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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:
01 November 2017 to 25 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Physical state/Appearance: clear, yellow liquid
- CAS Number: 13539-13-4
- EC Number: 236-912-2
- Purity: >99% (UVCB)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed Whatman GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until two successive dry weights within 4% were attained, thereby indicating that complete dryness had been attained. The suspended solids concentration was equal to 4.0 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
10 other: mg C/L
Based on:
ThCO2
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Based on the results of a preliminary solubility/dispersibility trial and following the recommendations of the International Standards Organisation (ISO 10634, 1995), the test item was dispersed in the test medium using the high shear mixing method.

The following test preparations were prepared and inoculated in 5 liter glass test culture vessels each containing 3 liters of solution:

a) Duplicate inoculated control vessels consisting of inoculated mineral medium.
b) Duplicate procedure control vessels containing the reference item (sodium benzoate) in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) Duplicate test item vessels containing test item in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) A single toxicity control consisting of the test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L.

One additional vessel containing water only was incubated under the same conditions as the test vessels to allow the temperature to be checked on each sampling occasion.

Each vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room, in darkness, and the temperature of the additional vessel containing water which was incubated under the same conditions as the test vessels ranged from 20 to 24 °C.

Approximately 24 hours prior to addition of the test and reference items, the vessels were filled with 2400 mL of mineral medium and 22.5 mL of inoculum and aerated overnight. On Day 0, the test and reference items were added and the pH of all vessels measured using a handheld meter. The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.

For the duration of the study, the test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.

The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.

Observations
The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

pH Measurements
The pH of the test preparations was determined on Day 0 (pre- and post-adjustment) and on Day 28 using handheld meter.

Temperature Measurements
Temperature was measured daily in the additional water-only vessel using a Hanna Instruments HI 93510 digital thermometer.
Reference substance:
benzoic acid, sodium salt
Remarks:
10 mg C/L
Test performance:
The averaged total CO2 evolution in the two inoculum control vessels on Day 28 was 33.52 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.

The CO2 production at the end of the test for the replicate vessels was <20%, satisfying the validation criterion given in the OECD Test Guidelines.

Biodegradation in the procedure and toxicity controls satisfied the validation criterion given in the OECD Test Guidelines.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
Acidification of the test vessels on Day 28 prior to final analyses being performed on Day 29. This acidification effectively kills the micro-organisms present and drives of any dissolved CO2 present in the test vessels. Acidification of the test vessels was not performed during the study due to a concurrent study which shared the control and reference item vessels being extended past 28 days. The lack of acidification was considered not to have had an impact on this study as no significant biodegradation had been shown to have occurred, and past experience has shown that only limited amounts of dissolved CO2 are released by the acidification process in the test design employed.
Results with reference substance:
The reference item, sodium benzoate, attained 80% biodegradation after 14 days and 100% biodegradation after 28 days, thereby satisfying the validation criterion given in the OECD Test Guideline 301B and confirming the suitability of the inoculum and test conditions.

Statistical analysis of the Day 28 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.

The toxicity control attained 29% biodegradation after 14 days and 37% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable according to the criteria set forth in OECD Guideline No. 301B.

The reference item, sodium benzoate, attained 80% biodegradation after 14 days and 100% biodegradation after 28 days, thereby satisfying the validation criterion given in the OECD Test Guideline 301B and confirming the suitability of the inoculum and test conditions.

Percentage Biodegradation Values

Day

% Biodegradation

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

43

0*

22

6

55

0*

21

8

62

0*

24

10

81

0*

29

14

80

0*

29

21

96

0*

27

28

100

0*

37

*Negative biodegradation values reported as 0% biodegradation

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Executive summary:

A study was performed to the standardized guideline OECD 301B and EU Test Method C.4-C, under GLP conditions to assess the ready biodegradability of the test item in an aerobic aqueous medium.

 

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures between 20 and 24 °C for 28 days.

 

Based on the results of a preliminary solubility/dispersibility trial and following the recommendations of the International Standards Organisation (ISO 10634, 1995), the test item was dispersed in the test medium using the high shear mixing method.

 

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were included for validation purposes.

 

All validation criteria were met in this study, and the test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readilybiodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

Key study

A study was performed to the standardized guideline OECD 301B and EU Test Method C.4-C, under GLP conditions to assess the ready biodegradability of the test item in an aerobic aqueous medium. All validation criteria were met in this study, and the test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readilybiodegradable under the strict terms and conditions of OECD Guideline No. 301B (Envigo, 2018).

Key value for chemical safety assessment

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

Additional information