DETERMINATION OF READY BIODEGRADABILITY 

BY THE CARBON DIOXIDE EVOLUTION TEST METHOD 

 



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STUDY INFORMATION 

 

 

 

 

 

 

 
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TABLE OF CONTENTS 

Title/Cover Page..................................................................................................................................... 1 

Good Laboratory Practice Compliance Statement ................................................................................. 2

Quality Assurance Statement ................................................................................................................. 3 

Report Approval ..................................................................................................................................... 4 

Study Information................................................................................................................................... 5 

Table of Contents ................................................................................................................................... 6 

Summary ................................................................................................................................................ 8 

Introduction ............................................................................................................................................ 9 

Objective ................................................................................................................................................ 9 

Experimental Design .............................................................................................................................. 9 

Materials and Methods ........................................................................................................................... 9 

Test Substance ......................................................................................................................... 10 

Reference Substance................................................................................................................ 10 

Test Medium............................................................................................................................ 11 

Test Apparatus and Conditions................................................................................................ 11

Test Inoculum .......................................................................................................................... 11 

Preparation of Test Chambers ................................................................................................. 12

Biodegradation Test Initiation ................................................................................................. 12 

Sample Collection and Analysis.............................................................................................. 12 

Test Termination...................................................................................................................... 12 

Calculations ............................................................................................................................. 13 

Results and Discussion......................................................................................................................... 13 

Carbon Analysis....................................................................................................................... 13 

Observations and Measurements ............................................................................................. 13 

Conclusions .......................................................................................................................................... 14 

References ............................................................................................................................................ 15 
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TABLE OF CONTENTS 

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TABLES 

Table 1. Dissolved Organic Carbon (DOC) and pH of Test Solutions at Test Termination.............. 16 

Table 2. Measured Inorganic Carbon Concentration of Trapping Solutions (mg C/L) ..................... 17 

Table 3. Cumulative Milligrams of Carbon Dioxide Evolved ........................................................... 18 

Table 4. Cumulative Percent of Theoretical Carbon Dioxide Evolved.............................................. 19 

FIGURES 

Figure 1. Cumulative Percentage of Theoretical Carbon Dioxide Evolved ........................................ 20 

APPENDIX 

Appendix I. Protocol and Protocol Deviation ................................................................................ 21 
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SUMMARY 

The ready biodegradability of the test substance was determined by the Carbon Dioxide 

Evolution Test Method (OECD Guideline 301B). Tests of ready biodegradability are stringent tests 

that provide limited opportunity for acclimation and biodegradation to occur. In the CO2 test, 

inoculated mineral medium was dosed with a known amount of test substance as the nominal sole 

source of organic carbon and aerated with CO2-free air. The CO2 produced from the mineralization 

of organic carbon within the test chambers was displaced by the flow of CO2-free air and trapped as 

K2CO3 in KOH trapping solution. The amount of CO2 produced by the test substance (corrected for 

that evolved by the blank inoculum) is expressed as a percentage of the theoretical amount of CO2

(TCO2) that could have been produced if complete biodegradation of the test substance occurred. The 

test contained a blank control group, a reference group and a treatment group. Each group contained 

three replicate test chambers. The blank control was used to measure the background CO2 production 

of the inoculum and was not dosed with a carbon source. The reference chambers were dosed with 

sodium benzoate, a substance known to be biodegradable, at a nominal concentration of 10 mg C/L. 

The treatment group test chambers were used to evaluate the test substance at a nominal concentration 

of 10 mg C/L. The results indicated that the activated sludge inoculum was active, degrading the 

reference substance 98.0%. The average cumulative percent biodegradation for the test substance 

was 94.2%.

Test Substance 

Average 

Cumulative Percent 

Biodegradation 

Average 

Final 

pH 

Readily 

Biodegradable2

 
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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. 

This study was conducted by for at the 

 Ltd. biodegradation facility in Easton, Maryland. Original raw data generated 

by . and the final report are filed under Project Number 523E-101 in the 

archives located on the site. 

OBJECTIVE 

The objective of the study was to measure the amount of carbon dioxide (CO2) produced from 

the biodegradation of a nonvolatile organic test substance. This value is expressed as a percentage of 

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

the test substance occurred. 

EXPERIMENTAL DESIGN 

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

contained three replicate test chambers. The control group was used to measure the background CO2

production of the inoculum and was not dosed with a carbon source. The reference group chambers 

were used to check the viability of the inoculum and were dosed with sodium benzoate, a substance 

known to be biodegradable, at a nominal concentration of 10 mg C/L. The treatment group chambers 

were used to evaluate the test substance at a nominal concentration of 10 mg C/L. 

MATERIALS AND METHODS 

This study was conducted according to the procedures outlined in the protocol, “Test 

Substance: Determination of Ready Biodegradability by the Carbon Dioxide Evolution Test Method”. 

The protocol was based on the procedures specified in the OECD Guideline for Testing of Chemicals, 

Method 301B (1), and Annex to Commission Directive 67/548/EEC.1992, Method C.4-C, Carbon 

Dioxide (CO2) Evolution (2). 
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Test Substance 

 The test substance was received from . on April 19, 2005 and was assigned 

. identification number 7161. The following is a description of the test 

substance used in this study: 

 Lot Number: 8488/035 

 CAS Number: Not Given 

 Physical Description: Liquid 

 Purity: Not Given 

 Expiration Date: Not Given 

 Water Solubility: Not Given 

 Carbon Content: 72% 

 Storage Conditions: Ambient . 

 The test substance was administered to the treatment group test chambers by direct weight 

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

appropriate route of administration. The amount of test substance used to dose the treatment group 

test chambers were calculated based on the theoretical carbon content. 

Reference Substance 

The reference substance was received from Mallinckrodt Chemicals on January 08, 2002 and 

was assigned Identification number 5861. A stock solution of the 

reference substance was prepared in NANOpure® water at a nominal carbon concentration of 400 mg 

C/L. The stock solution was analyzed for total organic carbon (TOC). The reference substance was 

administered to the reference group test chambers by volumetric addition. Dosing amounts were 

based on the measured carbon content of the reference substance solution. Following is a description 

of the reference substance used in this study: 

Name: Sodium benzoate 

Manufacturer: Mallinckrodt Baker, Inc. 

 Paris, Kentucky 

Lot Number: 0168 V38617 

Physical Description: White powder 

Chemical Abstract Number: 532-32-1 

Expiration Date: January 8, 2007 

Purity: 99.4% 

Storage Conditions: Ambient 
 

 

 
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Preparation of Test Chambers 

The following were added to each chamber: 

1) 2470 mL of NANOpure® water 

2) 3 mL calcium chloride solution (2.75%) 

3 mL of ferric chloride solution (0.025%) 

3 mL of magnesium sulfate solution (2.25%) 

30 mL of phosphate buffer (pH 7.4) 

3) 30 mL of the activated sludge inoculum 

All chambers were aerated with CO2 free air for approximately 24 hours at a rate of 50 to 100 

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

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

to the exit air lines of each chamber. A volume of reference substance stock solution to achieve a 

nominal concentration of 10 mg C/L was added to the reference group chambers. An amount of test 

substance (approximately 0.0417 g) to achieve a nominal concentration of 10 mg C/L was added to 

the treatment group chambers. The final volume within all chambers was brought up to 3000 mL by 

the addition of NANOpure® water. 

Biodegradation Test Initiation 

The biodegradation test was started by bubbling CO2 free air through each of the test chambers 

at a rate of 50 to 100 mL per minute. The CO2 produced from the degradation of organic carbon 

sources within the test chamber was trapped as K2CO3 in the KOH solution and the amount of 

inorganic carbon in the trapping solution was measured at various intervals during the study, using a 

Shimadzu TOC-5000 carbon analyzer. 

Sample Collection and Analysis 

 On days 1, 5, 9, 13, 15, 20, 22 and 26, the CO2 trap nearest the test chamber was removed and 

analyzed for inorganic carbon. The two remaining traps were placed one position closer to the test 

chamber and a new trap was 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 was removed and 

the pH determined. The contents of all chambers were then acidified by the addition of 3 mL of 
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concentrated hydrochloric acid to drive off inorganic carbonate. All chambers were aerated overnight 

and then a sample from each test chamber was removed for dissolved organic carbon (DOC) analysis 

and then the trapping solutions closest to the test chambers were analyzed for inorganic carbon. 

Calculations 

The results of the inorganic carbon analyses of the CO2 traps were converted to mg CO2

produced using the following equation: 

mg CO2 = result (mg C/L) X vol. of KOH (L) X 3.67 mg CO2/mg C 

The cumulative mg of CO2 for the test and reference substances were corrected for the amount 

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

Cumulative mg CO2 produced = Σ mg CO2 test - mean Σ mg CO2 control 

The percentage of theoretical carbon dioxide (%TCO2) evolved was calculated as follows: 

% TCO2 = (mg of carbon in test) x ( . mg CO /mg Carbon)

mg CO produced

2

2

3 67

X 100 

RESULTS AND DISCUSSION 

Carbon Analysis 

The measured total organic carbon (TOC) concentration of the reference substance stock 

solution was 398.7 mg C/L. The volume of stock solution used to dose the reference chambers was 

adjusted based on the measured TOC value so that approximately 10 mg C/L was delivered. 

Observations and Measurements 

The temperature range recorded during the test was 18.6-26.3 oC. The mean result of the 

standard plate count performed on the inoculum was 6.60 x 105

 CFU/mL. The measured total 

suspended solids concentration of the test solution was 3.467 mg/L and was within the acceptable 

limit specified in the test guideline (1). 
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The measured dissolved organic carbon (DOC) on Day 29 and pH values of the test chamber 

contents on Day 28 are presented in Table 1. The measured concentrations of inorganic carbon in the 

carbon dioxide trapping solutions are presented in Table 2. The cumulative amounts of CO2 produced 

over the test period are presented in Table 3. The cumulative percent of theoretical carbon dioxide 

(% TCO2) evolved is presented in tabular and graphical forms in Table 4 and Figure 1, respectively. 

The control chambers evolved an average of 22.2 mg CO2 over the test period. This value has 

been corrected for the amount of CO2 in the trapping solution since potassium hydroxide solution, 

even when freshly prepared, contains carbonates. The amount of CO2 evolved by the control 

chambers did not exceed the 40 mg/L (120 mg total) value considered the acceptable limit for CO2

evolution tests (1). 

 The viability of the inoculum and validity of the test were supported by the results of the 

reference substance, sodium benzoate, from which an average of 98.0% of theoretical CO2 was 

evolved. An average percent biodegradation of greater than 60% was achieved by Day 13, thereby 

fulfilling the criteria for a valid test by reaching the pass level by Day 14 (1). 

 The final mean percent biodegradation for the test substance was 94.2%. The test Substance 

may be considered readily biodegradable, since the pass level of 60% TCO2 was achieved. 

CONCLUSIONS 

 Evidence of ready biodegradability in a Carbon Dioxide Evolution Test is 60% TCO2 within the 

28-day test period (1,2). In addition, the pass level must be reached within 10 days of achieving 10% 

TCO2 (1,2). The final mean percent biodegradation for the test substance was 94.2%. The test 

substance may be considered readily biodegradable since the pass level of 60% TCO2 was achieved. 
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REFERENCES 

1. Organisation for Economic Cooperation and Development. 1992. Guideline for the Testing 

of Chemicals. Guideline 301B: Ready Biodegradability: CO2 Evolution (Modified Sturm Test). 

Adopted by the Council on 17 July 1992. 

2. Annex to Commission Directive 67/548/EEC. 1992. Carbon Dioxide (CO2) Evolution. Official 

Journal of the European Communities No. L383. Method C.4-C, 

3. Standard Methods for the Examination of Water and Wastewater. Seventeenth Edition. 

Parts 2450 and 9215. American Public Health Association. 
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Table 1 

Dissolved Organic Carbon (DOC) and pH of Test Solutions at Test Termination 

Test Chamber/Nominal Concentration 

DOC1

(mg C/L) pH 

Control Rep. A <1.0 

Control Rep. B 

Control Rep. C 

Sodium Benzoate Rep. A (10 mg C/L) 

Sodium Benzoate Rep. B (10 mg C/L) 

Sodium Benzoate Rep. C (10 mg C/L) 

<1.0 

<1.0 

<1.0 

<1.0 

<1.0 

Test Substance Rep. A (10 mg C/L) 

Test Substance Rep. B (10 mg C/L) 

Test Substance Rep. C (10 mg C/L) 

1.0 

1.1 

0.9 

7.2 

7.2 

7.2 

7.3 

7.2 

7.3 

7.1 

7.2 

7.2 

 1

 Samples were filtered (0.45 μm) and purged with CO2 free air prior to analysis. 
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Table 2 

Measured Inorganic Carbon Concentration of Trapping Solutions (mg C/L) 

Date Day Control 

Rep. A 

Control 

Rep. B 

Control 

Rep. C 

Reference 

Rep. A 

Reference 

Rep. B 

Reference 

Rep. C 

DBAT 

Rep. A 

DBAT 

Rep. B 

16-Jun-05 20-Jun-05 24-Jun-05 28-Jun-05 30-Jun-05 

5-Jul-05 7-Jul-05 

11-Jul-05 14-Jul-05 

1 5 9 

13 15 20 22 26 29 

7.82 

19.57 14.79 15.96 12.42 13.13 

7.35 7.99 

14.40 

8.14 

15.52 15.50 12.83 

9.55 9.63 7.31 8.22 

16.80 

8.25 

17.46 15.55 13.61 

9.07 

11.37 

6.09 7.21 8.67 

4.99 

151.00 

91.06 57.11 19.97 31.14 12.05 16.10 17.89 

7.57 

62.42 69.01 43.26 71.68 62.75 

7.33 

21.65 54.68 

7.80 

123.20 111.70 

57.93 16.73 24.29 13.54 18.18 20.91 

10.29 90.69 

123.60 

57.11 15.02 23.31 12.94 12.86 34.23 

8.87 

105.00 

75.32 73.82 31.01 41.81 15.25 18.69 19.26 
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Table 3 

Cumulative Milligrams of Carbon Dioxide Evolved1,2

Date Day Control 

Rep. A 

Control 

Rep. B 

Control 

Rep. C 

Reference 

Rep. A

Reference 

Rep. B 

Reference 

Rep. C 

DBAT 

Rep. A 

DBAT 

Rep. B 

DBAT 

Rep. C 

Control3

Rep. A 

Control3

Rep. B 

16-Jun-05 20-Jun-05 24-Jun-05 28-Jun-05 30-Jun-05 

5-Jul-05 7-Jul-05 

11-Jul-05 14-Jul-05 

1 5 9 

13 15 20 22 26 29 

2.9 

10.1 15.5 21.3 25.9 30.7 33.4 36.3 41.6 

3.0 8.7 

14.4 19.1 22.6 26.1 28.8 31.8 38.0 

3.0 9.4 

15.1 20.1 23.5 27.6 29.9 32.5 35.7 

-1.1 

47.9 75.7 91.4 95.0 

102.2 104.1 107.2 108.8 

-0.2 

16.3 36.0 46.7 69.2 88.1 88.2 93.3 

108.5 

-0.1 

38.7 74.1 90.1 92.5 97.2 99.7 

103.5 106.3 

0.8 

27.7 67.4 83.2 84.9 89.3 91.5 93.4 

101.0 

0.3 

32.4 54.4 76.3 83.9 95.1 98.2 

102.1 104.3 

0.7 

35.0 67.1 83.0 88.2 98.4 

100.8 104.1 105.7 

0.7 6.1 9.7 

13.7 16.0 19.6 20.7 21.8 25.4 

0.8 4.7 8.6 

11.4 12.7 15.0 16.1 17.3 21.7 

 1The results of the inorganic carbon analyses of the CO2 traps were converted to mg CO2 produced using the following equation: 

 mg CO2 = cumulative result (mg C/L) X vol. of KOH (L) X 3.67 mg CO2/mg C 

 2Calculations performed in Excel 2000 full precision mode. Manual calculations may differ.  3Corrected for the CO2 attributed to the inoculum and the KOH by subtracting the average amount of CO2 evolved by the controls. 
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Table 4 

Cumulative Percent of Theoretical Carbon Dioxide Evolved1,2

Date Day 

Control 

Rep. A 

Control 

Rep. B 

Control 

Rep. C 

Reference 

Rep. A 

Reference 

Rep. B 

Reference 

Rep. C 

DBAT 

Rep. A 

D

R

16-Jun-05 20-Jun-05 24-Jun-05 28-Jun-05 30-Jun-05 

5-Jul-05 7-Jul-05 

11-Jul-05 14-Jul-05 

1 5 9 

13 15 20 22 26 

NA NA NA NA NA NA NA NA NA 

NA NA NA NA NA NA NA NA NA 

NA NA NA NA NA NA NA NA NA 

-1.0 

43.5 68.7 83.1 86.3 92.8 94.6 97.3 98.9 

-0.2 

14.8 32.7 42.4 62.9 80.0 80.1 84.7 98.5 

-0.1 

35.1 67.3 81.9 84.0 88.3 90.5 94.0 96.5 

0.7 

25.1 61.2 75.6 77.1 81.1 83.1 84.8 91.8 

Cumulative Average (N=3) Std. Dev. 

 NA 

 NA 

 98.0 

 1.3 

 

1The percentage of TCO2 was calculated using the following equation: 

% TCO2 = 

(mg of carbon in test) x ( . mg CO /mg Carbon)

mg CO produced

2 3 67

2 X 100 

2Calculations performed in Excel 2000 full precision mode. Manual calculations may differ. 

NA – Not Applicable 
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-10

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (days)

Percent of Theoretical CO2 Production

Sodium Benzoate Rep.A Sodium Benzoate Rep.B Sodium Benzoate Rep.C

Test Substance Rep.A Test Substance Rep.B Test Substance Rep.C

Figure 1. Cumulative Percentage of Theoretical Carbon Dioxide Evolved 
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APPENDIX I 

Protocol and Protocol Deviation 
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