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:
January to April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
pH of stock solution of sodium benzoate was 7.13 and not 7.4±0.2 as required, temperature in respirometer water baths ranged from 20.5°C - 22.9°C and not 22±1°C as required
GLP compliance:
yes
Remarks:
Study performed in compliance with OECD GLPs with following exceptions: test site for elemental analysis is not GLP-compliant facility

Test material

Reference
Name:
Unnamed

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Fresh activated sludge was used as the inoculum. The activated sludge was obtained from a domestic wastewater treatment facility in New Jersey, USA. This treatment facility was selected because it deals predominantly with domestic sewage as specified in the guideline. There were no known contaminants in the fresh activated sludge believed to be present at levels high enough to interfere with this study. Fresh activated sludge was obtained on day -1 of the test. The activated sludge was homogenized in a blender for two minutes at medium speed. The homogenated sample was allowed to settle for 1 hour, after which the supernatant was decanted (avoiding carry-over of sludge solids). An aliquot of the supernatant was used to determine microbial activity. The microbial activity was determined using an Easicult®-TTC dip slide. The agar stick was removed from the culturing tube and the agar dipped into the supernatant aliquot. Excess supernatant was blotted off with a clean paper towel, and the agar stick was then placed back into the culture tube. The whole unit was placed into an environmental chamber for 48 hours at approximately 25.9 degrees C. The microbial activity was determined to be 10E5 CFU/mL. The remaining decanted sludge supernatant was used for final preparation of the test medium on day -1.
Duration of test (contact time):
49 d
Initial test substance concentration
Initial conc.:
ca. 25.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Biodegradation was based on oxygen consumption and the theoretical oxygen demand of the test substance as calculated using results of an elemental analysis of the test substance. The empirical formula and the theoretical oxygen demand (ThOD) of the test substance were calculated from elemental analysis data (assuming 100 gram test substance). Sodium benzoate ThOD was calculated using the empirical formula and was determined to be 1.67 mg O2/mg sodium benzoate. The ThOD calculation of the test and positive control substance was based on Annex IV of OECD 301F guideline.

The microbial count of the activated sludge was 10E5 CFU/mL. The sludge supernatant was added at a 1% loading volume of to test medium. Test medium consisted of glass distilled water and mineral salts (phosphate buffer, ferric chloride, magnesium sulfate, calcium chloride). Test medium and activated sludge, which was aerated for 24 hours with carbon dioxide free air, was added to each respirometer flask.

The test substance was added to the flasks by weighing onto glass fiber filters.

Test vessels were glass flasks placed in a water bath and electronically monitored for oxygen consumption. Triplicate test systems were run for the test substance, positive control, and the blanks.

Test substance concentration was ~24-27 mg/L. Test temperature was 20.5 – 22.9 deg C.

All test vessels were stirred constantly for 49 days using magnetic stir bars.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
The pH of the stock solution of sodium benzoate was 7.13 and not 7.4±0.2 as required by the protocol. The temperature in the respirometer water baths ranged from 20.5°C - 22.9°C, and not 22±1°C as required in the protocol. These deviations did not have an effect on the study. No other excursions from the protocol were noted. By day 2, >60% biodegradation of positive control was observed, which meets the guideline requirement. Oxygen uptake of the inoculum blank was within guideline limits.
% Degradationopen allclose all
Parameter:
% degradation (O2 consumption)
Value:
11.5
St. dev.:
0.96
Sampling time:
10 d
Remarks on result:
other: Based on ThOD
Parameter:
% degradation (O2 consumption)
Value:
50.6
St. dev.:
1.6
Sampling time:
28 d
Remarks on result:
other: Based on ThOD
Parameter:
% degradation (O2 consumption)
Value:
62.3
St. dev.:
0.62
Sampling time:
49 d
Remarks on result:
other: Based on ThOD
Details on results:
Substance inherently biodegradable. Greater than 60% biodegradation was achieved on day 49.

BOD5 / COD results

Results with reference substance:
Sodium benzoate biodegraded 62.83% after 2 days (St. dev. = 0.21).

Any other information on results incl. tables

 

Percent biodegradation of test substance

Day

Rep 1

Rep 2

Rep 3

Mean

SD

9

6.39

6.05

8.25

6.90

1.18

10

10.43

12.08

12.09

11.5

0.96

14

31.88

33.90

34.42

33.4

1.34

21

43.18

42.45

45.47

43.7

1.58

28

52.04

48.89

50.99

50.6

1.60

35

56.41

53.70

54.81

55.0

1.36

42 60.16 58.54 58.73 59.1 0.89
49 62.26 61.65 62.88 62.3 0.62

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
The registered substsance can be considered inherently biodegradable having reached 62% in 49 days.
Executive summary:

In a readily biodegradable screening test using non-adapted inoculum, the registered substance achieved 62% biodegradation when the test duration was extended to 49 d (since the plateau had not been reached at 28 d). As such, it can be considered inherently biodegradable, and is not expected to persist in the environment under aerobic conditions.