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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 May - 04 Jun 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
- Source of inoculum: Taken from the secondary effluent of a domestic sewage treatment plant Odenthal, Germany (collected on 30 Apr 1999).
- Laboratory culture: no
- Pretreatment: Separation of coarse particles by filtration. Aerated for 7 d.
- Concentration of inoculum: 5 mL/L
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline
- Test temperature: 20 ± 1 °C
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2

SAMPLING
- Sampling frequency: On day 0, 7, 14, 21 and 28.
- Sampling method: 2 bottles were removed on each sampling day to measure the oxygen consumption.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 10 bottles
- Abiotic sterile control: no
- Toxicity control: yes, 10 bottles
- Other: Reference substance: yes, 10 bottles
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
24
Sampling time:
28 d
Remarks on result:
other: Mean of two replicates
Results with reference substance:
77% after 14 d and 91% after 28 d. The result with the reference substance is valid.

BIOLOGICAL RESULTS:

Table 1: Degradation of the test substance

 

Degradation [%]

 

7 d

14 d

21 d

28 d

Replicate 1

6

19

17

24

Replicate 2

10

20

18

23

Mean

8

20

18

24

Reference substance 1

68

77

77

92

Reference substance 2

67

77

76

89

Mean

68

77

77

91

Toxicity control 1

31

39

38

39

Toxicity control 2

31

39

39

39

Mean

31

39

39

39

VALIDITY CRITERIA:

Table 2: Validity criteria for OECD 301.

Criterion from the guideline

Outcome

Validity criterion fulfilled

Percentage degradation of the reference compound has reached the pass levels by day 14.

77% by day 14

yes

The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.

39% by day 14

yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
24% biodegradation was observed after 28 d in the study according to OECD guideline 301 D showing that the substance is not readily biodegradable.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Non-GLP guideline study
Qualifier:
according to
Guideline:
other: BOD test for insoluble substances (BODIS), preliminary guideline of the Bundesumweltamt Berlin (1990)
Qualifier:
equivalent or similar to
Guideline:
other: ISO 10708: Water quality -- Evaluation in an aqueous medium of the ultimate aerobic biodegradability of organic compounds -- Determination of biochemical oxygen demand in a two-phase closed bottle test
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was collected from the aeration tank of the municipal sewage treatment plant, Pforzheim, Germany.
- Laboratory culture: no
- Preparation of inoculum for exposure: Washed twice by centrifugation (10 min, 6000 rpm) with mineral solution. Supernatant was decanted and the pellet was mixed with mineral solution to give a concentration of 3.0 g/L. 2 L of mineral medium were inoculated with 20 mL of the activated sludge. 200 mL were transferred into 9 test vessels together with magnetic stirring bars. Bottles were closed with glass stoppers and shaken without test substances for 1 week in the dark at 20 °C and 100 rpm.
- Concentration of sludge: 3 g/L
Duration of test (contact time):
28 d
Initial conc.:
>= 4.15 - <= 4.45 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline
- Test temperature: 20 °C
- pH: 6.8 - 7.5 (7 d, before aeration), 7.0 - 7.2 (15 d, before aeration), 7.1 - 7.4 (21 d, before aeration), 7.6 - 7.8 (28 d, before aeration)
- Continuous darkness: yes
- Other: incubated on a rotary shaker at 100 rpm

TEST SYSTEM
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: 15 min prior to incubation the dilution water was aerated
- Measuring equipment: Oximeter (WTW Oxi 191)

SAMPLING
- Sampling frequency: On day 7, 15, 21 and 28.
- Other: After the measurements the test assays were aerated for 15 min, measured once more and were incubated for the remaining test period.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 3 bottles
- Abiotic sterile control: no
- Toxicity control: no
- Other: Reference substance: yes, 3 bottles
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
24.1
Sampling time:
28 d
Remarks on result:
other: Mean of three replicates
Results with reference substance:
The reference substance was degraded to 65.1% after 28 d. After 14 d 60% degradation was reached.

Table 1: Degradation rates after 7, 15, 21 and 28 d

 

Biodegradation [%]

7 d

15 d

21 d

28 d

Test substance 1

18.1

28.9

31.1

31.7

Test substance 2

4.6

12.6

16.5

16.5

Test substance 3

10.4

17.1

24.1

24.1

Mean

10.9

19.5

23.9

24.1

 

 

 

 

 

Reference substance 1

55.5

63.5

63.5

63.5

Reference substance 2

52.0

59.9

59.9

59.9

Reference substance 3

59.0

72.0

72.0

72.0

 Mean

55.5

65.1

65.1

65.1

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
24.1% biodegradation was observed after 28 d in the study according to the preliminary guideline of the German Environmental Agency (BODIS test) showing that the substance is not readily biodegradable.

Description of key information

Not readily biodegradable: 24% biodegradation after 28 d (EU Method C.4-E)

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

Two studies are available, testing the ready biodegradability of the substance by non-adapted sewage treatment plant microorganisms.

The key study was performed according to the EU Method C.4-E and GLP. The test was run for 28 days and microorganisms were exposed to 3 mg/L test material. O2 consumption was followed for the estimation of the biodegradation. Sodium benzoate was used as a reference substance. A toxicity control was set up with 3 mg/L test item and 2.9 mg/L reference substance in order to examine the toxicity of the substance on microorganisms. 24% of the substance was biodegraded after 28 d, and therefore it is not considered readily biodegradable. The toxicity control attained 39% degradation after 14 d, verifying that the tested item was not inhibitory to the inoculated microorganisms.

An available BODIS test (according to the preliminary guideline of the German Environmental Agency) confirms the result of the key study. The microorganisms were exposed to 4.15 – 4.45 mg/L substance for 28 d and the O2 consumption was followed as an indicator for biodegradation. Benzoic acid (0.104 mg/mL ThOD) was used as a reference substance while no toxicity control was run in parallel. 24.1% biodegradation was observed after 28 d showing that the substance is not readily biodegradable.

In conclusion all available results indicate that the substance is not rapidly biodegradable.