[No public or meaningful name is available]

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:

15 Jan - 02 Jul 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

other: US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q))

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: STP Effluent

Details on inoculum:

- Source of inoculum/activated sludge: A mixed population of sewage treatment micro-organisms was obtained on 03 June 2021 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.
- Storage conditions: The effluent was filtered (coarse filter paper, first 200 mL were discarded) and subsequently aerated in a temperature controlled room (20 °C).
- Pretreatment: no
- Effluent concentration: 1% v/v

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

307 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: according to guideline
- Additional substrate: no
- Solubilising agent/ Application of test material: Acetone test item solution: 1000 mg/ 10 mL (nominal); 500 µl of this acetone test item solution (mixed to ensure homogeneity of the solution) was applied to filter paper (Whatman GF/A (70 mm)) and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) and allowed to cool to temperatures of approximately 21 °C prior to addition of inoculum. The volume was then adjusted to 500 mL to give a final concentration of 100 mg/L. Additionally, all controls received a filter to which pure acetone was applied and the solvent allowed to evaporate to dryness for approximately 15 minutes.
- Test temperature: 20-22 °C
- pH: 7.5 to 7.6 in the inoculated test item vessels after 28 d
- pH adjusted: no
- pH Measurement: prior to the addition of the inoculum; 28 d (all vessels)
- Aeration of dilution water: yes, approximately 20 h prior to the start of the test (9 mg O2/L)
- Effluent concentration: 1% v/v
- Continuous darkness: no, diffuse light

TEST SYSTEM
- Culturing apparatus: CES Multi-Channel Aerobic Respirometer
- Number of culture flasks/concentration: 2 (+1) /test item concentration; To assure the possibility of prolonging the test, 3 replicates were prepared for all test item concentrations. Since the test was not prolonged only 2 replicates were measured.
- Test performed in closed vessels due to significant volatility of test substance: The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.
- Details of trap for CO2 and volatile organics if used: ethanolamine solution (50% v/v)
- Data generation: The data generated from the respirometer’s own battery backed memory was collected four times a day on the hard disk drive of a nondedicated computer.
- Measurement of BOD values: daily

CONTROL AND BLANK SYSTEM
- Inoculum blank: n=2 (+1), inoculum + filter paper (Whatman GF/A (70 mm diameter)); To assure the possibility of prolonging the test, 3 replicates were prepared as control. Since the test was not prolonged only 2 replicates were measured.
- Abiotic sterile control: no
- Toxicity control: n= 1 (+1),100 mg/L aniline, freshly distilled and 100 mg/L test item + filter paper (Whatman GF/A (70 mm diameter)); 2 replicates were prepared as toxicity control. Since the test was not prolonged only 1 replicate was measured.
- Procedure control: n= 1 (+1), 100 mg/L aniline, freshly distilled + filter paper (Whatman GF/A (70 mm diameter)); 2 replicates were prepared as procedure control. Since the test was not prolonged only 1 replicate was measured.

TOC Measurement diluted aniline:
- Measurement: Shimadzu TOC-VCPH (Software Version 2.30)
- Result: Total Organic Carbon of the diluted aniline confirmed that it had been prepared correctly.

INITIAL EXPERIMENTS
1. Initial experiment:
- Test item concentration: 10 mg C/L
- Vailidity criteria: procedure control failed the validation criteria on day 14

2. Initial experiment:
- Test item conenctration: 10 mg C/L
- Results: All validity criteria were fullfilled. Test item degradation did not achieve 60% degradation. Due to a technical error, the test was terminated on day 28 although the sponsor requested an extension to 60 d. Therefore, the test was repeated.

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

71

Sampling time:

28 d

Remarks on result:

other:

Remarks:

Test item UVCB, the 10-day window criteria does not apply

Details on results:

Toxicity control: test item not toxic to the tested microorganisms; 67% biodegradation after 14 d and 73% biodegradation after 28 d

Results with reference substance:

Procedure control (Aniline): 65% biodegradation after 14 d, > 60% degradation within the 10 d window; 73% degradation after 28 d

VALIDITY CRITERIA OECD 301 F

Table 1: Validity criteria for OECD 301 F

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	< 20%	yes
Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).	65%	yes
The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.	67%	yes
The oxygen uptake of the inoculum blank is normally 20-30 mg O2/L and should not be greater than 60 mg/L in 28 days.	29.61 mg O2/L after 28 d	yes

Table 2 Percentage Biodegradation Values – Definitive Test

Day		Biodegradation (%)
	Procedure Control	Test Item
		R1	R2	Mean	Toxicity Control
0	0	0	0	0	0
1	0	0	0	0	0
2	0	11	10	11	7
3	0	14	14	14	22
4	0	18	17	18	37
5	7	27	25	26	46
6	31	34	32	33	47
7	55	39	36	38	48
8	57	43	40	42	49
9	58	46	44	45	53
10	59	50	48	49	59
11	61	53	51	52	62
12	62	56	53	55	64
13	64	57	55	56	66
14	65	59	57	58	67
15	66	60	58	59	67
16	66	61	60	61	68
17	67	62	61	62	69
18	67	63	62	63	69
19	68	64	63	64	69
20	68	65	64	65	70
21	69	66	65	66	71
22	70	67	66	67	71
23	71	67	66	67	72
24	72	68	67	68	72
25	72	68	68	68	73
26	72	69	69	69	73
27	73	69	70	70	73
28	73	70	71	71	73

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Interpretation of results:

readily biodegradable, but failing 10-day window

Remarks:

According to chapter R. 7b of the ‘Guidance on information requirements and chemical safety assessment - Endpoint specific guidance' (ECHA, 2017) the 10 d window does not apply for UVCB substances.

Description of key information

Readily biodegradable according to OECD criteria (71% after 28 d, OECD 301F).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater

Additional information

The available key study on the ready biodegradability of the UVCB test substance was performed according to OECD guideline 301 F (GLP). The biodegradability of the substance was assessed by following the O2 consumption of non-adapted effluent microorganisms over 28 days at a test substance concentration of 100 mg/L (nominal). A mean biodegradation of 71% was observed after 28 d. The ten-day window criterion was not met. According to chapter R. 7b of the ‘Guidance on information requirements and chemical safety assessment - Endpoint specific guidance' (ECHA, 2017) the 10 d window does not apply for UVCB substances. The toxicity control achieved 67 % degradation after 14 days. Thus, inhibitory effects of the test substance on activated sludge microorganisms can be excluded. For these reasons, the test substance is stated to be readily biodegradable according to OECD criteria.