# FINAL REPORT

Study Title



# DETERMINATION OF 'RE ADY' BIODEGRADABILIT Y: CARBON

DIOXIDE (CO 2 

# ) EVOLUTION TEST (MO DIFIED STURM TEST) O F

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 CO 2



### .................................................................................................... 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 CO 2 

### 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

T able 2 HCl titrated in duplicate blank bottles

........................................................................... 14

Table 3 HCl titrated in Ba(OH) 2 

### solution (background bottles)

.................................................. 14

Table 4 CO 2 

### production in the blank.

......................................................................................... 15

Table 5 CO 2 

### production and percentage biodegradation of the positive control substance.

..... 15

Table 6 CO 2 

### production and percentage biodegradation of the test substance (bottle A).

....... 15

Table 7 CO 2 

### production and percentage biodegradation of the test substance (bottle B).

....... 16

Table 8 CO 2 

### 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

### - Page 5 -

4. SUMMARY

Determination of ‘ready’ biodegradability: carbon dioxide (CO 2 

### ) 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 CO 2 

### production (ThCO 2 

### ) of was calculated to be 3.36 mg CO 2 

### /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 CO 2 

### -measurement on the 29 th 

### 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.

### - 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: CO 2 

### 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 (CO 2 

### ) 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.

### - 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 (ThCO 2 

### ) 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

Identificatio n

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 con tainer with nitrogen after handling

Stability in water At least 24 hours

Solubility in water No

### - 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 CH 3 

### COONa (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.

### - Page 9 -

6.4. Test procedure and conditions

Test duration 28 days (last CO 2 

### -measurement on the 29 th 

### 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 KH 2 

### PO 4 

### mineral components 21.75 g K 2 

### HPO 4 

### 67.20 g Na 2 

### HPO 4 

### .12H 2 

### O

0.50 g NH 4 

### Cl

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

pH 7.4 ± 0.2

B) 22.50 g MgSO 4 

### .7H 2 

### O dissolved in Milli-Q water

and made up to 1 litre.

C) 36.40 g CaCl 2 

### .2H 2 

### O dissolved in Milli-Q water and

made up to 1 litre.

D) 0.25 g FeCl 3 

### .6H 2 

### O 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 CO 2 

### from the air.

Synthetic air (CO 2 

### < 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 CO 2 

### 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 CO 2



### .

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).

### - 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 CO 2 

### -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 CO 2



### Experimental CO 2 

### production The CO 2 

### produced in each test bottle reacted with the

barium hydroxide in the gas scrubbing bottle and

precipitated out as barium carbonate. The amount of CO 2 

### 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

28 th 

### day, for the inoculum blank and test suspension.

Titrations for the positive and toxicity control were made at

least 14 days.

Each time the CO 2 

### -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 CO 2 

### -absorber was placed at the far end of the series.

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

as pH-indicator.

On the 28 th 

### 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

CO 2 

### present in the test suspension. The final titration wa s made on day 29.

Theoretical CO 2 

### production The theoretical CO 2 

### 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 28 th 

### 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.

### - Page 11

-

6.6. Interpretation

6.6.1. Data evaluation

ThCO 2 

### , expressed as mg CO 2 

### /mg test substance, that can be generated by a test substance was

calculated as follows:

The first step in calculating the amount of CO 2 

### produced is to correct for background (endogenous)

CO 2 

### production. Thus the amount of CO 2 

### 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 CO 2 

### produced:

Relative biodegradation values were calculated from the cumulative CO 2 

### production relative to the

total expected CO 2 

### 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 ThCO 2 

### ) occurred within 14 days, the test

substance was assumed to be inhibitory.

The total CO 2 

### 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. Th e 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 CO 2 

### release in the blank at the end of the test did not exceed 40 mg/l (59.3 mg CO 2 

### per 2 litres of medium, corresponding to 29.7 mg CO 2 

### /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.

### - 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 BaOH 2



### . On day 23 it was noted that not all washing bottles titrated contained exactly 100 ml BaOH 2



### . The deviation in the volume of BaOH 2 

### 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 CO 2 

### 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 CO 2 

### production

The ThCO 2 

### of was calculated to be 3.36 mg CO 2 

### /mg.

The ThCO 2 

### of sodium acetate was calculated to be 1.07 mg CO 2 

### /mg.

The ThCO 2 

### per test bottle are given in Tables 5- 8 ( APPENDIX 1

. )

7.2. Biodegradation

All data are presented in APPENDIX 1. The results of CO 2 

### 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

### - 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 ThCO 2 

### ).

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

To xicity control 7.6 7.8



### 8. CONCLUSION

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

presently performed.

### - Page 14

-

APPENDIX 1 TABLES

Notes: All calculations are performed without rounding off.

Produced CO 2 : 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

### - Page 15

-

APPENDIX 1 TABLES

– CONTINUED

Table 4 CO 2 production in the blank.

Day HCl (0.05 N) titrated (ml) Produced

CO 2 (ml HCl) Produced

CO 2 (mg) Cumulative

C O 2 (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 CO 2 production and percentage biodegradation of the positiv e control substance.

Day HCl (0.05 N) titrated (ml) Produced

CO 2 (ml HCl) Produced

CO 2 (mg) Cumulative

CO 2 (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 CO 2 produced (cumulative) and the ThCO 2 of sodium acetate: 85.6 mg CO 2 /2l

Table 6 CO 2 production and percentage b iodegradation of the test substance (bottle A).

Day HCl (0.05 N) titrated (ml) Produced

CO 2 (ml HCl) Produced

CO 2 (mg) Cumulative

CO 2 (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 48.2 0 48.33 0.00 0.0 12.1 14

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

### - Page 16

-

APPENDIX 1 TABLES

– CONTINUED

Table 7 CO 2 production and percentage biodegradation of t he test substance (bottle B).

Day HCl (0.05 N) titrated (ml) Produced

CO 2 (ml HCl) Produced

CO 2 (mg) Cumulative

CO 2 (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 CO 2 produced (cumulative) and the ThCO 2 of the test substance: 86.7 mg CO 2 /2l

Table 8 CO 2 production and percentage biodegradation of the toxicity control.

Day HCl (0.05 N) titrated (ml) Produced

CO 2 (ml HCl) Produced

CO 2 (mg) Cumulative

CO 2 (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 CO 2 produced (cumulative) and the sum of the ThCO 2 of the test substance and

positive control: 173.3 mg CO 2 /2l (ThCO 2 test substance: 87.7 mg CO 2 /2l + ThCO 2 sodium acetate: 85.6 mg CO 2 / 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