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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
adopted on 17 July 1992
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
January 1998
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
anaerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of activated sludge: Balatonfüred, Hungary, on 27 July 2018 (six days before the main test)
- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. 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 5 g dry material per litre was mixed with test water (mineral medium, see above) and then aerated under test conditions (for 6 days) until use. The pH of the activated sludge inoculum after preparation was 7.51, just before use: 7.63. A pH adjustment of the activated sludge inoculum was not performed.
- Pretreatment: Pre-conditioning (27 July – 02 August 2018) consisted of aerating (1 L/minute) activated sludge (in mineral medium) for 6 days at the test temperature (the actual temperature: 20.0 – 20.9 °C). Before use the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating 0.1 mL of the different, 10^-1, 10^-2, 10^-3 and 10^-4 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by these plating experiments by manual colony counting. During the pre-conditioning the approximately cell count of aerated inoculum was in the range of ~10^8/L; therefore, before the test the inoculum was further diluted 30 000x with mineral medium to reach the necessary ~10^4 – 10^6 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improved the precision of the test methods by reducing blank values. The inoculum was not pre-adapted to the test chemical.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium according to Guidelines
- Additional substrate: no
- Solubilising agent: no
- Test temperature: 20.1 - 21.1 °C
- pH: 7.42
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, with special neck and glass stoppers)
- Number of culture flasks/concentration: 16 (measurement in duplicate)
- Measuring equipment: O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method]

SAMPLING
- Sampling frequency: in all groups on days 0, 2, 5, 7, 12, 14, 21 and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 16 bottles
- Positive control: yes, 16 bottles
- Toxicity control: yes, 16 bottles
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The test item solubility was investigated in ultrapure water. 0.02 g test item was dispersed in 100 mL of ultrapure water by 15 min. ultrasonic treatment. At the concentration of 200 mg/L homogeneous stable opalescent emulsion was obtained.
Test performance:
Inoculum Control: The oxygen depletion in the inoculum control did not exceed 1.5 mg O2/L after 28 days. It was 1.19 mg O2/L in average.
Oxygen Concentration: The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. (The lowest value was 1.81 mg O2/L, it was measured on the 28th day of the experiment in the toxicity control.)
Parallels: The difference of duplicate values for the degradation at the plateau, at the end of the test or at the end of the 10-d window was not greater than 20 %. The highest difference (18.3 %) between the duplicate values from the 12th day to the 28th day (at about the biodegradation plateaus) of the test for degradation was calculated in the test item group. It was observed on the 14th day.
Reference Item: The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. (The percentage degradation of the reference item was 66.5% on the 14th day.)
Key result
Parameter:
% degradation (O2 consumption)
Value:
33.3
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of the test item reached a mean of 33.3% after 28 days based on its COD. The pass level for ready biodegradability is the removal of 60 % ThODNH4 (in the present case COD) in a 10-day window. Minimal biodegradation of the test item occurred in this study; the value remained far below the pass level; therefore, the test item was considered to be not ready biodegradable. The exact nitrogen content of the test item is not known; but supposedly no nitrification occurs during the test and total oxidised nitrogen (nitrate and nitrite) concentration determinations was not performed after each oxygen measurement.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to a mean of 66.5 % after 14 days, and to a mean of 71.8% after 28 days of incubation, based on its ThODNH4. The biodegradability reached its plateau on about the 7th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.

Table 1.: 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

2

5

7

12

14

21

28

Test item

2.0

1a

7.1

12.0

20.9

35.1

37.0

34.5

33.0

1b

4.1

9.8

19.9

31.7

30.8

31.7

33.6

mean

5.6

10.9

20.4

33.4

33.9

33.1

33.3

Reference item

3.0

2a

45.7

47.8

60.6

64.6

64.9

68.6

71.1

2b

49.1

52.0

65.6

66.2

68.1

70.4

72.5

mean

47.4

49.9

63.1

65.4

66.5

69.5

71.8

Toxicity control

Test item: 2.0 Reference item: 3.0

4a

17.9

26.4

35.3

45.9

47.3

49.2

49.9

4b

21.3

25.6

37.2

48.7

47.6

48.4

49.2

mean

19.6

26.0

36.3

47.3

47.4

48.8

49.6

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In a Closed Bottle Test the test substance was not readily, but primary inherent biodegradable (33% after 28 days).
Executive summary:

In a Closed Bottle Test according to OCED guideline 301 D the ready biodegradability of the test item was determined. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 2.0 mg/L. The test item concentration was chosen based on the results of preliminary solubility test (and an oxygen demand calculation performed based on a typical molecular structure detail within this UVCB test item). The exact chemical oxygen demand (COD) of the test item (2.49 mg O2/mg test item) was determined in frame of the main experiment. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the applied test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of the test item reached a mean of 33.3 % after 28 days based on its chemical oxygen demand. The maximum calculated biodegradation of 33.9 % was noticed on the 14th day of the test. The biodegradability reached its plateau on about the 12th day and from this day onwards the observed slight changes were considered as being within the biological variability range of the applied test system. The Nitrogen content of the test item is not known; however measurements of total oxidised nitrogen (nitrate and nitrite) concentrations were considered as not necessary, as in the production process N is not used. Therefore, the biodegradability value of the test item was calculated based on its COD; any correction, based on nitrite and/or nitrate development was not performed. The reference item, sodium benzoate, was sufficiently degraded to a mean of 66.5 % after 14 days, and to a mean of 71.8 % after 28 days of incubation, based on ThODNH4. (The biodegradability reached its plateau on about the 7th day and from this day onwards the observed slight changes were considered as being within the biological variability range of the applied test system.) In the toxicity control containing both, the test item and the reference item, a mean of 47.4 % biodegradation was noted within 14 days and 49.6 % biodegradation after 28 days of incubation (from about the 12th day of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system).The test item is considered to be not readily, but primary inherent biodegradable, since the pass level for ready biodegradability is removal of 60 % chemical oxygen demand (COD) in a 10-day window. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.

Description of key information

In a Closed Bottle Test the test substance was not readily, but primary inherent biodegradable (33% after 28 days).

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria
Type of water:
freshwater

Additional information

In a Closed Bottle Test according to OCED guideline 301 D the ready biodegradability of the test item was determined. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 2.0 mg/L. The test item concentration was chosen based on the results of preliminary solubility test (and an oxygen demand calculation performed based on a typical molecular structure detail within this UVCB test item). The exact chemical oxygen demand (COD) of the test item (2.49 mg O2/mg test item) was determined in frame of the main experiment. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the applied test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of the test item reached a mean of 33.3 % after 28 days based on its chemical oxygen demand. The maximum calculated biodegradation of 33.9 % was noticed on the 14th day of the test. The biodegradability reached its plateau on about the 12th day and from this day onwards the observed slight changes were considered as being within the biological variability range of the applied test system. The Nitrogen content of the test item is not known; however measurements of total oxidised nitrogen (nitrate and nitrite) concentrations were considered as not necessary, as in the production process N is not used. Therefore, the biodegradability value of the test item was calculated based on its COD; any correction, based on nitrite and/or nitrate development was not performed. The reference item, sodium benzoate, was sufficiently degraded to a mean of 66.5 % after 14 days, and to a mean of 71.8 % after 28 days of incubation, based on ThODNH4. (The biodegradability reached its plateau on about the 7th day and from this day onwards the observed slight changes were considered as being within the biological variability range of the applied test system.) In the toxicity control containing both, the test item and the reference item, a mean of 47.4 % biodegradation was noted within 14 days and 49.6 % biodegradation after 28 days of incubation (from about the 12th day of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system).The test item is considered to be not readily, but primary inherent biodegradable, since the pass level for ready biodegradability is removal of 60 % chemical oxygen demand (COD) in a 10-day window.The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.