test Reagent:
NOTE 1—For thermophilic organisms use a 45 °C incubator.
7.5.1 Ferric Chloride Stock Solution (FeCl ·6H O)—
3 2
6.2 Pipets, sterile, 1 mL and 10 mL, “calibrated” to deliver.
Dissolve 13.5 g of ferric chloride in a mixture of 250 mL of
6.3 Test Tubes, with close fitting or airtight caps; 16 mm by
water and 250 mL of HCl (sp gr 1.19). Store in an airtight
150 mm and 20 mm by 150 mm.
amber container. Prepare fresh monthly.
7.5.2 p-Aminodimethylaniline Dihydrochloride Stock Solu-
6.4 Test Tube Racks, of sufficient size to contain 16 mm and
tion:
20 mm tubes.
p-Aminodimethylaniline dihydrochloride 1.0 g
(C H N ·2HCl)
8 12 2
7. Reagents
HCl (6 N) 500 mL
7.1 Purity of Reagents—Reagent grade chemicals shall be
Dissolve 1 g of p-aminodimethylaniline dihydrochloride in
used in all tests. Unless otherwise indicated, it is intended that
500 mL of 6 N HCl. Store for up to 1 month in an amber
all reagents conform to the specifications of the Committee on
airtight container.
Analytical Reagents of the American Chemical Society, when
7.6 Liquid Paraffın—Heavy, sterile, or sterile mineral oil.
such specifications are available.
7.7 Buffered Dilution Water, Stock Solution:
7.2 Purity of Water—Unless otherwise indicated, references
7.7.1 Dissolve 34.0 g of KH PO in 500 mL of water, adjust
2 4
to water shall be understood to mean Reagent Water Type II
pH to 7.2 with 1 N NaOH and dilute to 1 L with distilled water.
conforming to Specification D1193. In addition, reagent water
This is called the stock phosphate solution.
used for these test methods must be sterile.
7.7.2 Dissolve 38 g of MgCL i
...