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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November 2007-20 December 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: R507-2
Molecular weight: 553.05
CAS number: 15792-20-8
Description: Red powder
Batch: 070711
Purity: >/= 96.4 %
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 11 July 2011
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands, receiving predominantly domestic sewage.

The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.2 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle for 65 minutes and the liquid was decanted for use as inoculum at the amount of 10 ml/L of mineral medium.
Duration of test (contact time):
28 d
Initial conc.:
26.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test concentration and preparation of test solutions
The batch of R507-2 was a red powder with a purity of at least 96.4 %. R507-2 was tested in duplicate at 53 mg per 2 litres, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula.

Since R507-2 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts of R507-2 were added to the test bottles (2 litres) containing medium with microbial organisms and mineral components (test substance bottle A: 52.91 mg; test substance bottle B: 53.10 mg and toxicity control bottle: 52.89 mg). To this end, 1O 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. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface.

Reference substance concentration and preparation of test solutions
A solution of sodium acetate was prepared by dissolving 799.48 mg in Milli-RO water and making this up to a total volume of 200 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/L sodium acetate (12 mg TOC/L).

Test duration
28 days (last CO2-measurement on the 29th day) for the inoculum blank and test suspension and at least 14 days for the positive and toxicity control.
During the test period the test media were aerated and stirred continuously.

Test vessels
2 litre all-glass brown coloured bottles

Preparation of bottles
Pre-incubation medium - 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).
lnoculum 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).

Preparation - The test substance and positive control were added to the bottles containing the microbial organisms and mineral components (ca. 80 % of total volume).
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.
Reference substance:
other: Sodium acetate
Test performance:
In the toxicity control more than 25 % degradation occurred within 14 days (35 %, based on ThCO2) . Therefore, the test substance was assumed not to inhibit microbial activity.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
6
Sampling time:
29 d
Remarks on result:
other: Average result from 2 runs
Details on results:
Theoretical CO2 production

The ThC02 of R507-2 was calculated to be 1.67 mg CO2/mg.
The ThC02 of Sodium Acetate was calculated to be 1.07 mg CO2/mg.

Table CO2 production and percentage biodegradation of the test substance (bottleA).

 

 

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2 (mg)

Cumulative CO2 (mg)

Degradation1 (%)

Blank (mean)

bottle A

2

43.40

43.21

0.18

0.2

0.2

0

5

43.54

43.77

0.00

0.0

0.2

0

7

44.18

43.09

1.08

1.2

1.4

2

9

46.75

46.74

0.00

0.0

1.4

2

14

44.45

44.28

0.16

0.2

1.6

2

19

39.21

36.62

2.59

2.8

4.4

5

23

42.06

40.06

2.00

2.2

6.6

8

27

45.02

44.08

0.94

1.0

7.7

9

29

46.20

46.43

0.00

0.0

7.7

9

29

46.58

48.54

0.00

0.0

7.7

9

29

47.77

47.46

0.31

0.3

8.0

9

1l :Calculated as the ratio between CO2 produced (cumulative) and the ThC02 of the test substance: 88.4 mg CO/21

 

 

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

 

 

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Degradation (%)

Blank (mean)

bottle B

2

43.40

42.48

0.91

1.0

1.0

1

5

43.54

43.26

0.28

0.3

1.3

1

7

44.18

42.48

1.70

1.9

3.2

4

9

46.75

46.93

0.00

0.0

3.2

4

14

44.45

45.35

0.00

0.0

3.2

4

19

39.21

42.36

0.00

0.0

3.2

4

23

42.06

43.12

0.00

0.0

3.2

4

27

45.02

45.78

0.00

0.0

3.2

4

29

46.20

45.96

0.24

0.3

3.4

4

29

46.58

47.62

0.00

0.0

3.4

4

29

47.77

48.11

0.00

0.0

3.4

4

1l :Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 88.7 mg C02

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
R507-2 was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

Determination of 'ready' biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with R507-2.

 

The study procedure was based on EEC directive 92/69, C.4-C, December 1992, OECD guideline No. 301 B July 17, ·1992 and ISO Standard 9439 (1999).

 

The batch of R507-2 was a red powder with a purity of at least 96.4 %. R507-2 was tested in duplicate at 53 mg per 2 litres, corresponding to 12 mg TOC/I. The organic carbon content was based on the molecular formula. The Theoretical CO2 production (ThCO2) of R507-2 was calculated to be 1.67 mg CO2/mg.

 

Since R507-2 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts of R507 -2 were added to the test bottles (2 litres) 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. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface.

 

The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of R507-2.

In the toxicity control, R507-2 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, R507-2 was not readily biodegradable.

Description of key information

Determination of 'ready' biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with R507-2.

 

The study procedure was based on EEC directive 92/69, C.4-C, December 1992, OECD guideline No. 301 B July 17, ·1992 and ISO Standard 9439 (1999).

 

The batch of R507-2 was a red powder with a purity of at least 96.4 %. R507-2 was tested in duplicate at 53 mg per 2 litres, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula. The Theoretical C02production (ThCO2) of R507-2 was calculated to be 1.67 mg CO2/mg.

 

Since R507-2 was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1g/L, weighed amounts of R507-2 were added to the test bottles (2 litres) 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. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface.

 

The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of R507-2.

In the toxicity control, R507-2 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, R507-2 was not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information