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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:
From October 4, 2004 to November 3, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD Guideline 301 B, with GLP.
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge was collected from a sewage treatment works A-2500 Baden, which waste-water catchment is predominantly domestic.
- Preparation of inoculum for exposure: On arrival in the laboratory, the sample was aerated by means of a filtered compressed air before being used for the study. The suspended solids concentration was determined by filtering a 5 mL sample through a pre-dried and pre-weighed glass filter (Whatman GF/C). The filter with solids were dried at 105 °C and re-weighed and the sludge solids determined by difference.

- Pretreatment:
- Concentration of sludge: not more than 30 mg of suspended solids/L
Duration of test (contact time):
28 d
Initial conc.:
15 other: mg/L (of organic carbon)
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Test temperature: 21.6 ± 0.5 ºC
- pH: 7.35 – 7.64 (No major deviations were seen during the study time)
- pH adjusted: no
- Suspended solids concentration: in all vessels was about 20 mg of suspended solids of the sludg/L.
- Continuous darkness: yes


TEST SYSTEM
- Method used to create aerobic conditions: The air flow was regulated for each vessel individually. Air flow was determinated volumetrically in intervals. Adjustments were made as necessary to maintain a flow rate in the range of 30 to 100 mL per minute (1 – 3 bubbles per sec). As an added precaution, a 0.0125 M barium hydroxide solution was used as CO2 absorber.

- Measuring equipment: The amount of CO2 produced was determined by titrating the remaining Barium hydroxide with 0.05 M HCl (1.0997.3 Titrisol®, Merck KGaA, 64271 Darmstadt, Germany). Phenolphtalein (ACS Merck, 64271 Darmstadt, Germany) was used as a pH indicator. A factor of 5 was used to convert titres for 20 mL samples to values for the entire 100 mL wash-bottle contents and a second factor of 1.1 to convert volume-corrected titres to milligrams CO2.
- Details of trap for CO2 and volatile organics if used: Barium hydroxide was used to trap the CO2 formed by the degradation of the test substance.

SAMPLING
- Sampling frequency: The CO2 evolution was determined on Days 1, 2, 4, 6, 8, 10, 15, 20, 25 and 28/29
- Sample storage before analysis: The samples were analysed on the same day, except for Day 28, at which the incubation media were acidified with 1 mL 32 % HCl (Merck KGaA, 64271 Darmstadt, Germany) to release any residual CO2. CO2 was removed by continuous air flow overnight and absorber solutions were titrated on Day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, the medium plus inoculum was used as negative control.
- Toxicity control: yes, this contained sodium benzoate and the test substance.
- Other: sodium benzoate was used as positive control substance.


Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
23.6
Sampling time:
28 d
Details on results:
Toxicity control: Degradation in the toxicity control, which contained sodium benzoate and the test substance, was not indicative of a significant inhibition of the microbial activity by the test substance. The final biodegradation was 49.6 %.
Results with reference substance:
The plateau of biodegradation was reached on about Day 10 and the degradation of the positive control substance sodium benzoate exceeded the pass level of 60 % on about Day 6.
Biodegradation as percentage of the theoretical CO2 yield

Elapsed time

test substance

positive control

toxicity control

Days

A13

A14

mean

PK5

TK16

1

0.2

0.0

0.1

6.5

3.9

2

1.7

1.7

1.7

25.3

12.5

4

3.2

3.7

3.5

47.6

22.2

6

6.6

7.0

6.8

63.9

30.1

8

9.3

9.9

9.6

75.3

34.8

10

10.8

10.5

10.7

81.5

37.9

15

11.8

11.7

11.8

87.1

40.4

20

14.3

12.1

13.2

92.2

42.5

25

16.2

14.0

15.1

95.8

45.0

28

23.7

23.5

23.6

103.5

49.6
Validity criteria fulfilled:
yes
Remarks:
(The percentage degradation of the positive control exceeded 60 % within ten days of first reaching 10 %. The total mean blank CO2 production in this study was 32.1 mg/L).
Interpretation of results:
not readily biodegradable
Conclusions:
The carbon dioxide evolution from the test substance with a nominal concentration of 15 mg Carbon/L reached a maximum of 23.6 % after 28 days of incubation at 21.6 ± 0.5 °C. According to the results of this study he test substance is not readily biodegradable.

Executive summary:

The biodegradability of the test substance was analyzed by the determination of carbon dioxide evolution at frequent intervals over a 28 days period ( intervals: 1, 2, 4, 6, 8, 10, 15, 20, 25 and 28/29 days).

The test substance was suspended in buffered mineral salts medium at a nominal concentration of 15 mg organic carbon per liter. The medium was inoculated with microorganisms derived from a sample of activated sludge not previously exposed to the test substance. The mixtures were incubated in the darkness at 21.6 ºC ± 0.5 ºC for 28 days under constant stirring.

The reference substance sodium benzoate was degraded by 81.5 % within 10 days

Degradation in the toxicity control, which contained sodium benzoate and the test substance, was not indicative of a significant inhibition of the microbial activity by the test substance.

The carbon dioxide evolution from the test material reached a maximum of 23.6 % after 28 days of incubation at 21.6 ± 0.5 °C. According to the results of this study the test substance is not readily biodegradable.

Description of key information

According to the results of this study the test substance is not readily biodegradable.

Key value for chemical safety assessment

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

Additional information

The biodegradability of the test substance was analyzed by the determination of carbon dioxide evolution at frequent intervals over a 28 days period (intervals:1, 2, 4, 6, 8, 10, 15, 20, 25 and 28/29 days).

The test substance was suspended in buffered mineral salts medium at a nominal concentration of 15 mg organic carbon per liter. The medium was inoculated with microorganisms derived from a sample of activated sludge not previously exposed to the test substance. The mixtures were incubated in the darkness at 21.6 ºC ± 0.5 ºC for 28 days under constant stirring.

The reference substance sodium benzoate was degraded by 81.5 % within10 days

Degradation in the toxicity control, which contained sodium benzoate and the test substance, was not indicative of a significant inhibition of the microbial activity by the test substance.

The carbon dioxide evolution from the test material reached a maximum of 23.6 % after 28 days of incubation at 21.6 ± 0.5 °C.