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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to international guideline(s), GLP-compliant, performed in recognized contract research organization, no restrictions, fully adequate for assessment.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: sewage plant for domestic sewage in 8200 Veszprém, Hungary
- Method of cultivation / Preparation of inoculum for exposure: The activated sludge was washed by centrifugation (for 10 min.) and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged (for 10 min.). This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre were mixed with test water and then aerated until use (not later than an hour). Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial conc.:
2.9 mg/L
Based on:
test mat.
Initial conc.:
ca. 6 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral salts medium according to guidelines, prepared using deionised water.
- Test temperature: 21-23 °C
- pH: The pH value of the test water was checked prior study start. The pH of the test water was 7.4.
- Aeration of dilution water: The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was 8.9 mg/L at about 22 °C.
- Suspended solids concentration: 0.5 mL microbial inoculum/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BOD bottles (300 mL) with glass stoppers, temperature-controlled incubator
- Number of culture flasks/concentration and sampling date: 2
- Measuring equipment: oxygen meter with a stirring O2 electrode

SAMPLING
- Sampling frequency: Day 0, 7, 14, 21 and 28
- Sampling method: Two bottles from each study group were removed for measurement of the oxygen content.

CONTROL AND BLANK SYSTEM
- Inoculum blank (2 flasks/sampling date): inoculated mineral salts medium (MSM)
- Reference (2 flasks/sampling date): inoculated MSM + 3.6 mg sodium benzoate/L (corresponding to 6.0 mg O2/L)
- Toxicity control (2 flasks/sampling date): inoculated MSM + 2.9 mg test material/L (6.0 O2/L) + 3.6 mg sodium benzoate/L (6.0 mg O2/L)
Reference substance:
benzoic acid, sodium salt
Preliminary study:
During a 14-day preliminary test, the concentration of dissolved oxygen resulted a mean of 7.40 mg O2/L after 14 days of incubation in the test item flasks, which was nearly at the control level (7.80 mg O2/L). Therefore, measurement of the total oxidised nitrogen (nitrate and nitrite) concentrations was not performed in the main test.
Key result
Parameter:
% degradation (O2 consumption)
Value:
13
Sampling time:
28 d
Remarks on result:
other: mean of duplicates
Details on results:
Biodegradation of the test material at test end: 11% and 14% based on COD (Table 1).

In the toxicity control (test item + sodium benzoate), a mean of 36% biodegradation was noted within 14 days and 45% biodegradation after 28 days of incubation indicating that the test substance was not inhibitory to the microbial inoculum (Table 1).
Results with reference substance:
The reference substance sodium benzoate had achieved 73% of the ThOD after 14 days of incubation and 91% by Day 28 (Table 1), thus confirming the suitability of the used activated sludge inoculum.

Table 1 Biodegradation of the test and reference substance

Treatment

Flask

Percent of biodegradation after n days of exposure

 

No.

7

14

21

28

Test item

a

3.3

4.2

10.0

10.9

b

1.7

5.9

10.0

14.2

mean

2.5

5.0

10.0

12.6

Reference item

a

56.7

70.8

85.0

89.2

b

58.3

75.8

88.3

92.5

mean

57.5

73.3

86.7

90.8

Toxicity control

a

28.9

35.9

43.8

45.0

b

28.1

36.7

44.6

45.0

mean

28.5

36.3

44.2

45.0

 

Validity criteria fulfilled:
yes
Remarks:
Difference of extremes of replicate values of the removal of the test substance <20%; degradation of the reference compound ≥60% by 14 days; residual O2 content ≥ 0.5 mg/L; oxygen uptake of the inoculum blank < 1.5 mg/L after 28 days.
Interpretation of results:
under test conditions no biodegradation observed

Description of key information

Biodegradation in water: screening tests: aerobic biodegradation 13% (COD) in 28 days (OECD 301D, EU C.4-E)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information