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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum: Aereation stage of the HRC Limited sewage treatment plant treating predominantly domestic sewage.
- Preparation of inoculum for exposure: The sample was allowed to settle and the supernatant filtered through Whatman GFA filtrer paper (first 250 mL discarded).
Duration of test (contact time):
28 d
Initial conc.:
40 mg/L
Based on:
test mat.
Initial conc.:
31.5 mg/L
Based on:
IC (inorganic carbon)
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Standard nutrient medium
Solution 1:
KH2PO4 8.5 g/L
K2HPO4 21.75 g/L
Na2HPO4 . H2O 33.3 g/L
NH4Cl 1.7 g/L

Solution2:
MgSO4 . 7 H2O 22.5 g/L

Solution 3:
CaCl2 27.5 g/L

Solution 4:
FeCl3 . 6 H2O 0.25 g/L

1 mL of each solution 1 – 4 were added to each litre of aerated reverse osmosis purified and glass-distilled water. The “dilution water” was left at room temperature (ca. 20 °C), under gentle agitation for 24 h prior to use.

- Test temperature: 21 ± 1 °C

TEST SYSTEM
- Culturing apparatus: BOD bottles (280 mL, darkened glass)
- Number of culture flasks/concentration: 2
- Measuring equipment: Yellow Springs BOD Probe (model 54)
- Test performed in closed vessels

SAMPLING
- Sampling frequency: at day 0, 5, 15 and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: No

STATISTICAL METHODS:
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
5
Sampling time:
28 d
Details on results:
No breakdown products were determined. In a second replicate with the same concentration a biodegradation was only 1 %. Higher test levels could not be obtained because of the limited solubility of the test material in chloroform. This test was conducted from the notifier instead of a test on inherent biodegrability. The German C.A. did not insist on the conduction of the inherent test as the substance also will not be inherent biodegradable for structural reasons.
Results with reference substance:
Sodium benzoate attained 89 % biodegradation within 28 days. Oxygen depletions in the inoculated and non-inoculated control series were within the prescribed limits.

Table 1: Dissolved Oxygen Measurement (mgO2/L)

Treatment

Day

0

5

15

28

Dilution water, no inoculum

R1

8.50

8.40

8.35

8.20

R2

8.50

8.40

8.30

8.25

Mean

8.500

8.400

8.325

8.225

Dilution water, inoculum

R1

8.50

8.35

8.25

8.25

R2

8.50

8.30

8.25

8.15

Mean

8.500

8.325

8.250

8.200

Dilution water, inoculum and filter paper

R1

8.50

8.25

8.10

8.05

R2

8.50

8.30

8.10

8.10

Mean

8.500

8.275

8.100

8.075

Test Item (2 mg/L) with inoculum

R1

8.50

8.05

8.00

7.90

R2

8.50

8.10

7.90

7.70

Mean

8.500

8.075

7.950

7.800

Standard Substance (3 mg/L) with inoculum

R1

8.50

4.80

4.20

3.80

R2

8.50

4.90

3.90

3.70

Mean

8.500

4.850

4.050

3.750

Table 2: Mean Oxygen Depletion and Percentage Biodegradation Values

Treatment

Day

5

15

28

Dilution water, no inoculum

O2 depletion (mgO2/L)

0.100

0.175

0.275

% Degradation

-

-

-

Dilution water, inoculum

O2 depletion (mgO2/L)

0.175

0.2500

0.300

% Degradation

-

-

-

Dilution water, inoculum and filter paper

O2 depletion (mgO2/L)

0.225

0.400

0.425

% Degradation

-

-

-

Test Item (2 mg/L) with inoculum

O2 depletion (mgO2/L)

0.200

0.150

0.275

% Degradation

4

3

5

Standard Substance (3 mg/L) with inoculum

O2 depletion (mgO2/L)

3.475

4.200

4.450

% Degradation

70

84

89

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance can not be regarded as readily biodegradable.
Executive summary:

In a ready biogradability study according to OECD guideline 301D, an inoculum prepared from non-adapted activated sludge from the aereation stage of the HRC Limited sewage treatment plant (predominantly domestic sewage) was exposed to the test substance. Dissolved oxygen measurements for the test substance and standard substance solutions, together with the inoculated and non- inoculated blank controls, after 0, 5, 15 and 28 days were performed. The positive control sodiumbenzoate attained 89 % degradation within 28 days. The test substance attained 5 % biodegradation after 28 days. Oxygen depletions in the inoculated and non-inoculated control series were within the prescribed limits.

Based on these results, the test substance cannot be considered as readily biodegradable.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
sewage inoculum was pre-exposed to test substance
Qualifier:
according to guideline
Guideline:
EU Method C.5 (Degradation: Biochemical Oxygen Demand)
Version / remarks:
as cited in Directive 67/548/EEC
GLP compliance:
yes
Key result
Parameter:
% degradation (CO2 evolution)
Value:
16.5
Sampling time:
28 d
Results with reference substance:
Experimental values (reference substance):
62 % degradation after 10 d
77 % degradation after 14 d
84 % degradation after 24 d
87 % degradation after 28 d

The low solubility of the test material meant the usual SCAS and Zahn-Wellens methods were inappropriate. Instead the modified Sturm test was used with sewage inoculum which had been pre-exposed to the test substance.

Validity criteria fulfilled:
not specified
Interpretation of results:
not inherently biodegradable
Conclusions:
The test substance cannot be regarded as inherently biodegradable.
Executive summary:

Inherent biodegradation has been assessed in accordance with EU method C.5 (ECHA RSS, 2017). The test substance showed 16.5 % degradation until day 28 based on CO2 evolution. No inherent biodegradation can be seen for the test substance.

Description of key information

not readily or inherently biodegradable

Key value for chemical safety assessment

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

Additional information

Key Study


In a ready biogradability study according to OECD guideline 301D (Huntingdon, 1989), an inoculum prepared from non-adapted activated sludge from the aeration stage of the HRC Limited sewage treatment plant (predominantly domestic sewage) was exposed to the test substance. Dissolved oxygen measurements for the test substance and standard substance solutions, together with the inoculated and non- inoculated blank controls, after 0, 5, 15 and 28 days were performed. The positive control sodiumbenzoate attained 89 % degradation within 28 days. The test substance attained 5 % biodegradation after 28 days. Oxygen depletions in the inoculated and non-inoculated control series were within the prescribed limits.


Based on these results, the test substance cannot be considered as readily biodegradable.


 


Supporting study


The above result is further supported by the results of an inherent biodegradation study according to EU method C.5 (ECHA RSS, 2017). Although the sewage sludge was pre-exposed to the test substance only 16.5 % biodegradation was observed until study day 28. Thus, the test substance cannot be regarded as biodegradable.