Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas
Details on test material:
Purity: 99.89%

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
other: Activated sludge of city sewage plant
Details on inoculum:
- Source of activated sludge: Tsubuku city sewage plant, Japan
- Preparation of activated sludge: The sample was filtered through a filter (No.2), the first 200 mL being discarded and the rest was used as inoculum. The inoculum was kept under aerobic conditions until used.
- Number of bacteria measured: 9.5 x 10e4 cells/mL
- Preparation of mineral medium: One milliliter of each solution 1, solution 2, solution 3 and solution 4, were made up to 1 L with purified water.
Solution 1: 8.5 g of KH2PO4, 21.75 g of K2HPO4, 33.4 g of Na2HPO4.2H2O, and 0.5 g of NH4Cl were dissolved in 500 mL of purified water. The solution was made up to 1 L with purified water.
Solution 2: 22.5 g MgSO4.7H2O/L
Solution 3: 27.5 g CaCl2/L
Solution 4: 0.25 g FeCl3.6H2O/L
- Inoculation of activated sludge: The mineral medium was inoculated with one drop of activated sludge from a pointed pipette per liter of final volume
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 mg/L
Parameter followed for biodegradation estimationopen allclose all
Parameter followed for biodegradation estimation:
other: BOD
Parameter followed for biodegradation estimation:
other: GC analysis
Details on study design:
Preparation of test solutions:
The following test solutions were prepared and incubated under the conditions described below.
- (Water + test substance) (2 bottles): To each test bottle, the test substance saturated solution which was diluted with purified water to be about 10 mg/L as the test substance concentration was filled.
- (Sludge + test substance) (8 bottles): To each test bottle, the test substance saturated mineral medium which was diluted with mineral medium to be about 10 mg/L as the test substance concentration and was added activated sludge was filled.
- (Sludge + sodium dodecylsulfate) (6 bottles): 3 mg/L of sodium dodecylsulfate solution was prepared by inoculated mineral medium, and then the solution was filled to each test bottle.
- (Control blank) (9 bottles) : To each test bottle, inoculated mineral medium was filled.

Test substance saturated solution: The test substance gas was introduced into 1001 mL vacuum vessel containing about 200 mL of purified water for about 30 minutes to be saturated enough. The concentration of the test substance in the saturated solution was determined as 10 mg/L.
Test substance saturated mineral medium: The test substance gas was introduced into 1004 mL vacuum vessel containing about 600 mL of mineral solution for about 30 minutes to be saturated enough. The concentration of the test substance in the saturated solution was determined as 12 mg/L.

Instruments and conditions of cultivation:
- Instruments for incubation: Incubator
- Concentration of test substance: 12 mg/L
- Concentration of activated sludge: One drop/L
- Vessel: 102 mL BOD bottle with Teflon stopper
- Incubation temperature: 20 ± 1°C
- Incubation method: Kept in closed bottle
- Duration of incubation: 28 days

Measurement of dissolved oxygen (DO): DO of each test solution without (water + test substance) was analyzed in accordance with "JIS K 0102-1993-32.1 Winkler Azide modified method", and biochemical oxygen demand (BOD) was determined.

Frequency of measurement:
- (Sludge + test substance) and (sludge + sodium dodecylsulfate): After 5, 15 and 28 days
- (Control blank): At the initiation of incubation, after 5, 15 and 28 days
Reference substance
Reference substance:
other: Sodium dodecylsulfate

Results and discussion

% Degradationopen allclose all
Key result
Parameter:
other: % BOD
Value:
14
Sampling time:
5 d
Key result
Parameter:
other: % BOD
Value:
22
Sampling time:
15 d
Key result
Parameter:
other: % BOD
Value:
20
Sampling time:
28 d
Key result
Parameter:
other: % biodegradtion by GC analysis
Value:
21
Sampling time:
28 d
Details on results:
Both the average percentage biodegradation by BOD and GC analysis of the test substance were about 20%. However, it was considered that these were not due to the biodegradation of the test substance by microorganisms, each result was due to the following causes.
- The loss of dissolved oxygen from the test solutions of (sludge + test substance): At the preparation of the solution, the test solutions of (sludge + test substance) were prepared by introducing the test substance gas into the mineral medium. At this time, the concentrations of dissolved oxygen in the test solutions of (sludge + test substance) than that of (control blank), and the dissolved oxygen was maintained until the termination of the incubation. Therefore, it was considered that the decreases of the dissolved oxygen concentration in the test solutions of (sludge + test substance) based on the percentage biodegradation by BOD were due to the loss of dissolved oxygen by the introduction of the test substance gas.
- The loss of test substance from the test solutions of (sludge + test substance): As the test substance in a state of gas had high volatility, it was estimated that the loss of the test substance resulted from volatilization of a part of the test substance at the preparation of the test substance solution. And the lost amount of the test substance from the test solutions of (sludge + test substance) was a little larger than that of the test solutions of (water + test substance), because there were some influences by mineral compositions and so on. Therefore, it is considered that the decreases of the residual amount of the test substance in the test solutions of (sludge + test substance) based on the GC analysis were due to the difference of the lost amount of the test substance between the both test systems.

BOD5 / COD results

Results with reference substance:
BOD of sodium dodecylsulfate: 80% after 28 d

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance was not biodegraded by microorganisms under the test conditions.
Executive summary:

This test was performed to evaluate the biodegradability of the test substance by microorganisms according to OECD guideline for testing of chemicals "Ready biodegradability: 301D, closed bottle test".

The average percentage of biodegradation by BOD was found to be 20% and the average percentage of biodegradation by GC was found to be 21%.

Both the average percentage biodegradation by BOD and GC analysis of the test substance were about 20%. However, it was considered that these were not due to the biodegradation of the test substance by microorganisms, each result was due to the loss of dissolved oxygen from the test solutions of (sludge + test substance) and the loss of test substance from the test solutions of (sludge + test substance).

It was considered that the test substance was not biodegraded by microorganisms under the test conditions.

Percentage biodegradation of sodium dodecylsulfate calculated by BOD value was 80% after 28 days. It was concluded that these test conditions were valid.