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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May to June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed two times by settlement and resuspension 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.
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using a Buchner funnel

No additional data

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

other: carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Solution a KH2PO4 8.50 g/L, K2HPO4 21.75 g/L, Na2HPO4.2H2O 33.40 g/L, NH4Cl 0.50 g/L
Solution b CaCl2 27.50 g/L
Solution c MgSO4.7H2O 22.50 g/L
Solution d FeCl3.6H2O 0.25 g/L
To 1 liter (final volume) of purified water was added the following volumes of solutions a – d.
10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d
- Test temperature: approximately 21 ºC
- pH: 7.4 ± 0.2
- pH adjusted: yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 liter test culture vessels
- Method used to create aerobic conditions: The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 14, 17, 21, 28 and 29. The second absorber vessels were all sampled on Days 0 and 29.

CONTROL AND BLANK SYSTEM
- Toxicity control: For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge microorganisms used in the test.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The test item is not soluble in water therefore pre-study solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test performance:

For the purpose of the test, the test item was dispersed directly in mineral medium.
An amount of test item (37.2 mg) was dispersed in approximately 400 mL of minerai medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 12.4 mg/L, equivalent to 10 mg carbon/L.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

26

Sampling time:

28 d

Details on results:

The test material attained 26% degradation 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 58% degradation after 14 days and 90% degradation after 28 days thereby confirming that N-octadecylstearamide was not toxic to the sewage treatment micro-organisms used in the test.

Results with reference substance:

Sodium benzoate attained 71% degradation after 14 days and 84% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Percentage Biodegradation Values :

Day	% Degradation Sodium Benzoate Procedure Control	% Degradation Test Item	% Degradation Test Item plus Sodium Benzoate Toxicity Control
0	0	0	0
2	43	0	25
6	62	0	31
8	67	0	42
14	71	10	58
17	77	9	67
21	74	13	74
28	73	22	83
29	84	26	90

 

pH Values of the Test Preparations on Days 0 and 28 :

 

Test Vessel	pH prior to adjustment on Day 0	pH after adjustment on Day 0	pH on Day28
Inoculum Control R1	7.7	7.5	7.3
lnoculum Control R2	7.7	7.5	7.2
Procedure Control    R1	7.7	7.4	7.2
Procedure Control  R2	7.7	7.5	7.3
Test Item  R1	7.6	7.4	7.2
Test Item R2	7.6	-	7.3
Toxicity Control	7.6	7.5	7.3

 

Total and Inorganic Carbon Values in the Culture Vessels on Day 0 : 

Test vessel	Total Carbon* (mg/L)	Inorganic Carbon* (mg/L)	IC/TC Ratio (%)
Test Item 10mgC/L R1	10.71**	0.36	3
Test Item 10 mg C/L R2	10.43**	0.31	3

 

R1 - R2 = Replicate 1 and 2

* Corrected for control values.

** Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test material attained 26% degradation after 28 days and is not considered to be readily biodegradable under the conditions of OECD Guideline No. 301B.

Executive summary:

An experimental study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium according the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" and GLP. 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 approximately 21 °C for 28 days. The degradation 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 used for validation purposes.

The test item attained 26% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

An experimental study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium according the OECD Guidelines No. 301B and GLP. The test material attained 26% degradation after 28 days therefore is not considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater

Additional information

The REACH guidance document R7B indicates that biodegradation above 20% may be regarded as evidence of inherent biodegradation (for more details, please see the REACH gidance document R7b v4, June 2017, page 216)