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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:
10 August 2016 to ****
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:
- CAS Numer: 15337-18-5
- Description: Yellow viscous liquid
- Purity: 96.6%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
- Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on
14 November 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Pre-adaptation of the inoculum to the test item was not performed.

- Preparation of Inoculum
The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. The suspended solids concentration was equal to 3.0 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
5 other: mg C/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparation of Test System
An initial experiment was conducted with the test item and procedure control vessels prepared at a concentration of 10 mg C/L, and a concurrent toxicity control containing 10 mg C/L each of test and reference item, resulting in a total concentration of 20 mg C/L. The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25% biodegradation after 14 days, indicating that, under the strict terms and conditions of the OECD Guidelines, the test item was exhibiting inhibitory effects. A repeat test was therefore performed in which the test item was exposed at a reduced concentration of 5 mg C/L.

Based upon the results of the preliminary solubility work and according to testing guidelines, the test item was weighed onto a filter paper and added with the filter to the inoculated mineral medium.

The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) Duplicate inoculated control vessels consisting of inoculated mineral medium plus a filter paper.
b) Duplicate procedure control vessels containing the reference item (sodium benzoate) in inoculated mineral medium plus a filter paper to give a final concentration of 10 mg carbon/L.
c) Duplicate test item vessels containing the test item on a filter paper in inoculated mineral medium to give a final concentration of 5 mg carbon/L.
d) A single toxicity control consisting of the test item on a filter paper plus the reference item in inoculated mineral medium to give a final concentration of 15 mg carbon/L.

A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
Each test 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 at measured temperatures of between 22 and 24 ºC, in darkness.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 30 mL of inoculum and aerated overnight.

The pH was adjusted to pH 7.4 ± 0.2, as necessary, 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.

The 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 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH-absorbing solutions, which were prepared using purified water.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
21
Sampling time:
28 d
Details on results:
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of inoculum control R1. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The pH in the control and test solutions ranged from 7.7 to 7.8 prior to adjustment to 7.5 with using diluted hydrochloric acid on Day 0, and ranged from 7.4 to 7.5 on Day 28 (prior to acidification).

The test item attained 21% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 55% biodegradation after 14 days and 65% biodegradation after 28 days thereby satisfying the validation criterion given in the OECD Test Guidelines and confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

The reference item, sodium benzoate, attained 86% biodegradation by Day 14 days (and within 10 days of biodegradation exceeding 10%) and 114% biodegradation after 28 days thereby satisfying the validation criterion given in the OECD Test Guidelines and confirming the suitability of the inoculum and test conditions. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation.

Percentage Biodegradation Values

Day

% Biodegradation

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

51

0

29

6

72

0

48

8

75

0

50

10

83

0

55

14

86

12

55

21

99

11

54

28

103

5

53

29*

114

21

65

*Day 29 values corrected to include any carry-over of CO2detected in Absorber 2

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 21% 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 assess the ready biodegradability of the test item in an aerobic aqueous medium.  The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability: CO2 Evolution Test", Method C.4-C of Commission Regulation (EC) No. 440/2008, and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110.

An initial experiment was conducted with the test item and procedure control vessels prepared at a concentration of 10 mg C/L, and a concurrent toxicity control containing 10 mg C/L each of test and reference item, resulting in a total concentration of 20 mg C/L.  The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25% biodegradation after 14 days, indicating that, under the strict terms and conditions of the OECD Guidelines, the test item was exhibiting inhibitory effects.  

Following the recommendations of the test guidelines, a repeat test was performed in which the test item was exposed at a reduced concentration of 5 mg C/L to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 and 24 ºC for 28 days.

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 tested concurrently for validation purposes.

The test item attained 21% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. All validation criteria were met in this study.  

Description of key information

The test item attained 21% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Key value for chemical safety assessment

Additional information