4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-19 - 2017-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also conducted according to GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): a fresh sample of activated sludge was collected on Dec 8th, 2016 from the aeration tank of the Long Hua Wastewater Treatment Plant in Shanghai which treats predominantly domestic sewage with A/O process. Sludge was kept under aeration.

- Preparation of inoculum for exposure: According to the dry weight of 7.37%, 40.7 g of concentrated sludge needed to be suspended in 1L mineral medium to yield a concentration of 3g dry matter/L. The final concentration of the activaged sludge in the test medium was 30 mg suspended solids/L.

Duration of test (contact time):

28 d

Initial conc.:

> 24.36 - < 24.8 mg/L

Based on:

test mat.

Initial conc.:

> 51.4 - < 52.33 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Test temperature: 22.0 - 22.6 °C

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes

Reference substance:

benzoic acid, sodium salt

Test performance:

* mean total uptake in the inoculum blanks at the end of the test was less than 60 mg O2/L in 28 days
* reference substance was 84% by day 14
* different of extremes between replicates < 20%
* substance was not inhibitory to the inoculum

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Details on results:

pH 6.87 - 7.07

Results with reference substance:

Toxicity control: 63% biodegradation in 14 days
Sodium benzoate: 84% in 14 days

Day (d)	Test substance			Reference control			Toxicity control
	TS1	TS2	Average	PC1	PC2	Average	TC
1	-4	-4	-4	11	16	14	11
2	-6	-6	-6	54	52	53	41
3	-2	-5	-4	65	65	65	49
4	-2	-2	-2	69	71	70	53
5	-2	-2	-2	73	73	73	56
6	-11	-8	-10	75	75	75	56
7	-15	-15	-15	75	76	76	57
8	-1	-1	-1	79	80	80	60
9	-12	-14	-13	79	80	80	59
10	-10	-7	-9	80	82	81	61
11	-4	-4	-4	81	85	83	62
12	-8	-5	-7	83	86	85	62
13	-1	-1	-1	82	86	84	64
14	-10	-7	-9	82	86	84	63
15	-10	-5	-8	82	87	85	64
16	-9	-3	-6	82	86	84	64
17	-2	0	-1	85	88	87	65
18	-4	-1	-3	84	89	87	64
19	4	-4	0	84	92	88	67
20	1	-7	-3	86	93	90	69
21	5	-1	2	82	84	83	63
22	4	1	3	84	91	88	67
23	1	1	1	87	92	90	70
24	1	1	1	87	92	90	70
25	-1	-1	-1	90	93	92	70
26	3	0	2	87	92	90	68
27	0	0	0	87	92	90	68
28	-1	5	2	87	90	89	66

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The biodegradation of the test item was 2% after 28 days. The pass level of 60% was not achieved, so the test item cannot be considered as readily biodegradable.

Executive summary:

The ready biodegradability of the test item was investigated according to OECD 301F TG and in compliance with GLP. The biodegradation was followed during 28 days, with an initial concentration of ca. 24.5 mg/L using a non-adapted inoculum. Dissolved oxygen consumption was measured every day. After 28 days, the average percentage of biodegradation of the test item was 2%. It is therefore concluded that the test item is not readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Mar - 18 Apr 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Also conducted according to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge bacteria were obtained on 21 Mar 1992 from the aeration stage of the treatment plant at Huntingdon Research Centre Ltd. treating predominantly domestic sewage.
- Preparation of inoculum for exposure: The sample was allowed to settle and the supernatant filtered through Whatman GFA filter paper. The first 250 mL were discarded.
- Concentration of inoculum in the test: 1 drop of inoculum per litre

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Initial conc.:

4.83 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Standard nutrient medium as prescribed by the guideline 301 D.
- Solubilising agent: Chloroform
- Test temperature: 20 ± 1 °C

TEST SYSTEM
- Culturing apparatus: 280 mL BOD bottles, darkened glass, with ground stoppers. BOD bottles were filled, by siphon, and firmly stoppered to exclude all air bubbles.
- Number of culture flasks/concentration: The test and control series were prepared in duplicates.
- Measuring equipment: Yellow Springs BOD Probe (Model 54)

SAMPLING
- Sampling frequency: Dissolved oxygen concentrations for each test medium were determined in duplicate at 0, 5, 15 and 28 days.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, with and without filter paper
- Toxicity control: no
- Reference control: yes, 2 bottles with 2 mg/L aniline plus filter paper, 2 bottles with sodium benzoate without filter paper
- Other: non-inoculated dilution water

Reference substance:

aniline

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

-2

Sampling time:

28 d

Remarks on result:

other: Negative result

Details on results:

- Oxygen depletions in the inoculated and non-inoculated control series were within the prescribed limits.
- The test item attained 0, 5 and -2% degradation within 5, 15 and 28 days, respectively.

Results with reference substance:

- Sodium benzoate (prepared without filter paper) attained 82% degradation within 15 days and 94% within 28 days.
- Aniline (prepared with filter paper) attained 36% degradation within 15 days and 65% within 28 days.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The biodegradation of the test item was -2% after 28 days. The pass level of 60% was not achieved, so the test item cannot be considered as readily biodegradable.

Executive summary:

The ready biodegradability of the test item was investigated according to OECD 301D TG and in compliance with GLP. The biodegradation was followed during 28 days, with an initial concentration of 2 mg/L using a non-adapted inoculum. Dissolved oxygen consumption was measured after 0, 5, 15 and 28 days. Because the substance was added with filter paper, a standard using aniline and filter paper was concurrently run in addition to the regular sodium benzoate test. After 28 days, the percentage of biodegradation of the test item was -2%. It is therefore concluded that the test item is not readily biodegradable.

Description of key information

0% Biodegradation in 28 days (OECD 301D)

2% Biodegradation in 28 days (OECD 301F)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Two tests on the biodegradability of 4,4´-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS No. 107934-68-9) are available.

The first study was conducted according to OECD 301D using filter paper as carrier (Douglas, 1991). There was no biodegradation (0%) observed within 28 days.

The second test was conducted according to OECD 301F (2017). There was barely any biodegradation (2%) observed within 28 days.

Finally, A QSAR screening using BIOWIN v4.10 on 4,4´-(9H-fluoren-9-ylidene)bis(2-chloroaniline) indicated the substance even to be recalcitrant to ultimate biodegradation. Level III Fugacity modeling (not explicitely presented in this dossier) indicated soil and sediment as target compartments for environmental distribution (soil 53%, sediment 47%) for this substance with half-lives of 360 days for soil and 1620 days for sediment. Therefore, with regard to the PBT and vPvB criteria as presented in the Guidance on Information Requirements and Chemical Safety Assessment R.11 PBT-assessment (ECHA 2014), 4,4´-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS No. 107934-68-9) is regarded as very persistent (vP).