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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:
11 June 2010 - 9 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The inoculum: Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The activated sludge used for this study was washed by centrifugation (for 10 min.) and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged (for 10 min.). This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre were mixed with test water (see below) and then aerated until use. Before use the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
3.3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
RECONSTITUTED WATER:

Stock solutions In deionised water analytical grade salts were added to prepare the following stock solutions:
a) Solution: KH2PO4 2.125g K2HPO4 5.4375g Na2HPO4 x 12H2O 16.795g NH4Cl 0.125g Deionised water 250 mL
b) Solution: MgSO4 x 7 H2O 5.625g Deionised water 250 mL
c) Solution: CaCl2 x 2 H2O 9.10 g Deionised water 250 mL
d) Solution: FeCl3 x 6 H2O 0.25 g Deionised water 1000 mL

Ratio of ingredients
1 mL of the stock solutions a) - d) were combined and filled to a final volume of 1000 mL with deionised water. The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was about 8.4 mg/L at about 22 °C.

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 22.0-23.4°C, in the dark. The temperature was measured on weekdays during the experiment. The oxygen concentration of test water was 8.4 mg/L at the start of the test. The pH value of the test water was checked prior study start. The pH of the test water was 7.24. The test conditions were measured with suitable instruments and documented in the raw data.

PREPARATION OF THE TEST SOLUTIONS
The respective amount of the test material was weighed in directly to reach the required test item concentration of 3.3 mg/L. During the performance of the test the test solutions were ultrasonicated for 5 min. to ensure a good dispersion. The chosen test item concentration was based on the measured chemical oxygen demand (COD): 1.84 ±0.04 mg O2/mg test item and on the performed 14-d preliminary test. The components were applied in the amounts/volumes following ratio in the test flasks:

1.) Test Item (flasks 1a and 1b)
Based on the chemical oxygen demand (COD) of 1.84 mg O2/mg test item, 11.4 mg of test material was thoroughly mixed into 3.46 litres of aqueous test medium (corresponding to 3.3 mg/L test item, respectively a COD of about 6.072 mg O2/L).

2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), stock solution* corresponding to 12.096 mg of Sodium benzoate was mixed into 3.36 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 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 3.40 litres of aqueous test medium.

4.) Toxicity Control (flasks 4a and 4b)
11.4 mg of test material and reference item stock solution* (34.6 mL) were mixed into 3.46 litres of aqueous test medium corresponding to 3.3 mg/L test item (COD of 6.072 mg O2/L) and 3.6 mg/L reference item (ThODNH4 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 standing 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.

Test Bottles
The number of test bottles was the follows:
- 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

COD Measurement
The COD (chemical oxygen demand) of the test item were determined in using Lovibond® COD Measuring System.

Measurement of Oxygen
The incubation period of the closed bottle test was 28 days. The oxygen concentrations were measured with 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
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (O2 consumption)
Value:
10.7
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of the test material reached a mean of 10.7 % after 28 days based on the measured COD of the test item. The test item can therefore be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD.
The reference item Sodium benzoate was sufficiently degraded to a mean of 85.8% after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.
In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 41.3 % biodegradation was noted within 14 days and 44.6% 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 3.3 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.

BOD5 / COD results

Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 85.8% after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum

Any other information on results incl. tables

Table 1. 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

3.3

1a

8.1

6.6

6.4

6.4

6.0

1b

8.0

6.7

6.5

6.4

6.0

mean

8.5

6.65

6.45

6.40

6.00

Reference item

3.6

2a

8.1

2.2

1.5

1.5

1.4

2b

8.1

2.2

1.7

1.6

1.4

mean

8.10

2.20

1.60

1.55

1.40

Inoculum control

-

3a

8.2

7.5

6.8

6.8

6.8

3b

8.2

7.5

6.9

6.8

6.8

mean

8.20

7.50

6.85

6.80

6.80

Toxicity control

Test item: 3.3

Ref. item: 3.6

4a

8.0

1.9

1.7

1.6

1.3

4b

8.1

1.9

1.7

1.6

1.2

mean

8.05

1.90

1.70

1.60

1.25

Table 2. Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration

(mg/L)

Flask No.

Percent of biodegradation after n days of exposure

7

14

21

28

Test item

3.3

1a

13.2

5.8

4.9

11.5

1b

9.9

2.5

3.3

9.9

mean

11.5

4.1

4.1

10.7

Reference item

3.6

2a

86.7

87.5

86.7

88.3

2b

86.7

84.2

85.0

88.3

mean

86.7

85.8

85.8

88.3

Toxicity control

Test item: 3.3

Ref. item: 3.6

4a

44.6

40.9

41.3

43.8

4b

45.5

41.7

42.2

45.5

mean

45.0

41.3

41.7

44.6

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable
Conclusions:
Under the test conditions the percentage biodegradation of the test material reached a mean of 10.7 % after 28 days based on the measured COD of the test item. The test item can therefore be considered to be not ready biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD.
The reference item Sodium benzoate was sufficiently degraded to a mean of 85.8% after 14 days, and to a mean of 88.3 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.
In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 41.3 % biodegradation was noted within 14 days and 44.6% 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 3.3 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.
Executive summary:

In an assessment of ready biodegradability study, closed bottle test (10/095-322AN), the test material was determined to not be readily biodegradable according to EU test method C.4-E given that the degradation rates calculated from COD values were less than 60 %.

Under the test conditions, the percentage biodegradation of the test material reached a mean of 10.7 % after 28 days.