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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:
04 Mar - 01 Apr 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
adopted 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim
Oxygen conditions:
aerobic
Inoculum or test system:
other: supernatant from the centrifuged activated sludge of a local municipal STP
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant, D-31137 Hildesheim
- Storage conditions: by aeration with CO2 free air
- Storage length: 4 days
- Preparation of inoculum for exposure: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration for 4 hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air for 4 days.
- Concentration of sludge: 1.7 x 10^9 cfu/L (corresponding to cfu concentration of the inoculum in the final test solution)
- Initial cell/biomass concentration: 0.2 mL inoculum per test vessel was used to initiate inoculation. The incubation bottles were closed without air bubbles.
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
ThOD
Remarks:
2.57 mg O2/mg
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline (Mineral salts medium according to guideline)
- Test temperature: 20 - 21 °C
- pH: 7.37
- Aeration of dilution water: yes. One day before test start the demineralised water for the test medium was aerated until oxygen saturation and was then left standing at room temperature for at least 20 h
- Continuous darkness: yes
- Other: Detailes of parameters of the test media is listed in Table 1 in the section "Any other information on material and methods incl. tables"

TEST SYSTEM
- Culturing apparatus: lncubator (RUBARTH), BOD bottles 300 mL with glass stoppers
- Number of culture flasks/concentration: 5 x 2 per test item, reference item, toxicity control and control
- Method used to create anaerobic conditions: the activated sludge was maintained in an aerobic condition by aeration for 4 hours
- Measuring equipment: Oximeter, HQ 40d multi, HACH
- Test performed in closed vessels due to significant volatility of test substance: yes

SAMPLING
- Sampling frequency: 0, 7, 14, 21 and 28 days
- Sampling method: oxygen concentration was measured

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 bottles per sampling
- Toxicity control: yes, 2 bottles per sampling
- Other: Reference control: yes, 2 bottles per sampling

STATISTICAL METHODS: no statistics
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
80
Sampling time:
28 d
Details on results:
The test item reached the 10% level (end of lag-phase) after 3 days. The pass level of a biodegradation of > 60% was reached after 12 days. The biodegradation came to a maximum of 87% after 21 days. The test item is classified as readily biodegradable within the 10-d-window.
The biodegradation rate of the toxicity control came to 87% after 14 days. The biodegradation of the reference item was not inhibited by the test item. The reference control reached the pass level > 60% after 7 days.
Results in details are listed in Table 1 and 2 within the section "any other information on results incl. tables".
Results with reference substance:
The reference control reached the pass level > 60% after 7 days.

Table 1: Biodegradation of the Test ltem in Comparison to the Reference Control and the Toxicity Control

 

biodegradation [%]

 

study day [d]

 

 

 

7

 

14

 

21

 

28

Test Item

3.0 mg/L

 

25

 

75

 

87

 

80

Reference control 10 mg/L

74

86

86

78

 

Toxicity Control

1.5 mg/L Test ltem +

2.5 mg/L Reference Item

 

50

 

87

 

89

 

88

 

Table 2: Oxygen Determination of the lnoculum Control and Functional Control 

Study day

Inoculum Control

 

Functional Control, 5 mg/L

 

ThOD: 1.67 mg 02/mg

O2 [mg/L]

O2 [mg/L]

BOD

Degr.

[d]

C1

C2

mv

O2-depl.

R1

R2

mv

[mg O2/L]

[mg O2/mg]

[%]

0

9.07

9.13

9.10

-

9.08

9.08

9.08

-

-

-

7

8.10

8.03

8.07

1.04

2.05

1.61

1.83

6.22

1.24

74

14

8.54

7.73

8.14

0.97

0.96

(6.49)

0.96

7.16

1.43

86

21

8.84

7.51

8.18

0.92

1.31

0.70

1.01

7.15

1.43

86

28

7.42

7.55

7.49

1.62

(8.61)

0.97

0.97

6.50

1.30

78

  

Table 3: Oxygen Determination of the Test ltem and the Toxicity Control 

study day

 

Test Item, 3.0 mg/L

Taxicity Control

1.5 mg/L test item + 2.5 mg/L reference item

ThOD: 2.57 mg O2/mg

ThOD: 2.03 mg O2/mg

[d]

O2 [mg/L]

BOD

Degr.

O2 [mg/L]

BOD

Degr.

 

P1

P2

mv

[mgO2/L]

[mgO2/mg]

[%]

T1

T2

mv

[mg O2/L]

[mg O2/mg]

[%]

0

9.00

9.05

9.03

-

-

-

9.06

9.05

9.06

-

-

-

7

6.15

6.02

6.09

1.91

0.64

25

4.00

3.91

3.96

4.07

1.02

50

14

2.55

1.94

2.25

5.82

1.94

75

0.71

1.38

1.05

7.05

1.76

87

21

1.58

1.15

1.37

6.74

2.25

87

1.37

0.47

0.92

7.21

1.80

89

28

1.31

1.13

1.22

6.19

2.06

80

0.28

(8.41)

0.28

7.16

1.79

88

 mv degr. depl.

mv= mean value

dgra.= degradation

depl.= depletion

ThOD = Theoretical oxygen demand

BOD   = Biological oxygen demand

( )   = Outlier, not included in calculations

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Readily biodegradable (80% in 28 days, 10 day window passed); Closed Bottle Test; Toxicity control: Test substance not inhibitory at tested concentration of 1.5 mg/L

Description of key information

Readily biodegradable (80 % within 28 d, OECD 301 D)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

One GLP guideline study according to OECD 301 D is available investigating the ready biodegradability of the substance. A mixed microbial population of the supernatant from the centrifuged activated sludge of a local municipal STP was inoculated with a nominal concentration of 3.0 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 10.0 mg reference substance/L) and a toxicity control were run in parallel. The derived 28 d degradation rate was 80 % for the test substance and 88 % for the toxicity control. Since the test substance reached the pass level for ready biodegradability of > 60% removal of ThOD within 28 d (meeting the 10 d – window) it could be classified as readily biodegradable. Due to the fact that the degradation rate of the toxicity control was > 25% after 14 d, toxic effects of the test substance to the inoculum can be excluded.