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
Study period:
24 July 2017 to 23 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK obtained on 24 July 2017
- Storage conditions: room temperature in the dark
- Preparation of inoculum for exposure: The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and the filtrate maintained on continuous aeration in a temperature controlled room at approximately 21 °C prior to use.
- Pretreatment: The test item was dissolved directly in mineral medium.
- Water filtered: yes
- Type and size of filter used, if any: The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium
- Test temperature: 20 °C
- pH: 7.4
- pH adjusted: no
- Continuous darkness: yes, The BOD bottles were incubated in darkness in a temperature controlled water bath at measured temperatures of approximately 20 °C.

TEST SYSTEM
- Culturing apparatus: Test preparations were prepared and inoculated in 250 mL Biological Oxygen Demand (BOD) bottles (darkened glass) with ground glass stoppers
- Number of culture flasks/concentration: Test media inoculated with sewage treatment micro-organisms at a rate of 1 drop of inoculum per liter.
- Method used to create aerobic conditions: not reported
- Measuring equipment: YSI 54A dissolved oxygen meter and BOD Probe
- Test performed in closed vessels due to significant volatility of test substance: not reported
- Test performed in open system: no, closed bottle


SAMPLING
- Sampling frequency: Days 0, 2, 4, 7, 10, 12, 14, 21 and 28
- Sampling method: YSI 54A dissolved oxygen meter and BOD Probe

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the study. An aliquot (30 mL) of the 200 mg/L test item stock solution plus an aliquot (3 mL) of the sodium benzoate stock solution were dispersed in a final volume of 3 liters of mineral medium to give a final concentration of 2.0 mg test item/L plus 1.0 mg sodium benzoate/L. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the solutions.


STATISTICAL METHODS: Major Computerized Systems: Shimadzu TOC for TOC measurement
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
Validation Criteria: The results of the degradation test are considered valid if in the same test the reference item yields ≥ 60 % biodegradation (in a 10-Day window) by Day 14. Test items giving a measured BOD value which is ≥ 60 % of the ThOD or value (within 28 days) are regarded as being readily biodegradable. This level must be reached within 10 days of biodegradation exceeding 10%.
% Degradation
Key result
Parameter:
% degradation (O2 consumption)
Value:
48
Sampling time:
28 d
Details on results:
The oxygen depletion of the inoculated control did not exceed 4.60 mg O2/L after 28 days, the residual oxygen concentration in the test bottles remained at 6.20 mg O2/L or greater in all test vessels and the difference between the extremes of replicate oxygen depletion values at the end of the test was less than 20% in all vessels thereby satisfying the validation criteria.
The test item attained 48% biodegradation after 28 days and, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301D.
Variation in biodegradation rates on different sampling days was considered to be due to normal biological variation in respiration rates between control and test vessels.
Nitrate and nitrite analysis of the test solutions at each sampling occasion confirmed that no significant oxygen consumption as a result of nitrification occurred during the test.
The toxicity control attained 59% biodegradation after 14 days and 67% biodegradation after 28 days therefore confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

BOD5 / COD results

Results with reference substance:
The reference item, sodium benzoate, attained 63% biodegradation after 14 days and 60% biodegradation after 28 days thereby confirming the suitability of the test method and culture conditions.

Any other information on results incl. tables

Table 1: Dissolved oxygen

Test Series

Dissolved Oxygen (mg O2/L)

Day

0

2

4

7

10

12

14

21

28

Inoculum Control

R1

8.40

8.40

8.15

8.35

7.75

8.00

8.35

7.80

7.85

R2

8.45

8.35

8.10

8.35

8.10

8.10

8.20

7.80

7.70

Procedure Control

R1

8.40

5.60

4.85

5.10

4.85

4.90

5.05

4.50

4.50

R2

8.40

5.60

520

5.95

4.70

4.75

5.20

4.40

4.45

Test Item

R1

8.40

8.30

7.20

6.75

6.70

6.65

6.80

6.60

6.20

R2

8.40

8.25

7.40

6.85

6.55

6.55

6.85

6.50

6.55

Toxicity Control

R1

8.40

-

-

-

-

-

5.40

-

4.75

R2

8.45

-

-

-

-

-

5.35

-

4.60

R1-R2 = Replicates 1 and 2

Table 2: Oxygen depletion due to nitrification

Test Series

Oxygen Depletion (mg O2/L)

Day

2

4

7

10

12

14

21

28

Inoculum Control

(Mean Oxygen Depletion)

0.47

0.94

0.47

0.47

0.94

0.94

0.00

0.94

Procedure Control

R1

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.33

R2

0.47

0.00

0.00

0.00

0.00

0.00

0.00

0.19

Test Item

R1

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

R2

0.00

0.00

0.00

0.00

0.00

0.00

0.24

0.10

Toxicity Control

R1

-

-

-

-

-

0.61

-

0.19

R2

-

-

-

-

-

0.00

-

0.10

R1-R2 = Replicates 1 and 2

 

Table 3: Oxygen Depletion and Mean Percentage Biodegradation Values

Test Series

Day

2

4

7

10

12

14

21

28

Inoculum Control

Mean O2

Depletion (mg O2/L)*

-0.420

-0.640

-0.395

0.030

-0.565

-0.790

0.625

-0.290

Procedure Control

O2 Depletion

(mg O2/L)*

R1

2.750

3.250

3.225

3.050

3.050

3.200

3.275

2.920

R2

2.280

2.900

3.375

3.200

3.200

3.050

3.375

3.110

% Biodegradation (mean)

51

62

66

63

63

63

66

60

Test Item

O2 Depletion

(mg O2/L)*

R1

0.050

0.900

1.575

1.200

1.200

1.450

1.175

1.550

R2

0.100

0.700

1.475

1.350

1.350

1.400

1.035

1.100

% Biodegradation (mean)

3

29

55

46

46

52

41

48

Toxicity Control

O2 Depletion

(mg O2/L)*

R1

-

-

-

-

-

2.240

-

2.810

R2

-

-

-

-

-

2.950

-

3.100

% Biodegradation (mean)

-

-

-

-

-

59

-

67

R1-R2 = Replicates 1 and 2

pH 7.4

* Corrected for oxygen depletion due to nitrification

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 48 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301D.
Executive summary:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301D, "Ready Biodegradability; Closed Bottle Test” referenced as Method C.4-E of Commission Regulation (EC) No. 440/2008, and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 Paragraph (o).

The test item, at a concentration of 2.0 mg/L, was exposed to sewage treatment microorganisms with mineral medium in sealed culture vessels in the dark at measured temperatures of 20 °C for 28 days. The degradation of the test item was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The test item attained 48 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301D.