Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February 2018 to 23 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Method C.4-E “Closed Bottle Test"
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
OECD Guideline for Testing of Chemicals No. 301 D: "Ready Biodegradability: Closed Bottle Test", adopted July 17, 1992
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
EPA Guideline 712-C-98-076: OPPTS 835.3110, "Ready Biodegradability", January 1998
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No further details specified in the study report.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
The inoculum: Secondary effluent, microorganisms from a domestic waste water treatment plant.
Origin: The secondary effluent was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The secondary effluent used for this study was allowed to settle approximately for an hour, then the decanted effluent was aerated until use (not later than an hour).
Duration of test (contact time):
28 d
Initial conc.:
11.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST UNITS
Type and Size: BOD bottles (300 ml) with special neck and glass stoppers.
Identification: Each test flask was uniquely identified with study code, test group, days of measurement and replicate number.

TEST CONDITIONS
The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 ± 2°C according to guideline.
The test flasks were placed into an incubator and kept at 20.4 - 23.3°C, in the dark. The temperature was measured on weekdays during the experiment.
The oxygen concentration of test water was 9.0 mg/L at the start of the test.
The pH value of the test water was checked prior start of the experiment. The pH of the test water was 7.22.
The test conditions were measured with suitable instruments and documented in the raw data.

PREPARATION OF THE TEST SOLUTIONS
The respective amount of α, α’-Dichloro-p-xylene was weighed in directly to reach the required test item concentration of 3.4 mg/L.
The chosen test item concentration was based on theoretical oxygen demand (ThOD) of 1.74 mg O2/mg test item (calculated according to equation given in the guidelines) and on the performed 14-d preliminary test.
The components were applied in the amounts/volumes in the test flasks:
1.) Test Item (flasks 1a and 1b)
Based on the theoretical oxygen demand (ThOD) of 1.74 mg O2/mg test item, 11.6 mg of α, α’-Dichloro-p-xylene was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 3.4 mg/L test item, with a ThOD of about 5.916 mg O2/L).
2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThOD) of Sodium benzoate (1.67 mg O2 per mg; details on calculation are given in the guidelines), stock solution* corresponding to 12.168 mg of Sodium benzoate was mixed into 3.38 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThOD of about 6.012 mg O2/L).
* The concentration of the stock solution was: 360 mg/L.
3.) Inoculum Control (flasks 3a and 3b)
Only filtered inoculum was added to 3.38 litres of aqueous test medium.
4.) Toxicity Control (flasks 4a and 4b)
The Test item (11.6 mg) and the reference item stock solution* (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 3.4 mg/L test item (ThOD of 5.916 mg O2/L) and 3.6 mg/L reference item (ThOD of 6.012 mg O2/L).
* The concentration of the reference item stock solution was: 360 mg/L.
Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.

COURSE OF THE TEST
Preparation of Test Flasks
Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water.
Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.

The Test Bottles
The number of test bottles was the follow:
10 bottles containing the test item and inoculum
-10 bottles containing the reference item and inoculum (procedure control)
-10 bottles containing only inoculum (inoculum control)
-10 bottles containing the test item, reference item and inoculum (toxicity control)

MEASUREMENTS
Measurement of Oxygen
The incubation period of the closed bottle test was 28 days.
The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.

Measurement of Temperature
Temperature was measured continuously and registered on weekdays.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
7.6
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of α, α’-Dichloro-p-xylene reached a mean of 7.6 % after 28 days based on the ThOD of the test item.
In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 39.0 % biodegradation was noted within 14 days and 42.0% biodegradation after 28 days of incubation.
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 76.7 % after 14 days, and to a mean of 82.5 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

[mg/L]

Flask No.

mg O2/L after n days of exposure

0

7

14

21

28

Test item

3.4

1a

1b

mean

8.6

8.5

8.55

7.7

7.9

7.80

7.3

7.4

7.35

7.1

7.2

7.15

6.8

6.8

6.80

Reference item

3.6

2a

2b

mean

8.8

8.7

8.75

4.4

4.4

4.40

3.2

3.4

3.30

2.8

2.9

2.85

2.4

2.6

2.50

Inoculum control

-

3a

3b

mean

8.8

8.8

8.80

8.6

8.5

8.55

7.9

8.0

7.95

7.7

7.8

7.75

7.6

7.4

7.50

Toxicity control

Test item: 3.4

Reference item: 3.36

4a

4b

mean

8.7

8.6

8.65

4.3

4.3

4.30

3.2

3.1

3.15

2.6

2.6

2.60

2.3

2.4

2.35

 

Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration [mg/L]

Flask No.

mg O2/L after n days of exposure

7

14

21

28

Test item

3.4

1b

1b

0.65

0.35

0.45

0.25

0.45

0.25

0.50

0.40

Reference item

3.6

2a

2b

4.15

4.05

4.75

4.45

4.95

4.75

5.10

4.80

Toxicity control

Test item: 3.4

Reference item: 3.6

4a

4b

4.15

4.05

4.65

4.65

5.05

4.95

5.10

4.90

Oxygen depletion: (mt0 – mtx) – (mb0 – mbx), where:

mt0: oxygen concentration (mg/L) of test group on day 0

mtx: oxygen concentration (mg/L) of test group on day x

mb0: oxygen concentration (mg/L) of inoculum blank on day 0

mbx: oxygen concentration (mg/L) of inoculum blank on day x

 

BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration [mg/L]

Flask No.

mg O2/L after n days of exposure

7

14

21

28

Test item

3.4

1b

1b

0.19

0.10

0.13

0.07

0.13

0.07

0.15

0.12

Reference item

3.6

2a

2b

1.15

1.13

1.32

1.24

1.38

1.32

1.42

1.33

Toxicity control

Test item: 3.4

Reference item: 3.6

4a

4b

0.59

0.58

0.66

0.66

0.72

0.71

0.73

0.70

 

BOD = (mg O2/L of T.i. and/or R.i. – mg O2/L if i.control) / mg Ti. And/or R.i./L in flask = mg O2/mg T.i. and/or R.i.

where:

T.i. = test item

R.i. = reference item

i.control = inoculum control

 

Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration [mg/L]

Flask No.

mg O2/L after n days of exposure

7

14

21

28

Test item

3.4

1b

1b

mean

11.0

5.9

8.5

7.6

4.2

5.9

7.6

4.2

5.9

8.5

6.8

7.6

Reference item

3.6

2a

2b

mean

69.2

67.5

68.3

79.2

74.2

76.7

82.5

79.2

80.8

85.0

80.0

82.5

Toxicity control

Test item: 3.4

Reference item: 3.6

4a

4b

mean

34.8

34.0

34.4

39.0

39.0

39.0

42.4

41.6

42.0

42.8

41.1

42.0

 

Biodegradation% = [BOD (mg O2/mg T.i. or R.i.) / ThOD (mg O2/mg T.i.) or ThOD (mg O2/mg R.i.)] x 100

where:

T.i. = test item

R.i.= reference item

i.control = inoculum control

 

ThOD of test item = 1.74 mg O2/mg test item

ThOD of reference item = 1.67 mg O2/mg reference item

 

The biodegradation in the toxicity control was calculated according to the following formula:

(BOD (mg O2/mg T.i. and R.i.) / [ThOD (mg O2/mg T.i.) + ThOD (mg O2/mg R.i.)] * ½ ) x 100

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item α, α’-Dichloro-p-xylene was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.
Under the test conditions the percentage biodegradation of α, α’-Dichloro-p-xylene reached a mean of 7.6 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore the test item is considered not readily biodegradable.
The reference item Sodium benzoate was sufficiently degraded to a mean of 76.7 % after 14 days, and to a mean of 82.5 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.
In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 39.0 % biodegradation was noted within 14 days and 42.0% biodegradation after 28 days of incubation.
According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 3.4 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.
The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.
Based on the results of this study, the test item is considered not readily biodegradable.
Executive summary:

The test item α,α’-Dichloro-p-xylene was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

 

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

 

Under the test conditions the percentage biodegradation of α, α’-Dichloro-p-xylene reached a mean of 7.6 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore, the test item is considered not readily biodegradable.

 

The reference item Sodium benzoate was sufficiently degraded to a mean of 76.7 % after 14 days, and to a mean of 82.5 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

 

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 39.0 % biodegradation was noted within 14 days and 42.0% biodegradation after 28 days of incubation.

 

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 3.4 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

 

The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.

 

Based on the results of this study, the test item is considered not readily biodegradable.

Description of key information

The test item is considered not readily biodegradable.

Key value for chemical safety assessment

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

Additional information

The test item α,α’-Dichloro-p-xylene was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

 

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

 

Under the test conditions the percentage biodegradation of α, α’-Dichloro-p-xylene reached a mean of 7.6 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore, the test item is considered not readily biodegradable.

 

The reference item Sodium benzoate was sufficiently degraded to a mean of 76.7 % after 14 days, and to a mean of 82.5 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

 

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 39.0 % biodegradation was noted within 14 days and 42.0% biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 3.4 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

 

The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.

Based on the results of this study, the test item is considered not readily biodegradable.