FINAL REPORT

Study Title

DETERMINATION OF 'READY' BIODEGRADABILITY: CARBON 

DIOXIDE (CO2) EVOLUTION TEST (MODIFIED STURM TEST) OF 

Author

Test Facility

 

Laboratory Project Identification

- Page 1 of 16 -
- Page 2 - 

1. CONTENTS 

1. CONTENTS ...................................................................................................................................... 2

2. STATEMENT OF GLP COMPLIANCE ............................................................................................. 3

3. QUALITY ASSURANCE STATEMENT ............................................................................................ 4

4. SUMMARY ........................................................................................................................................ 5

5. INTRODUCTION .............................................................................................................................. 6

5.1. Preface ...................................................................................................................................... 6

5.2. Aim of the study ........................................................................................................................ 6

5.3. Guidelines ................................................................................................................................. 6

5.4. Storage and retention of records and materials ........................................................................ 6

5.5. Definitions.................................................................................................................................. 7

6. MATERIALS AND METHODS .......................................................................................................... 7

6.1. Test Substance ......................................................................................................................... 7

6.1.1. Test substance information ............................................................................................. 7

6.1.2. Study specific test substance information ...................................................................... 7

6.1.3. Test concentration and preparation of test solutions ...................................................... 8

6.2. Reference substance ................................................................................................................ 8

6.2.1. Reference substance ...................................................................................................... 8

6.2.2. Reference substance concentration and preparation of test solutions........................... 8

6.3. Test system ............................................................................................................................... 8

6.4. Test procedure and conditions .................................................................................................. 9

6.4.1. Preparation of bottles ...................................................................................................... 9

6.4.2. Determination of CO2....................................................................................................10

6.4.3. Measurements and recording .......................................................................................10

6.5. Electronic data capture ...........................................................................................................10

6.6. Interpretation ...........................................................................................................................11

6.6.1. Data evaluation .............................................................................................................11

6.6.2. Acceptability of the test .................................................................................................11

6.7. List of deviations .....................................................................................................................12

6.7.1. List of protocol deviations .............................................................................................12

6.7.2. List of standard operating procedures deviations .........................................................12

7. RESULTS .......................................................................................................................................12

7.1. Theoretical CO2 production .....................................................................................................12

7.2. Biodegradation ........................................................................................................................12

7.3. Monitoring of temperature and pH ..........................................................................................13

8. CONCLUSION ................................................................................................................................13

TABLES 

Table 1 pH values of different test media. .................................................................................13

Table 2 HCl titrated in duplicate blank bottles ...........................................................................14

Table 3 HCl titrated in Ba(OH)2 solution (background bottles) ..................................................14

Table 4 CO2 production in the blank. .........................................................................................15

Table 5 CO2 production and percentage biodegradation of the positive control substance. .....15

Table 6 CO2 production and percentage biodegradation of the test substance (bottle A). .......15

Table 7 CO2 production and percentage biodegradation of the test substance (bottle B). .......16

Table 8 CO2 production and percentage biodegradation of the toxicity control. .......................16

Table 9 Comparison of biodegradation of the test substance in bottles A and B. .....................16

FIGURES 

Figure 1 Biodegradation of and sodium acetate in the modified Sturm 

test ................................................................................................................................12

APPENDICES 

 APPENDIX 1 TABLES ........................................................................................................................14

Final Report


- Page 5 - 

4. SUMMARY 

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) 

with 

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In 

addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) 

No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C and the ISO International Standard 

9439, 1999 and ISO Standard 10634, 1995. 

was a clear colourless liquid with a purity ≥ 95%. The test substance was tested in 

duplicate at 13 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the 

molecular formula. 

The Theoretical CO2 production (ThCO2) of was calculated to be 3.36 mg CO2/mg. 

The study consisted of six bottles: 

- 2 inoculum blanks (no test substance), 

- 2 test bottles ( 

- 1 positive control (sodium acetate) and 

- 1 toxicity control ( plus sodium acetate). 

Since was not sufficiently soluble to allow preparation of an aqueous solution at a 

concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with 

microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each 

weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension 

was added quantitatively to the test medium. The test solutions were continuously stirred during the 

test, to ensure optimal contact between the test substance and the test organisms. Test duration was 

28 days (last CO2-measurement on the 29th day). 

The relative biodegradation values calculated from the measurements performed during the test 

period revealed 14 and 13% biodegradation of for the duplicate bottles tested. 

Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) 

was not met. 

In the toxicity control, was found not to inhibit microbial activity. 

Since all criteria for acceptability of the test were met, this study was considered to be valid. 

In conclusion, was designated as not readily biodegradable. 

Final Report
- Page 6 - 

5. INTRODUCTION 

5.1. Preface 

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

Technical Coordinator

Study Plan Start : 16 November, 2011

Completion : 15 December, 2011

5.2. Aim of the study 

The purpose of the study was to evaluate a non-volatile test substance for its ready biodegradability in 

an aerobic aqueous medium with microbial activity introduced by inoculation with the supernatant of 

activated sludge. 

5.3. Guidelines 

The study procedures described in this report were based on the Organization for Economic Cooperation and Development (OECD), OECD guidelines for Testing of Chemicals, Section 3, 

Degradation and Accumulation, guideline No. 301 B: "Ready Biodegradability: CO2 Evolution Test" 

adopted July 17, 1992. 

In addition, the procedures were designed to meet the test methods prescribed by the following 

guidelines: 

 Commission Regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the 

determination of ecotoxicity, Publication No. L142, C.4. "Biodegradation: determination of the 

'ready' biodegradability, C.4-C: Carbon dioxide (CO2) evolution test (Modified Sturm Test). 

ISO Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic 

compounds in aqueous medium - carbon dioxide evolution test (1999). 

 ISO Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly 

water-soluble organic compounds for the subsequent evaluation of their biodegradability in an 

aqueous medium" (1995). 

5.4. Storage and retention of records and materials 

Records and materials pertaining to the study including protocol, raw data, specimens (except 

specimens requiring refrigeration or freezing) and the final report are retained in the archives 

for a period of at least 2 years after finalization of the report. After this period, the sponsor will be 

contacted to determine how the records and materials should be handled. will retain 

information concerning decisions made. 

Final Report
- Page 7 - 

Those specimens requiring refrigeration or freezing will be retained by for as long as the 

quality of the specimens permits evaluation but no longer than three months after finalization of the 

report. 

will retain a test substance sample until the expiry date, but no longer than 10 years after 

finalization of the report. After this period the sample will be destroyed. 

5.5. Definitions 

Readily biodegradable are those test substances giving a result of at least 60% biodegradation 

within 28 days. This pass level must be reached within the 10 days immediately following the 

attainment of 10% biodegradation (10-day window). 

Theoretical carbon dioxide (ThCO2) is the quantity of carbon dioxide calculated (mg) to be produced 

from the known or measured carbon content of the test substance when fully mineralized; also 

expressed as mg carbon dioxide evolved per mg test substance. 

Total Organic Carbon (TOC) of a sample is the sum of the organic carbon in solution and in 

suspension. 

6. MATERIALS AND METHODS 

6.1. Test Substance 

6.1.1. Test substance information 

Identification 

Structure

Molecular formula

Molecular weight

CAS Number

Description

Batch

Purity

Test substance storage

Stability under storage conditions

Expiry date 17 October 2012 

6.1.2. Study specific test substance information 

Hygroscopic No

Volatile No

Test substance handling Flush container with nitrogen after handling

Stability in water At least 24 hours

Solubility in water No

Final Report
- Page 8 - 

6.1.3. Test concentration and preparation of test solutions 

was a . The test substance was tested in 

duplicate at 13 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the 

molecular formula. 

Since was not sufficiently soluble to allow preparation of an aqueous solution at a 

concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with 

microbial organisms and mineral components (test substance bottle A: 25.9 mg; test substance bottle 

B: 25.8 mg and toxicity control bottle: 26.1 mg). To this end, 10 ml of Milli-RO water was added to 

each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting 

suspension was added quantitatively to the test medium. The test solutions were continuously stirred 

during the test, to ensure optimal contact between the test substance and the test organisms. 

6.2. Reference substance 

6.2.1. Reference substance 

Identification number RS186

Name Sodium acetate

Description White powder (determined at 

Molecular formula CH3COONa (taken from label)

Molecular weight 82.03 (taken from label)

Batch number K34333668

Article number 1.06268.0250

Purity 99.0%

Expiry Date 28 February 2015

Certified Yes

Storage conditions At room temperature in the dark

Supplier Merck, Darmstadt, Germany

6.2.2. Reference substance concentration and preparation of test solutions 

A solution of sodium acetate was prepared by dissolving 400.1 mg in Milli-RO water and making this 

up to a total volume of 100 ml. Volumes of 20 ml from this stock solution were added to 2 litres of the 

test medium of the positive control bottle and the toxicity control bottle, resulting in a final 

concentration of 40 mg sodium acetate per litre (12 mg TOC/l). 

6.3. Test system 

Source The source of test organisms was activated sludge freshly 

obtained from a municipal sewage treatment plant: 

' 

, receiving predominantly domestic sewage. 

Treatment The freshly obtained sludge was kept under continuous 

aeration until further treatment. The concentration of 

suspended solids was 4.7 g/l in the concentrated sludge 

(information obtained from the municipal sewage treatment 

plant). Before use, the sludge was allowed to settle 

(35 minutes) and the supernatant liquid was used as 

inoculum at the amount of 10 ml/l of mineral medium. 

Reason for selection The test has been accepted internationally for determining 

the 'ready' biodegradability of test substances under aerobic 

conditions. 

Final Report
- Page 9 - 

6.4. Test procedure and conditions 

Test duration 28 days (last CO2-measurement on the 29th day). 

During the test period the test media were aerated and 

stirred continuously. 

Test vessels 2 litre all-glass brown coloured bottles. 

Milli-RO / Milli-Q water Tap-water purified by reverse osmosis (Milli-RO) and 

subsequently passed over activated carbon and ionexchange cartridges (Milli-Q) (Millipore Corp., Bedford, 

Mass., USA). 

Stock solutions of A) 8.50 g KH2PO4

mineral components 21.75 g K2HPO4

67.20 g Na2HPO4.12H2O 

0.50 g NH4Cl

 dissolved in Milli-Q water and made up to 1 litre, 

 pH 7.4 ± 0.2 

B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water 

 and made up to 1 litre. 

C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and 

 made up to 1 litre. 

D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and 

 made up to 1 litre. 

Mineral medium 1 litre mineral medium contains: 10 ml of solution (A), 

1 ml of solutions (B) to (D) and Milli-RO water. 

Barium hydroxide 0.0125 M Ba(OH)2 (Boom, The Netherlands), stored in a 

sealed vessel to prevent absorption of CO2 from the air. 

Synthetic air (CO2 < 1 ppm) A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was 

passed through a bottle, containing 0.5 - 1 litre 0.0125 M 

Ba(OH)2 solution to trap CO2 which might be present in small 

amounts. The synthetic air was sparged through the 

scrubbing solutions at a rate of approximately 1-2 bubbles 

per second (ca. 30-100 ml/min). 

6.4.1. Preparation of bottles 

Pre-incubation medium The day before the start of the test (day -1) mineral 

components, Milli-RO water (ca. 80% total volume) and 

inoculum (1% final volume) were added to each bottle. This 

mixture was aerated with synthetic air overnight to purge the 

system of CO2. 

Type and number of bottles Test suspension: containing test substance 

and inoculum (2 bottles). 

Inoculum blank: containing only inoculum (2 bottles) 

Positive control: containing reference substance and 

inoculum (1 bottle). 

Toxicity control: containing test substance, reference 

substance and inoculum (1 bottle). 

Final Report
- Page 10 - 

Preparation At the start of the test (day 0) test and reference substance 

were added to the bottles containing the microbial organisms 

and mineral components. 

 The volumes of suspensions were made up to 2 litres with 

Milli-RO water, resulting in the mineral medium described 

before. 

Three CO2-absorbers (bottles filled with 100 ml 

 0.0125 M Ba(OH)2) were connected in series to the exit air 

line of each test bottle. 

6.4.2. Determination of CO2

Experimental CO2 production The CO2 produced in each test bottle reacted with the 

barium hydroxide in the gas scrubbing bottle and 

precipitated out as barium carbonate. The amount of CO2

produced was determined by titrating the remaining Ba(OH)2

with 0.05 M standardized HCl (1:20 dilution from 1 M HCl 

(Titrisol® ampul), Merck, Darmstadt, Germany). 

Measurements Titrations were made every second or third day during the 

first 10 days, and thereafter at least every fifth day until the 

28th day, for the inoculum blank and test suspension. 

Titrations for the positive and toxicity control were made at 

least 14 days. 

Each time the CO2-absorber nearest to the test bottle was 

removed for titration; each of the remaining two absorbers 

was moved one position in the direction of the test bottle. A 

new CO2-absorber was placed at the far end of the series. 

Phenolphthalein (1% solution in ethanol, Merck) was used 

as pH-indicator. 

On the 28th day, the pH of all test suspensions was 

measured and 1 ml of concentrated HCl (37%, Merck) was 

added to the bottles of the inoculum blank and test 

suspension. The bottles were aerated overnight to drive off 

CO2 present in the test suspension. The final titration was 

made on day 29. 

Theoretical CO2 production The theoretical CO2 production was calculated from the 

molecular formula. 

6.4.3. Measurements and recording 

pH At the start of the test (day 0) and on the 28th day. 

Temperature of medium Continuously in a vessel with Milli-RO water in the same 

room. 

6.5. Electronic data capture 

Observations/measurements in the study were recorded electronically using the following programme: 

REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, 

NJ, USA): Temperature.

Final Report
- Page 11 - 

6.6. Interpretation 

6.6.1. Data evaluation 

ThCO2, expressed as mg CO2/mg test substance, that can be generated by a test substance was 

calculated as follows: 

Molecular substancetweight tes

No. of carbon atoms in test substance Molecular weight CO2 ThCO2

The first step in calculating the amount of CO2 produced is to correct for background (endogenous) 

CO2 production. Thus the amount of CO2 produced by a test substance is determined by the 

difference (in ml of titrant) between the experimental and blank Ba(OH)2 traps. 

The amount of 0.05 N HCl titrated is converted into mg of CO2 produced: 

44 1.1 ml titratedHCl

2

.0 05 ml titratedHCl

mg CO2

Relative biodegradation values were calculated from the cumulative CO2 production relative to the 

total expected CO2 production based on the total carbon content of the amount of test substance 

present in the test bottles. A figure of more than 10% biodegradation was considered significant. 

The relative biodegradation values were plotted versus time together with the relative biodegradation 

of the positive control. If applicable, the number of days is calculated from the attainment of 10% 

biodegradation until 60% biodegradation. Should this period be ≤ 10 days (10-day window), then the 

test substance is designated as readily biodegradable. 

Toxicity control: if less than 25% biodegradation (based on ThCO2) occurred within 14 days, the test 

substance was assumed to be inhibitory. 

The total CO2 evolution in the inoculum blank was determined by the cumulative difference (in ml of 

titrant) between the blank Ba(OH)2 traps and fresh Ba(OH)2. 

6.6.2. Acceptability of the test 

1. The positive control substance was biodegraded by 69% within 14 days. 

2. The difference of duplicate values for %-degradation of the test substance was always less than 

20.

3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/l (59.3 mg CO2 per 

2 litres of medium, corresponding to 29.7 mg CO2/l). 

4. The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral medium at 

the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test 

medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., 

Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from 

the test substance, 12 mg TOC/l). 

Since all criteria for acceptability of the test were met, this study was considered to be valid. 

Final Report
- Page 12 - 

6.7. List of deviations 

6.7.1. List of protocol deviations 

1. Due to a problem with the dispensor used for filling the washing bottles with 100 ml BaOH2. 

On day 23 it was noted that not all washing bottles titrated contained exactly 100 ml BaOH2. 

The deviation in the volume of BaOH2 is estimated as maximally 10% (minimal 90 ml). 

 Evaluation: As the amounts of HCl titrated for the the duplicate test substance bottles were 

higher than in the blank (this means that no CO2 was produced by the test substance), this 

technical failure had no effect on the final outcome of the test. 

The study integrity was not adversely affected by the deviation. 

6.7.2. List of standard operating procedures deviations 

Any deviations from standard operating procedures were evaluated and filed in the study file. There 

were no deviations from standard operating procedures that affected the integrity of the study. 

7. RESULTS 

7.1. Theoretical CO2 production 

The ThCO2 of was calculated to be 3.36 mg CO2/mg. 

The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg. 

The ThCO2 per test bottle are given in Tables 5-8 (APPENDIX 1.)

7.2. Biodegradation 

All data are presented in APPENDIX 1. The results of CO2 production and biodegradation in blank 

bottles, background bottles and each test bottle are listed in Table 2 to 8. Table 9 contains the 

comparison of biodegradation of in bottles A and B. 

Figure 1 shows the curves for biodegradation of the two bottles with the positive 

control and the toxicity control. 

Figure 1 Biodegradation of and sodium acetate in the modified Sturm test 

Final Report
- Page 13 - 

The relative biodegradation values calculated from the measurements performed during the test 

period revealed 14 and 13% biodegradation of for the duplicate bottles tested. 

Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) 

was not met. 

In the toxicity control more than 25% biodegradation occurred within 14 days (37%, based on ThCO2). 

Therefore, the test substance was assumed not to inhibit microbial activity. 

Functioning of the test system was checked by testing the reference substance sodium acetate, which 

showed a normal biodegradation curve (see also section 6.7.1). 

7.3. Monitoring of temperature and pH 

The temperature recorded in a vessel with water in the same room varied between 21.7 and 22.9°C. 

The pH values of the different test media are presented in Table 1.

Table 1 pH values of different test media.

Test medium: At the start of the test: On day 28:

Blank control (A) 7.6 7.6

Blank control (B) 7.7 7.6

Positive control 7.6 7.8

(A) 7.6 7.6

(B) 7.6 7.6

Toxicity control 7.6 7.8

8. CONCLUSION 

was not readily biodegradable under the conditions of the modified Sturm test 

presently performed. 

Final Report
- Page 14 - 

APPENDIX 1 TABLES 

Notes: All calculations are performed without rounding off. 

 Produced CO2 : negative values are expressed as 0.00 ml HCl. 

Table 2 HCl titrated in duplicate blank bottles

Day HCl (0.05 N) titrated (ml)

Blank A Blank B Mean Value

2 46.69 47.00 46.85

5 46.80 47.11 46.96

7 43.90 45.41 44.66

9 45.67 45.55 45.61

14 44.60 45.06 44.83

19 43.51 43.90 43.71

23 36.84 38.81 37.83

27 43.83 45.14 44.49

29 43.82 44.06 43.94

29 48.05 46.98 47.52

29 48.03 48.36 48.20

Table 3 HCl titrated in Ba(OH)2 solution (background bottles)

Day HCl (0.05 N) titrated (ml)

Bottle A Bottle B Mean value

2 49.87 49.94 49.91

k5 50.37 50.38 50.38

7 50.00 50.00 50.00

9 50.49 48.53 49.51

14 49.41 49.80 49.61

19 51.16 51.09 51.13

23 50.57 49.68 50.13

27 49.18 50.27 49.73

29 48.61 48.65 48.63

29 49.24 49.30 49.27

29 50.63 49.84 50.24

Final Report
- Page 15 - 

APPENDIX 1 TABLES– CONTINUED 

Table 4 CO2 production in the blank.

Day

HCl (0.05 N) titrated (ml) Produced 

CO2

(ml HCl)

Produced 

CO2

(mg)

Cumulative 

CO2

(mg) Ba(OH)2 

1) Blank (mean)

2 49.91 46.85 3.06 3.4 3.4

5 50.38 46.96 3.42 3.8 7.1

7 50.00 44.66 5.35 5.9 13.0

9 49.51 45.61 3.90 4.3 17.3

14 49.61 44.83 4.78 5.3 22.6

19 51.13 43.71 7.42 8.2 30.7

23 50.13 37.83 12.30 13.5 44.2

27 49.73 44.49 5.24 5.8 50.0

29 48.63 43.94 4.69 5.2 55.2

29 49.27 47.52 1.76 1.9 57.1

29 50.24 48.20 2.04 2.2 59.3

1): "Strength" of untreated 0.0125 M Ba(OH)2 solution

Table 5 CO2 production and percentage biodegradation of the positive control substance.

Day

HCl (0.05 N) titrated (ml) Produced 

CO2

(ml HCl)

Produced 

CO2

(mg)

Cumulative 

CO2

(mg)

Biodegradation 1)

(%) Blank 

(mean)

Positive 

control

2 46.85 38.25 8.60 9.5 9.5 11

5 46.96 25.19 21.77 23.9 33.4 39

7 44.66 34.61 10.05 11.0 44.4 52

9 45.61 39.79 5.82 6.4 50.8 59

14 44.83 37.51 7.32 8.1 58.9 69

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of sodium acetate: 85.6 mg CO2/2l

Table 6 CO2 production and percentage biodegradation of the test substance (bottle A).

Day

HCl (0.05 N) titrated (ml) Produced 

CO2

(ml HCl)

Produced 

CO2

(mg)

Cumulative 

CO2

(mg)

Biodegradation 1)

Blank (mean) bottle A (%)

2 46.85 46.62 0.23 0.2 0.2 0

5 46.96 45.47 1.49 1.6 1.9 2

7 44.66 44.14 0.52 0.6 2.4 3

9 45.61 43.12 2.49 2.7 5.2 6

14 44.83 42.84 1.99 2.2 7.4 8

19 43.71 42.20 1.51 1.7 9.0 10

23 37.83 39.66 0.00 0.0 9.0 10

27 44.49 43.29 1.20 1.3 10.3 12

29 43.94 43.05 0.89 1.0 11.3 13

29 47.52 46.82 0.70 0.8 12.1 14

29 48.20 48.33 0.00 0.0 12.1 14

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 87.0 mg CO2/2l

Final Report
- Page 16 - 

APPENDIX 1 TABLES– CONTINUED 

Table 7 CO2 production and percentage biodegradation of the test substance (bottle B).

Day

HCl (0.05 N) titrated (ml) Produced 

CO2

(ml HCl)

Produced 

CO2

(mg)

Cumulative 

CO2

(mg)

Biodegradation 1)

Blank (mean) bottle B (%)

2 46.85 46.99 0.00 0.0 0.0 0

5 46.96 45.49 1.47 1.6 1.6 2

7 44.66 44.13 0.52 0.6 2.2 3

9 45.61 45.16 0.45 0.5 2.7 3

14 44.83 42.90 1.93 2.1 4.8 6

19 43.71 42.25 1.46 1.6 6.4 7

23 37.83 41.38 0.00 0.0 6.4 7

27 44.49 42.50 1.99 2.2 8.6 10

29 43.94 42.29 1.65 1.8 10.4 12

29 47.52 46.87 0.65 0.7 11.1 13

29 48.20 47.93 0.27 0.3 11.4 13

1): Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 86.7 mg CO2/2l

Table 8 CO2 production and percentage biodegradation of the toxicity control.

Day

HCl (0.05 N) titrated (ml) Produced 

CO2

(ml HCl)

Produced 

CO2

(mg)

Cumulative 

CO2

(mg)

Biodegradation 1)

(%) Blank 

(mean)

toxicity 

control

2 46.85 36.97 9.88 10.9 10.9 6

5 46.96 26.81 20.15 22.2 33.0 19

7 44.66 32.34 12.32 13.5 46.6 27

9 45.61 39.20 6.41 7.1 53.6 31

14 44.83 35.41 9.42 10.4 64.0 37

1): Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test substance and

 positive control: 173.3 mg CO2/2l (ThCO2 test substance: 87.7 mg CO2/2l + ThCO2 sodium acetate: 85.6 mg CO2/2l)

Table 9 Comparison of biodegradation of the test substance in bottles A and B.

Day Biodegradation (%)

Bottle A Bottle B Mean A and B ∆ A-B 1)

2 0 0 0 0

5 2 2 2 0

7 3 3 3 0

9 6 3 5 3

14 8 6 7 3

19 10 7 9 3

23 10 7 9 3

27 12 10 11 2

29 13 12 13 1

29 14 13 13 1

29 14 13 14 1

1): Absolute difference in biodegradation between bottles A and B

Final Report