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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:
09 Nov - 09 Dec 1999
Reliability:
2 (reliable with restrictions)
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)
GLP compliance:
yes (incl. certificate)
Remarks:
Umweltministerium Baden-Württemberg, Stuttgart, Germany
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: effluent from municipal sewage treatment plant, Pforzheim, Germany
- Storage conditions: The effluent was kept under aerobic conditions in the period between sampling and application.
- Preparation of inoculum for exposure: The inoculum was filtered through a coarse filter (Schwarzband, Schleicher & Schüll), the first 200 mL was discarded.
- Storage length: the inoculum was used on the day of collection
- Initial cell concentration: The initial number of microorganisms was determined separately for each batch (blank, test substance, reference and toxicity control) and were as follows: Inoculum blank: 2.4E+02 cells/mL, 7.92 E+05 cells/bottle; Reference: 2.5E+02 cells/mL, 8.25E+04 cells/bottle; Toxicity test: 2.2E+02 cells/mL, 7.26E+04 cells/bottle; Test substance: 2.9E+02 cells/mL; 9.57E+04 cells/bottle
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium was prepared from 4 stock solutions containing 8.5 mg/L KH2PO4, 28.5 mg/L K2HPO4*3H2O, 33.3 mg/L Na2HPO4*2H2O, 0.5 mg/L NH4Cl, 22.5 mg/L MgSO4*7H2O, 34.2 mg/L CaCl2*2H2O, and 0.15 mg/L FeCl3.
- Test temperature: 20 ± 2 °C
- pH: 7.4 ± 0.2
- Aeration of dilution water: The medium was strongly aerated for 60 min and allowed to stand for 24 h at the test temperature. The O2 content was measured afterwards.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BOD flasks with ground-in-glass stoppers
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: The effluent was kept under aerobic conditions. The mineral medium was aerated for 60 min and allowed to stand for 24 h at the test temperatue.
- Measuring equipment: O2 concentrations were measured with a MTW Microprocessor Oximeter OXI 196 and a calibrated electrode EO 196-1,5. Each of the 3 parallel test assays was measured twice.
- Test performed in closed vessels due to significant volatility of test substance: The test was a Closed Bottle Test according to OECD guideline 301 D. The calculated vapor pressure of the test substance was estimated at 3.8 Pa.

SAMPLING
- Sampling frequency: 0 h, 7, 14, 21, and 28 d
- Sampling method: 3 bottles (assays) per measurement date. Each solution was immediately filled into the test vessels (volume approximately 330 mL) and the O2 concentrations were measured.
- Sample storage before analysis: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 3 replicates
- Toxicity control: yes, 3 replicates
- Reference substance: yes, 3 replicates
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
-8.5
Sampling time:
28 d
Details on results:
The negative values of degradation rates result from lower oxygen saturation rates in the test substance study group after aeration.

The determined 28 d degradation rate for the reference substance was 91.8% and 54.2% for the toxicity control (reference substance + test substance).
Results with reference substance:
The reference substance was degraded to 91.8% (mean) within 28 d.

The calculated theoretical oxygen demand (ThOD) of the test substance was 2.34 mg O2/mg test substance.

The calculated theoretical oxygen demand (ThOD) of the reference substance was 1.67 mg O2/mg reference substance.

The calculated theoretical oxygen demand (ThOD) of the toxicity control was 1.00 mg O2/mg substance mixture.

On average, the initial oxygen contents of the aerated test media were as follows:

- 8.9 mg/L inoculum blank

- 8.9 mg/L reference substance

- 8.6 mg/L toxicity control

- 8.6 mg/L test substance

Table 1: Individual calculations of the degradation rates and average values.

 

% degradation rates

Bottle n°

Reference

Test substance

7 d

14 d

21 d

28 d

7 d

14 d

21 d

28 d

1

70.1

73.7

69.5

80.8

-12.8

-16.7

-12.4

-6.4

2

70.1

66.5

64.7

125.7

-17.9

-13.2

-13.2

-9.4

3

68.9

77.2

65.3

68.9

-11.5

-9.0

-11.5

-9.4

average

69.7

72.5

66.5

91.8

-14.1

-13.0

-12.4

-8.4

Bottle n°

Toxicity test

 

 

 

 

7 d

14 d

21 d

28 d

 

 

 

 

1

54.9

56.9

52.9

54.9

 

 

 

 

2

50.9

53.9

50.9

53.9

 

 

 

 

3

57.9

53.9

48.9

53.9

 

 

 

 

average

54.5

54.9

50.9

54.2

 

 

 

 


Since the pass levels for ready biodegradability are > 60% removal of ThOD within the 28 d period of the test, the test substance cannot be classified as readily biodegradable.

The degradation rate of the toxicity control was > 25% (54.9%) after 14 d. Therefore, toxic efffects of the test substance to the inoculum can be excluded.


 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
No degradation of the substance was recorded after 28 d of incubation (-8.5% based on O2 consumption). Thus, the substance is not readily biodegradable according to the OECD criteria.

Description of key information

Not readily biodegradable (0% within 28 d, OECD 301 D)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

One GLP guideline study according to OECD 301D is available investigating the ready biodegradability of the substance. A mixed microbial population from the effluent of a local municipal STP was inoculated with a nominal concentration of 2 mg/L test substance and incubated for 28 d under controlled conditions in the dark. Degradation was followed by the determination of dissolved oxygen content after 0 h, 7, 14, 21 and 28 d. An inoculum blank, a procedure control (with 2 mg reference substance/L) and a toxicity control were run in parallel. The derived 28 d degradation rate was -8.5% for the test substance and 54.2% for the toxicity control. The negative value resulted from lower oxygen saturation rates in the test substance study groups after aeration. The pass level for ready biodegradability is > 60% removal of ThOD within 28 d, meeting the 10 d - window. Therefore, the test substance could not be classified as readily biodegradable. Since the degradation rate of the toxicity control was > 25% after 14 d, toxic effects of the test substance to the inoculum can be excluded.