Reaction mass of Phosphorous(1+), (N-ethylethanaminato) diphenyl(phenylmethyl)-(T-4)-salt with 4, 4' - [2,2,2-trifluoro-1-(trifluoromethyl) ethylidene bis[phenol] (1:1) and (4,4'-[2,2,2-Trifluoro-1-(trifluoromethyl)ethylidene]diphenol

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:

2015

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:

yes

Remarks:

The study integrity was not adversely affected by this deviation.

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by this deviation.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3706OB
- Expiration date of the lot/batch:31 March 2019
- Purity test date:> 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:At room temperature

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

-Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was determined to be 4.5 g/l in the concentrated sludge. Before use, the sludge was allowed to settle (42 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/l of mineral medium.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

TOC

Initial conc.:

17 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
The test item was a white powder with a purity of >99%. The test substance was tested in duplicate at 17 mg/l, corresponding to 10 mg TOC/l. The organic carbon content was based on the molecular formula taking into account the mole ratio of the components.
Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 34.21 mg; test substance bottle B: 33.98 mg and toxicity control bottle: 33.89 mg). To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface.

Stock solutions of A) 8.50 g KH2PO4
mineral components 21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli-RO water and made up to 1 litre,
pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water
and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and
made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and
made up to 1 litre.
Mineral medium 1 litre mineral medium contains: 10 ml of solution (A),
1 ml of solutions (B) to (D) and Milli-RO water.
Barium hydroxide 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
Synthetic air (CO2 < 1 ppm) A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
Illumination The test media were excluded from light

TEST SYSTEM
- Pre-incubation medium:
The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

- Type and number of bottles:
>Test suspension: containing test substance and inoculum (2 bottles).
>Inoculum blank: containing only inoculum (2 bottles)
>Positive control: containing reference substance and inoculum (1 bottle).
>Toxicity control: containing test substance, reference substance and inoculum (1 bottle).

-Preparation: At the start of the test (day 0), test and reference substance were added to the bottles containing the microbial organisms and mineral components.
The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.


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

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

3

Sampling time:

28 d

Details on results:

THEORETICAL CO2 PRODUCTION:
The ThCO2 of teh test item was calculated to be 2.12 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

BIODEGRADATION:
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation ofthe test item (1% and 3%, based on ThCO2).
In the toxicity control, more than 25% biodegradation occurred within 14 days (32%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve

TEMPERATURE AND PH
The temperature recorded in a vessel with water in the same room varied between 21.6 and 22.5°C.
The pH values ranged from 7.6 at the start of the test to 7.5 on day 28.

Table 1: CO₂production and percentage biodegradation of the positive control substance.

Day	HCl (0.05N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation * (%)
	Blank (mean)	Positive control
2	44.98	39.33	5.65	6.2	6.2	7
5	43.74	22.71	21.03	23.1	29.3	34
7	43.44	30.77	12.67	13.9	43.3	51
9	43.69	37.33	6.36	7.0	50.3	59
14	42.67	37.29	5.38	5.9	56.2	66

* Calculated as the ratio between CO₂produced (cumulative) and the ThCO₂of sodium acetate: 85.5 mg CO₂/2l

Table 2: CO₂production and percentage biodegradation of the test substance (bottle A)

Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation * (%)
	Blank (mean)	Bottle A
2	44.98	45.01	0.00	0.0	0.0	0
5	43.74	43.63	0.11	0.1	0.1	0
7	43.44	45.08	0.00	0.0	0.1	0
9	43.69	44.19	0.00	0.0	0.1	0
14	42.67	43.77	0.00	0.0	0.1	0
19	42.31	43.17	0.00	0.0	0.1	0
23	40.45	43.25	0.00	0.0	0.1	0
27	41.10	42.50	0.00	0.0	0.1	0
29	42.83	42.33	0.50	0.6	0.7	1
29	44.88	45.69	0.00	0.0	0.7	1
29	46.78	47.25	0.00	0.0	0.7	1

*Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 72.5 mg CO₂/2l

Table 3: CO₂production and percentage biodegradation of the test substance (bottle B).

Day	HCl (0.05 N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation* (%)
	Blank (mean)	Bottle B
2	44.98	44.15	0.83	0.9	0.9	1
5	43.74	43.11	0.63	0.7	1.6	2
7	43.44	43.28	0.16	0.2	1.8	2
9	43.69	44.80	0.00	0.0	1.8	2
14	42.67	43.10	0.00	0.0	1.8	2
19	42.31	43.23	0.00	0.0	1.8	2
23	40.45	42.30	0.00	0.0	1.8	2
27	41.10	42.85	0.00	0.0	1.8	2
29	42.83	42.96	0.00	0.0	1.8	2
29	44.88	45.83	0.00	0.0	1.8	2
29	46.78	46.24	0.54	0.6	2.4	3

*Calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 72.0 mg CO₂/2l

Table 4: CO₂production and percentage biodegradation of the toxicity control.

Days	HCl (0.05N) titrated (ml)		Produced CO2 (ml HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation*(%)
	Blank (mean)	Toxicity control
2 5 7 9 14	44.98 43.74 43.44 43.69 42.67	39.82 28.90 32.28 37.13 35.09	5.16 14.84 11.16 6.56 7.58	5.7 16.3 12.3 7.2 8.3	5.7 22.0 34.3 41.5 49.8	4 14 22 26 32

*Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test substance and positive control: 157.3 mg CO2/2l (ThCO2 test substance: 71.8 mg CO2/2l + ThCO2 sodium acetate: 85.5 mg CO2/2l)

Table 5 Comparison of biodegradation of the test substance in bottles A and B.

Day	Biodegradation (%)
	Bottle A	Bottle B	Mean A andB	∆ A-B*
2	0	1	1	1
5	0	2	1	2
7	0	2	1	2
9	0	2	1	2
14	0	2	1	2
19	0	2	1	2
23	0	2	1	2
27	0	2	1	2
29	1	2	2	1
29	1	2	2	1
29	1	3	2	2

*Absolute difference in biodegradation between bottles A and B

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Executive summary:

The determination of the "ready" biodegradability of the test item was performed using the carbon dioxide (CO2) evolution test (modified Sturm test) according to OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C, ISO 9439, 1999 and ISO 10634, 1995.

The test substance was tested in duplicate at 17 mg/l, corresponding to 10 mg TOC/l. The organic carbon content was based on the molecular formula taking into account the mole ratio of the components. The Theoretical CO2 production (ThCO2) of test item was calculated to be 2.12 mg CO2/mg.

The study consisted of six bottles:

- 2 inoculum blanks (no test substance),

- 2 test bottles (Test item),

- 1 positive control (sodium acetate) and

- 1 toxicity control (Test item plus sodium acetate).

Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface. Test duration was 28 days (last CO2 measurement on day 29). The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of the test item (1% and 3%, based on ThCO2). In the toxicity control, the test item Reaction mass of AminoPhosphonium salt and Bisphenol AF (XA 31) was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid.

Description of key information

The Reaction mass of AminoPhosphonium salt and BisphenolAF (XA 31) was not readily biodegradable under the conditions of the carbon dioxide (CO2) evolution test (modified Sturm test) (OECD guideline No. 301 B)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

One reliable study is available for the Reaction mass of AminoPhosphonium salt and BisphenolAF (XA 31) for this endpoint. The determination of the "ready" biodegradability of the test item was performed using the carbon dioxide (CO2) evolution test (modified Sturm test) according to OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C, ISO 9439, 1999 and ISO 10634, 1995.

The test substance was tested in duplicate at 17 mg/l, corresponding to 10 mg TOC/l. The organic carbon content was based on the molecular formula taking into account the mole ratio of the components. The Theoretical CO2 production (ThCO2) of test item was calculated to be 2.12 mg CO2/mg.

The study consisted of six bottles:

- 2 inoculum blanks (no test substance),

- 2 test bottles (Test item),

- 1 positive control (sodium acetate) and

- 1 toxicity control (Test item plus sodium acetate).

Test duration was 28 days (last CO2 measurement on day 29). The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of the test item (1% and 3%, based on ThCO2). In the toxicity control, the test item was found not to inhibit microbial activity.