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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
Study period:
2013-10-25 to 2013-12-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 301 C with GLP statement. All validity criteria were fulfilled and no deviations were observed.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
1992
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
GLP statement (December 20, 2013)
Specific details on test material used for the study:
No data
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Test inoculum was purchased from the Chemicals Evaluation and Research Institute (3-2-7 Miyanojin, Kurume, Fukuoka 839-0801, Japan), on October 23, 2013.
Duration of test (contact time):
28 d
Initial conc.:
100 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 by adding each 6 mL of stock solution A, B, C and D (as specified in JIS K 0102-2008 21) to 1976 mL of purified water (which was adapted to quality of Japanese pharmacopeia) and adjusted the pH to 7.0.
- Test temperature: 25 ± 1 °C
- pH: 7.0
- pH adjusted: Yes
- Suspended solids concentration: 30 mg/L (sludge)

TEST SYSTEM
- Culturing apparatus: Bottles
- Test substance was weighed and placed in a culture bottle, and then a fixed amount of mineral medium or purified water was added. It was confirmed that pH of the test solution was 7.0, subsequently, an activated sludge solution was added.
- Amount of the test solution: 300 mL
- Number of culture flasks/concentration: 3 bottles (test suspensions); 1 bottle (inoculum blank); 1 bottle (activity control); 1 bottle (abiotic control)
- Test suspensions: Test substance (No. 1: 30 mg, No.2: 30 mg; No.3: 30 mg) + mineral medium 290 mL + inoculum 900 mg/L solution 10 mL
- Measuring equipment: Test solution was cultivated in an oxygen consumption analyzer for 28 days, and the oxygen consumption was continuously measured by using a Coulometer (OM-3100A, Ohkura Electric Co., Ltd.).
- Test performed in closed vessels: Yes
- Test performed in open system: No
- Other: The following items were observed during the test period and recorded: leakage check of the equipment, temperature of the thermostatic equipment chamber, presence/absence of the growth of the activated sludge in the culture bottle, degree of dissolution of the test substance, and presence/absence of coloring due to biodegradation.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes; Mineral medium 290 mL + inoculum 900 mg/L solution 10 mL
- Abiotic sterile control: Yes; Test substance (30 mg) + purified water 300 mL
- Toxicity control: Yes; Aniline 29.5 µL (30.12 mg) + mineral medium 290 mL + inoculum 900 mg/L solution 10 mL
Reference substance:
aniline
Preliminary study:
None
Test performance:
No data
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Remarks on result:
other: The degrees of degradation of the test suspensions (test substance + sludge, n=3) calculated from the oxygen consumption were -2%, -3% and -3%, respectively. The mean degradability of the test substance was 0%
Details on results:
Biodegradability calculated from the oxygen consumption:
The degrees of degradation of the test suspensions (test substance + sludge, n=3) calculated from the oxygen consumption were -2%, -3% and -3%, respectively. The mean degradability of the test substance was 0%.

Biodegradability calculated by the direct quantitative analysis:
On the test solution after 28 days culture, the residual concentration of the test substance was measured by the GC method. According to the results of direct quantitative analysis by GC, the test substance in the abiotic control (only the test substance) was detected at 100% relative to the amount supplied and the test substance in the test suspensions (test substance + sludge) were detected at 99%, 100% and 100%. In the test suspensions, the degradabilities calculated from these values were 1%, 0% and 0%. Thus, the mean degradability of test substance by GC analysis was 0%.
Results with reference substance:
Biodegradabilities of aniline were 61% after 7 days and 74% after 14 days, and the oxygen consumption curve was normal. Thus, the degradation activity of the active sludge was normal. The difference of maximum value of the biodegradability and minimum value after 28 days became 1%, and it was under 20%. Therefore, the test was valid.

Table 5.2.1/1: Biodegradability calculated from oxygen consumption

 

Bottle No.

Groups

Theoretical oxygen demand (mg)

7th day

14th day

21st day

28th day

Measured value (mg)

Degradability (%)

Measured value (mg)

Degradability (%)

Measured value (mg)

Degradability (%)

Measured value (mg)

Degradability (%)

1

Test suspensions bottle 1

90.7

3.4

-2

4.2

-2

4.5

-2

5.1

-2

2

Test suspensions bottle 2

90.7

2.6

-2

3.4

-3

3.8

-3

3.8

-3

3

Test suspensions bottle 3

90.7

3.0

-2

3.8

-2

4.0

-3

4.1

-3

4

Activity control (Aniline)

90.6

60.0

61

72.9

74

73.8

74

74.2

74

5

Inoculum blank

-

4.8

-

6.0

-

6.5

-

6.8

-

6

Abiotic control

-

0.0

-

0.0

-

0.0

-

0.0

-

 

Mean degradability on the 28th day: 0%*

*: For the mean value of degradability calculated from BOD was negative, it was made to be 0%

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The degrees of degradation of the test suspensions (test substance + sludge, n=3) calculated from the oxygen consumption were -2%, -3% and -3%, respectively. The mean degradability of the test substance was 0%. Thus, the test item should be regarded as not readily biodegradable according to this test.
Executive summary:

The ready biodegradability of the test material has been determined by the Modified MITI Test according to the OECD Guideline No. 301 C with GLP compliance.

A nominal concentration of test material (100 mg/L) introduce in the system.

 

The degrees of degradation of the test suspensions (test substance + sludge, n=3) calculated from the oxygen consumption were -2%, -3% and -3%, respectively. The mean degradability of the test substance was 0%. According to the results of direct quantitative analysis by GC, the test substance in the abiotic control (only the test substance) was detected at 100% relative to the amount supplied and the test substance in the test suspensions (test substance + sludge) were detected at 99%, 100% and 100%. In the test suspensions, the degradabilities calculated from these values were 1%, 0% and 0%. Thus, the mean degradability of test substance by GC analysis was 0%. Biodegradabilities of aniline were 61% after 7 days and 74% after 14 days, and the oxygen consumption curve was normal; the activity of the inoculum is thus verified and the test is considered as valid.

 

Thus, the test item should be regarded as not readily biodegradable according to this test.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 301 D but was not performed according to GLP. However, this study is well documented and is considered reliable with restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to 2 mg DW/L and distributed over the appropriate biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992).
River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. Particles in the river water were removed by sedimentation.
Duration of test (contact time):
84 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: The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4•2H2O, 22.5 mg MgSO4•7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3•6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
- Solubilising agent (type and concentration if used): None
- Test temperature: 22-24 °C
- Suspended solids concentration: 2 mg DW/L
- Continuous darkness: Yes
- Deionized water containing no more than 0.01 mg/L copper was prepared in a water purification system.
- Other: None

TEST SYSTEM
- Culturing apparatus: 0.3 L Biological oxygen demand (BOD) bottles with glass stoppers
- Number of culture flasks/concentration: 3 bottles containing only inoculum (control) and 3 bottles for test substance with the inoculum
- Method used to create aerobic conditions: No data
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands). The pH was measured using a EUTECH instruments pH meter. The temperature was measured and recorded with a thermo couple connected to a data logger.
- Test performed in closed vessels: Yes
- Test performed in open system: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes

OTHERS:
- The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a pre-weighed 8 µm cellulose nitrate filter. This filter was dried for 1.5 hours at 104 °C and weighed after cooling. The dry weight was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume.
The biological oxygen demand (BOD) mg/mg of the test compound was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle. The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The dissolved oxygen (DO) in the BOD bottles was measured over time for a maximum period of 84 to 112 days. A test was terminated when biodegradation exceeded 60 %.
Reference substance:
not specified
Preliminary study:
None
Test performance:
No data
Key result
Parameter:
% degradation (O2 consumption)
Value:
2
Sampling time:
28 d
Remarks on result:
other: River
Parameter:
% degradation (O2 consumption)
Value:
8
Sampling time:
84 d
Remarks on result:
other: River
Details on results:
No degradation (<10%) was found with AKZ012 .

Test conditions: The validity of the tests is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item showed toxic to sludge up to 84 days; test item undergoes 2 and 8 % degradation in river after 28 and 84 days, respectively in the test conditions. Thus, the test item should be regarded as not readily biodegradable according to this test.
Executive summary:

This study was performed according to the OECD Guideline 301 D, to determine the biodegradability of the test substance by the Closed Bottle Test.

The test material was exposed to activated sewage sludge micro-organisms at a concentration of 2 mg/L with culture medium in closed bottles in the dark at 22-24 °C for 7, 14, 21, 28, 42, 56 and 84 days. The degradation of the test material was assessed by the measurement of oxygen consumption.

 

Test item is partially degradable. The validity of the test is demonstrated by oxygen concentrations > 0.5 mg/L in all bottles during the test period.

 

Test

substance

Inoculum

Biodegradation at day (%)

7

14

21

28

42

56

84

Sludge

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

River

Toxic

Toxic

2

2

2

4

8

 

The test item showed toxic to sludge up to 84 days; test item undergoes 2 and 8 % degradation in river after 28 and 84 days, respectively in the test conditions. Thus, the test item should be regarded as not readily biodegradable according to this test.

Description of key information

OECD Guideline 301C, GLP, Key study, validity 1:

0% biodegradation after 28d.

Not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

To assess the ready biodegradability of the registered substance, two valid studies are available.

The first study (Institute of Ecotoxicology Co. Ltd., 2013), assessed as the key study, was performed on the registered substance according to OECD Guideline 301C with GLP compliance. The test substance at a concentration of 100 mg/L was exposed to activated sludge with culture medium for 28 days, in aerobic condition. According to this test, the degree of degradation of the test suspensions (test substance + sludge, n=3) calculated from the oxygen consumption were -2%, -3% and -3%, respectively (mean of 0% after 28 days). These negative values can reflect a potential toxicity of the substance to micro-organisms at the tested concentration (100 mg/L). As no toxicity control was performed, this statement cannot be proved. According to the results of direct quantitative analysis by GC, the test substance in the abiotic control (only the test substance) was detected at 100% relative to the amount supplied and the test substance in the test suspensions (test substance + sludge) were detected at 99%, 100% and 100%. In the test suspensions, the degradabilities calculated from these values were 1%, 0% and 0%. Thus, the mean degradability of test substance by GC analysis was 0%. Biodegradabilities of aniline were 61% after 7 days and 74% after 14 days, and the oxygen consumption curve was normal; the activity of the inoculum is thus verified and the test is considered as valid. Thus, the test substance should be regarded as not readily biodegradable according to this test.

The second study (AkzoNobel, 2012), assessed as a supporting study, was performed on the registered substance according to OECD Guideline 301D without GLP compliance. The test substance was exposed to two inoculums (activated sewage sludge micro-organisms and river water) at a concentration of 2 mg/L with culture medium in closed bottles in the dark at 22-24 °C for 7, 14, 21, 28, 42, 56 and 84 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. The validity of the test was demonstrated by oxygen concentrations > 0.5 mg/L in all bottles during the test period. In activated sludge, the test substance was observed to be toxic up to 84 days, at the tested concentration (2 mg/L). In river water, the test substance was observed to be toxic up to 14 days only and undergoes 2 and 8% degradation after 28 and 84 days, respectively in the test conditions. The test substance is partially degradable but should be regarded as not readily biodegradable according to this test. This study supports the result of the OECD 301C key study.

In conclusion, the registered substance is not readily biodegradable and a potential toxicity to sludge micro-organisms is expected.