P R O T O C O L 

 READY BIODEGRADABILITY BY THE 

CARBON DIOXIDE EVOLUTION TEST METHOD 

Organisation for Economic Cooperation and Development 

OECD Guideline 301B 

and 

Council of the European Communities Directive 67/548/EEC 

Annex V, Guideline C.4-C 
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 READY BIODEGRADABILITY BY THE 

CARBON DIOXIDE EVOLUTION TEST METHOD 

SPONSOR: 

SPONSOR’S REPRESENTATIVE: 

TESTING FACILITY: 

STUDY DIRECTOR: 

LABORATORY MANAGEMENT: 

FOR LABORATORY USE ONLY

Proposed Dates: 

Experimental 

Start Date: ________________________ 

Experimental 

Termination Date: _________________ 

Project No.: ________________________ 

Test Concentrations: _________________________________________________________ 

Test Substance No.: ___________ Reference Substance No. (if applicable): ____________ 

PROTOCOL APPROVAL

 

STUDY DIRECTOR DATE 

 

LABORATORY MANAGEMENT DATE 

 

SPONSOR’S REPRESENTATIVE DATE 
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INTRODUCTION 

Tests of ready biodegradability, by definition, provide limited opportunity for acclimation and 

biodegradation to occur. A positive result in a test of ready biodegradability is an indication that the test 

substance will undergo rapid and ultimate biodegradation in the environment. A negative result in a test 

of ready biodegradability does not necessarily mean that the test substance will not be biodegraded 

under relevant environmental conditions but that additional testing may be needed. 

OBJECTIVE

The objective of the study will be to measure the amount of carbon dioxide (CO2) produced 

from the biodegradation of a nonvolatile organic test substance and express it as a percentage of the 

theoretical amount of CO2 (TCO2) that could have been produced assuming complete biodegradation of 

the test substance to CO2 and other inorganic constituents. 

EXPERIMENTAL DESIGN 

The test will contain a blank control group, a reference group, and a treatment group. Each 

group will contain three replicate test chambers. The blank control is used to measure the background 

CO2 production of the inoculum and will not be dosed with a carbon source. The reference chambers 

will be dosed with sodium benzoate, a substance known to be biodegradable, at a concentration of 

10 mg C/L. The treatment group will be used to evaluate the test substance at 10 mg C/L. 

MATERIALS AND METHODS 

Test methods are based on the procedures specified in the OECD Guideline for Testing of 

Chemicals, Guideline 301B (1) and Council of the European Communities, Guideline C.4-C, Carbon 

Dioxide (CO2) Evolution (2). 

Test Substance 

Information on the characterization of test, control or reference substances is required by

Good Laboratory Practice (GLP) Standards and Principles. The Sponsor is responsible for providing

verification that the test substance has been characterized according to 

GLPs prior to its use in the study. If verification of GLP test substance characterization is not 

provided to ., it will be noted in the compliance statement of the final 

report. 

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The Sponsor is responsible for all information related to the test substance and agrees to accept 

any unused test substance and/or test substance containers remaining at the end of the study. 

If the test substance is relatively water soluble, a stock solution of the test substance will be 

prepared at a nominal concentration of 400 mg C/L in high quality water. Volumetric addition of a test 

substance stock solution is the most appropriate route of administration for water-soluble materials. 

The dosing volume of the test substance stock solution will be calculated based on the measured TOC.

The stock solution may be stored in a refrigerator for a maximum of three days. 

For poorly soluble materials, the test substance will be administered by direct weight 

addition. Direct weight addition of a test substance that is relatively insoluble in water is the most 

appropriate route of administration. Dosing amounts will be based on the carbon content of the test 

substance. If the carbon content of the test substance is not provided by the Sponsor, the carbon 

content will be determined by elemental analysis and will be performed by Quantitative Technologies 

Inc. (Whitehouse, NJ). The carbon analysis will not be conducted in accordance with Good 

Laboratory Practice Standards. 

Reference Substance 

A stock solution of sodium benzoate will be prepared at a nominal concentration of 400 mg C/L 

in high quality water. The TOC of the stock solution must within 15% of the nominal concentration to 

be acceptable. If the TOC is not acceptable, a new stock solution will be prepared. The dosing volume

of the reference substance stock solution will be calculated based on the measured TOC. The stock

solution may be stored in a refrigerator for a maximum of three days. 

Test Medium 

The test medium is a modified biochemical oxygen demand (BOD) test dilution water and will 

be prepared using high quality water as described in Appendix I.

Test Apparatus and Conditions 

The test chambers will be amber 4-liter bottles. The air entering the chambers will be passed 

through Drierite to remove ambient moisture and then through Ascarite to produce CO2 free air. The air 

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exiting the test chambers will be passed through a series of three gas washing bottles each containing

approximately 100 mL of 0.5 N KOH to trap the CO2 that has evolved within the chamber. An

additional set of gas washing bottles that are not connected to a chamber will be maintained 

concurrently with the traps connected to the chambers. The amount of CO2 detected in these traps will 

be subtracted from the blank control traps to determine the amount of CO2 produced by the blank

control. The test will be incubated at 20±3°C. Test temperatures will be measured each working day

using a min/max thermometer. Magnetic stirrers will be employed to mix the contents of the 

chambers. The stirrers will be cycled on and off approximately every 15 minutes to prevent the 

transfer of heat from the stirrer motors to the test chambers. Test chambers will be identified by

project number, test substance ID, test concentration, and vessels number. 

Test Inoculum 

Activated sludge will be collected from Denton Wastewater Treatment Facility, Denton, 

Maryland. The sludge will be sieved using a 2 mm screen and then aerated for approximately four 

hours. After the aeration period, the sludge will be homogenized in a blender at medium speed for 

approximately 2 minutes and then allowed to settle for approximately 30 minutes. If in the opinion of 

the Study Director the supernatant above the settled solids contains high levels of suspended sludge 

solids after 30 minutes of settling, the sludge may be settled for an additional 30 minutes. The 

supernatant will be used as the inoculum the same day that it is prepared. A total suspended solids 

measurement and standard plate count will be performed on the inoculum. Plates will be incubated at 

20 ± 3°C for approximately 48 hours. 

Preparation of Test Chambers 

The following will be added to each chamber: 

1) 2470 mL of high quality water 

2) 3 mL calcium chloride solution 

3 mL of ferric chloride solution 

3 mL of magnesium sulfate solution 

30 mL of phosphate buffer 

3) 30 mL of the activated sludge inoculum

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All chambers will be aerated with CO2 free air for approximately 24 hours at a rate of 50 -100 

mL per minute to purge the systems of CO2. After the aeration period, the flow of CO2 free air will be 

stopped, and three CO2 traps each containing approximately 100 mL of 0.5 N KOH will be connected to 

the exit air lines of each chamber. A volume reference substance stock solution necessary to deliver 10 

mg C/L will be added to the reference group chambers. Treatment group test chambers will be dosed 

with sufficient test substance to deliver 10 mg C/L. The volume within all chambers will be brought up 

to 3000 mL by the addition of high quality water. 

Biodegradation Test Initiation 

The biodegradation test will be started by bubbling CO2 free air through the test media at a rate 

of 50-100 mL per minute. The CO2 produced from the degradation of organic carbon sources within the 

test chamber is trapped as K2CO3 in the KOH solution and measured using a carbon analyzer. 

Sample Collection and Analysis 

 The CO2 traps will be removed for analysis twice a week over the 4-week test period. More or 

less frequent removal and analysis of the traps may be conducted at the discretion of the study director 

with consultation with the Sponsor. The CO2 trap nearest the chamber will be removed and analyzed 

for inorganic carbon. The two remaining traps are placed one position closer to the test chamber and a 

new trap is placed on the end of the series. 

Test Termination

On the 28th day of the test an aliquot of the contents of each test chamber will be removed and 

the pH determined. The contents of chamber will then be acidified by the addition of 3 mL of 

concentrated hydrochloric acid to drive off inorganic carbonate. The chambers will be aerated 

overnight and then a sample from each chamber will be removed for dissolved organic carbon (DOC) 

analysis. The remaining trapping solutions closest to the test chambers will be analyzed for inorganic 

carbon. 

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Calculations

The results of the carbon analysis will be converted to mg CO2 produced and used to determine 

the percentage of theoretical CO2 (%TCO2) evolved at each sampling interval. The cumulative %TCO2

will be plotted against time. The results of the carbon analyses will be converted to mg CO2 using the 

following equation: 

mg CO2 = result ( ) mg C L x vol. of KOH ( L) x 3 6. 7 mg CO2 / mg C

The cumulative mg of CO2 for the test and reference substances will be corrected for the 

amount of CO2 evolved by control, using the following equation: 

Cumulative mg CO evolved = ∑ ∑ mg CO test − mean CO control 2 2 2

The percent of theoretical CO2 produced will be calculated using the following equation: 

%

( )( . /

TCO mg CO produced

mg carbon in test mg CO mg Carbon

2 X 2

3 67 2

= 100

)

Results 

No bias is expected in this study and statistical methods will not be used in the analysis of the 

data. Interpretation of the results will be based on the following: 

1. The test is considered valid if the reference substance produces at least 60% of its TCO2 within 

14 days, and the CO2 production of the blank control does not exceed 40 mg/L. 

2. Test substances achieving 60% of TCO2 within 10 days of reaching 10% TCO2 are regarded as 

readily biodegradable. If the 60% criteria is not met, biodegradation studies conducted under 

more favorable conditions should be performed to evaluate the substances biodegradation 

potential. 

RECORDS TO BE MAINTAINED 

Records to be maintained will include, but not limited to, the following: 

 1. A copy of the signed protocol. 

 2. Identification and characterization of the test substance as provided by Sponsor. 

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 3. Study initiation and termination dates. 

 4. Experimental initiation and termination dates. 

 5. Stock solution concentration calculations and solution preparation. 

 6. Inoculum source and pretreatment data. 

 7. Results of CO2 analysis. 

 8. Temperature range recorded during test period. 

 9. Copy of final report. 

FINAL REPORT 

A final report of the results of the study will be prepared by The 

report is to include, but is not limited to, the following, when applicable: 

 1. Name and address of facility performing the study. 

 2. Dates on which the study was initiated and completed. 

3. A statement of compliance signed by the Study Director addressing any exceptions to Good 

Laboratory Practice Standards. 

4. Objectives and procedures stated in the approved protocol, including any changes in the 

original protocol. 

 5. Identification and characterization of the test substance as provided by Sponsor including name, 

CAS number, percent active, percent carbon, theoretical TOC, and other characteristics, if 

provided by the Sponsor. 

 6. A description of the transformations and calculations performed on the data. 

 7. Results of the TOC analysis performed on test and reference substance stock solutions. 

 8. A description of the test system. 

9. A description of the preparation of the test solutions, the testing concentrations, the route of 

administration, and the duration of the test. 

10. A description of all circumstances that may of affected the quality or integrity of the data. 

11. The name of the study director, the names of other scientists or professionals, and the names of 

all supervisory personnel, involved in the study. 

12. The signed and dated reports of each of the individual scientists or other professionals involved 

in the study, if applicable. 

13. The location where the raw data and final report are to be stored. 

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14. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and 

audits were made and findings reported to the Study Director and Management. 

CHANGES TO THE FINAL REPORT 

If it is necessary to make corrections or additions to the final report after it has been accepted, 

such changes shall be made in the form of an amendment issued by the Study Director. The amendment 

shall clearly identify the part of the study that is being amended and the reasons for the alteration. 

Amendments shall be signed and dated by the Study Director and Laboratory QA. 

CHANGES TO PROTOCOL

Planned changes to the protocol will be in the form of written amendments signed by the 

Study Director and approved by the Sponsor’s Representative. Amendments will be considered as 

part of the protocol and will be attached to the final protocol. Any other changes will be in the form

of written deviations signed by the Study Director and filed with the raw data. All changes to the 

protocol will be indicated in the final report. 

GOOD LABORATORY PRACTICES 

This study will be conducted in accordance with Good Laboratory Practice Standards for EPA

(40 CFR Part 792) and OECD (ENV/MC/CHEM (98) 17). Each study conducted by 

s routinely examined by the Quality Assurance Unit for 

compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. 

A statement of compliance with Good Laboratory Practices will be prepared for all portions of the study

conducted by The Sponsor will be responsible for compliance with Good 

Laboratory Practices for procedures performed by other laboratories (e.g., residue analyses or 

pathology). Raw data for all work performed at . and a copy of the final 

report will be filed by project number in archives located on the . site or at an 

alternative location to be specified in the final report. 

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