[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:

17-10-2019 to 09-01-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test conducted in accordance with OECD TG 301F and OECD TG 301 : Annex III para. 3, 6 and 7 ; use of silicone oil emulsifying agent for poorly soluble compounds

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Test conducted in accordance with OECD TG 301F and OECD TG 301 : Annex III para. 3, 6 and 7 ; use of silicone oil emulsifying agent for poorly soluble compounds

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2016 ; signature: January 2017

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: See ‘Test material information’
- Expiration date of the lot/batch: See ‘Test material information’
- Purity test date: See ‘Test material information’

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: See ‘Test material information’
- Stability under storage conditions: Stable.
- Stability under test conditions: See other fields. Substance is stable in silicone oil dispersant/emulsifier - prior to the exposure to the biological inoculum as part of (bio)degradation test.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Soluble in silicone oil dispersant/emulsifier.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not applicable. Not pre-treated.
- Preliminary purification step (if any): Not applicable.
- Preparation of a nanomaterial dispersion (incl. dilution): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: Test conducted in accordance with OECD TG 301F and OECD TG 301 : Annex III para. 3, 6 and 7 ; use of silicone oil emulsifying agent for poorly soluble compounds

- Final preparation of a solid: Not applicable.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Test item was tested: (i) neat and/or (ii) pre-dispersed into : silicone oil [WACKER AR 20 SALBEN SILICONÖL / batch number: IG12700 / expiry date: 10-02-2020] ; corresponding controls were conducted. For specifics see ‘OTHER SPECIFICS – other information:’ for more information, below.

INFORMATION ON NANOMATERIALS
- Chemical Composition: Not applicable.
- Density: Not applicable.
- Particle size & distribution: Not applicable.
- Specific surface area: Not applicable.
- Isoelectric point: Not applicable.
- Dissolution (rate): Not applicable.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) Not applicable.

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: For pH see below. Separate replicates of test item and toxicity control were prepared for measurement of the pH-value at test start as indicated below. No precipitate in the culture medium was reported in the study.
- other information: The test solutions were prepared in measuring flasks and were placed in brown glass bottles as incubation vessels (inoculum control, functional control) or were prepared directly in brown glass bottles (functional control with silicone oil, test item with and without silicone oil, toxicity control with and without silicone oil). Controls and/or test item groups: were comprised as the following:
(a) Inoculum control (C) : test medium without test and / or reference item
(b) Inoculum control with silicone oil (CS) : test medium without test and / or reference item and 500 μL Silicone oil were pipetted directly into the inoculated test medium.
(c) Toxicity control (T) : test and reference item in test concentration
(d) Toxicity control with silicone oil (TS) : test item (incl. silicone oil as described above) and reference item in test concentration
(e) Functional control (R) : Sodium benzoate at 30 mg/L (ThOD 1.67 mg O2/mg or ThOD in test vessel: 50.1 mg O2/L)
(f) Functional control with silicone oil (RS) : 500 μL silicone oil [WACKER AR 20 SALBEN SILICONÖL / batch number: IG12700 / expiry date: 10-02-2020]
(g) Test Item (P) : test item at 17 mg/L (corresponding to 4.8 μL test item / 250 mL test solution) (test item ThOD cited in the full study report; ThOD in test vessel: 51.2 mg O2/L)
(h) Test item with Silicone oil (Ps) : test item at 17 mg/L concentration, pre-dispersed within silicone oil stock solution (85.0 mg test item / 10 mL silicone oil) and corresponding to 500 μL stock solution for 250 mL test solution (test item ThOD cited in the full study report; ThOD in test vessel: 51.2 mg O2/L)
Replicates were prepared as follows:
(i) two for the inoculum control (C1, C2)
(ii) two for the inoculum control with silicone oil (CS1, CS2)
(iii) one for the functional control (R1)
(iv) one for the functional control with silicone oil (RS1)
(v) two for the test item (P1, P2)
(vi) two for the test item with silicone oil (PS1, PS2)
(vii) one for the toxicity control (T1)
(viii) one for the toxicity control with silicone oil (TS1)
Separate replicates of test item and toxicity control were prepared for measurement of the pH-value at test start:
(a) one for the inoculum control with silicone oil (Cs pH)
(b) one for the functional control with silicone oil (Rs pH)
(c) one for the test item (P pH)
(d) one for the test item with silicone oil (PS pH)
(e) one for the toxicity control (T pH)
(f) one for the toxicity control with silicone oil (TS pH)
Further information on pH results is provided in further sections below.

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): Inoculum of the aqueous phase of non-adapted activated sludge, from the municipal sewage treatment plant, 31137 Hildesheim, Germany. This facility treats predominantly domestic/municipal sewage.
- Laboratory culture: See below.
- Method of cultivation: See below.
- Storage conditions: See preparation of inoculum for exposure and pretreatment fields.
- Storage length: < 1 week (actual: 3 days prior to exposure)
- Preparation of inoculum for exposure: The activated sludge was washed twice with tap water. After the second washing the settled sludge was resuspended in mineral
salts medium and was maintained in an aerobic condition by aeration with CO2 free air until test start (3 days). The amount of inoculum used to initiate inoculation was 4.01 mL/L (25.0 mg/L dw). Colony forming units in the test vessels was ca. 1x10^7 to 1x10^8 CFU/L
- Pretreatment: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. Determination of dry weight is made to inoculate final solution with 25.0 mg/L dry weight activated sludge.
- Concentration of sludge: The sludge was diluted in the BOD bottles to 25 mg DW/L.
- Initial cell/biomass concentration:
- Water filtered: Yes. Mineral salts medium according to OECD TG 301 F was utilised during the study.
- Type and size of filter used, if any: Not reported.

Duration of test (contact time):

28 d

Initial conc.:

17 mg/L

Based on:

test mat.

Remarks:

test item at 17 mg/L (P) and/or test item at 17 mg/L within silicone oill (Ps)

Initial conc.:

51.2 mg/L

Based on:

ThOD

Remarks:

in test vessel: 51.2 mg O2/L within (P) and/or (Ps)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: salts Medium according to OECD TG 301 F was utilised during the study. Consisting of the required volumes of mineral medium stock solutions, ultrapure water and inoculum, as applicable.
- Additional substrate: Not applicable.
- Solubilising agent (type and concentration if used): Test item was tested: (i) neat and/or (ii) pre-dispersed into : silicone oil [WACKER AR 20 SALBEN SILICONÖL / batch number: IG12700 / expiry date: 10-02-2020] dispersant/emulsifier ; corresponding controls were conducted.
- Test temperature: 22 ±1 °C [Nominal: 20 - 24 ± 1 °C ; Actual: 21.0 – 22.2 °C]
- pH: See table 1.
- pH adjusted: No (final pH was in the range of 7.52 to 7.54 for test item vessels with and without silicone oil ; and/or 7.51 to 7.67 for all controls: inoculum controls, functional controls, toxicity controls with and without silicone oil)
- CEC (meq/100 g): Not applicable.
- Aeration of dilution water: Yes.
- Suspended solids concentration: Determination of dry weight is made to inoculate final solution with 25.0 mg/L dry weight activated sludge.
- Continuous darkness: Yes.
- Other: Not applicable.

TEST SYSTEM
- Culturing apparatus: Brown glass bottles (volume 500 mL) with fill volume 250 mL ; continuous stirring on test
- Number of culture flasks/concentration: See ‘CONTROL AND BLANK SYSTEM’ for detailed information on replicates prepared.
- Method used to create aerobic conditions: Aeration of dilution water/mineral medium, prior to testing and continuous stirring on test.
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: See SAMPLING for further information. The respirometer used during this study is an Oxitop Control System (WTW Oxitop). Evolved carbon dioxide is absorbed by the CO2 trap.
- Test performed in closed vessels due to significant volatility of test substance: No. Bottles were closed with OxiTop measuring heads.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used: A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2.
- Other: See ‘CONTROL AND BLANK SYSTEM’ for detailed information on replicates prepared.

SAMPLING
- Sampling frequency: Daily (at least were reported) [Actual: 360 measuring points during the 28 d incubation period]
- Sampling method: The respirometer used during this study is an Oxitop Control System.
- Sterility check if applicable: Not applicable.
- Sample storage before analysis: Not applicable.
- Other: The oxygen consumption was determined in the incubation vessels by the OxiTop® OC110 measuring system at 360 measuring points during the 28 d incubation period. The concentration of nitrite and nitrate was determined by photometric measurement (on NANOCOLOR® UV/VIS, MACHEREY-NAGEL, using related nitrate and nitrite test kits) in the test item replicates and inoculum control replicates at test end. At test start the pH-values of the prepared solutions in the measuring flasks (inoculum control, functional control) and the prepared solutions in the brown glass bottle (test item and toxicity control), were determined. At test end the pH-values of the test solutions in the brown glass bottles were determined. pH readings were done with pH-meter WTW Multi 350i.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. Inoculum control (C)
- Abiotic sterile control: No.
- Toxicity control: Yes. Toxicity control (T) : test and reference item in test concentration and/or Toxicity control with silicone oil (TS) : test item (incl. silicone oil as described above) and reference item in test concentration
- Other: Controls and/or test item groups: were comprised as the following:
(a) Inoculum control (C) : test medium without test and / or reference item
(b) Inoculum control with silicone oil (CS) : test medium without test and / or reference item and 500 μL Silicone oil were pipetted directly into the inoculated test medium.
(c) Toxicity control (T) : test and reference item in test concentration
(d) Toxicity control with silicone oil (TS) : test item (incl. silicone oil as described above) and reference item in test concentration
(e) Functional control (R) : Sodium benzoate at 30 mg/L (ThOD 1.67 mg O2/mg or ThOD in test vessel: 50.1 mg O2/L)
(f) Functional control with silicone oil (RS) : 500 μL silicone oil [WACKER AR 20 SALBEN SILICONÖL / batch number: IG12700 / expiry date: 10-02-2020]
(g) Test Item (P) : test item at 17 mg/L (corresponding to 4.8 μL test item / 250 mL test solution) (test item ThOD cited in the full study report; ThOD in test vessel: 51.2 mg O2/L)
(h) Test item with Silicone oil (Ps) : test item at 17 mg/L concentration, pre-dispersed within silicone oil stock solution (85.0 mg test item / 10 mL silicone oil) and corresponding to 500 μL stock solution for 250 mL test solution (test item ThOD cited in the full study report; ThOD in test vessel: 51.2 mg O2/L)
Replicates were prepared as follows:
(i) two for the inoculum control (C1, C2)
(ii) two for the inoculum control with silicone oil (CS1, CS2)
(iii) one for the functional control (R1)
(iv) one for the functional control with silicone oil (RS1)
(v) two for the test item (P1, P2)
(vi) two for the test item with silicone oil (PS1, PS2)
(vii) one for the toxicity control (T1)
(viii) one for the toxicity control with silicone oil (TS1)
Separate replicates of test item and toxicity control were prepared for measurement of the pH-value at test start:
(a) one for the inoculum control with silicone oil (Cs pH)
(b) one for the functional control with silicone oil (Rs pH)
(c) one for the test item (P pH)
(d) one for the test item with silicone oil (PS pH)
(e) one for the toxicity control (T pH)
(f) one for the toxicity control with silicone oil (TS pH)
Further information on pH results is provided in further sections below.

STATISTICAL METHODS: Standard evaluation of data was conducted graphically, presented in the full study report.

Reference substance:

benzoic acid, sodium salt

Remarks:

30.0 mg/L

Test performance:

1. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item at end of 10-day window and on day 28. Therefore, the test is considered valid. (actual: 11% Test item vessels P1+P2 and 3% Test item with silicone oil vessels PS1+PS2)
2. The BOD of the (blank) Inoculum control was 19.6 mgO2/L and < 60 mgO2/L after 28 days and/or within the Inoculum control with silicone oil was 15.8 mgO2/L and < 60 mgO2/L after 28 days
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual 7.51 to 7.67 without silicon oil and/or 7.52 to 7.66 with silicone oil)
4. The toxicity test (T1) attained 74% degradation after 14 days and/or the toxicity control with silicone oil (TS1) attained 67% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment microorganisms used in the study.
5. Sodium benzoate (R1) attained 71% degradation after 3 days and/or 100% degradation after 14 days and/or sodium benzoate within silicone oil (RS1) attained 62% degradation after 5 days and/or 85% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
All validity criteria were considered to be met.
Full biological oxygen demand (BOD) data for all vessels is documented in the full study report. Biodegradation % information from the BOD data is summarised in the tables.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

mean (n=2) ; without silicone oil

Value:

75

Sampling time:

28 d

Remarks on result:

other: test P1 and P2 without silicone oil ; 10-day window met

Remarks:

day 28 values: P1 = 69% ; P2 = 80% and mean = 75%

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

mean (n=2) ; with silicone oil

Value:

93

Sampling time:

28 d

Remarks on result:

other: test PS1 and PS2 with silicone oil

Remarks:

day 28 values: PS1 = 91% ; PS2 = 94% and mean = 93%

Details on results:

Full biological oxygen demand (BOD) data for all vessels is documented in the full study report. Biodegradation % information from the BOD data is summarised in the tables.
The 10-day window criteria was met.
Both test item replicates (P1+P2, without silicone oil) reached the 10% level (beginning of biodegradation) within 9 days (mean of replicates). The 60 % threshold was reached within 19 days. The mean biodegradation on day 28 was 75%.
Both test item replicates with silicone oil (PS1+PS2) reached the 10% level (beginning of biodegradation) within 11 days. The 60 % threshold was reached within 17 days. The mean biodegradation on day 28 was 93%.
For further information see tables. All validity criteria were considered to be met.
Colony forming units (CFU) of the inoculum were determined prior to test start by standard dilution plate count: 1.83 x 10^9 CFU/L. The CFU concentration of the inoculum corresponds to approximately: 0.73 x 10^7 CFU/L in the final test solution.

Results with reference substance:

Sodium benzoate (R1) attained 71% degradation after 3 days and/or 100% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
Sodium benzoate within silicone oil (RS1) attained 62% degradation after 5 days and/or 85% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
The pass level for ready biodegradation (≥ 60% degradation) was reached within 3 days in the functional control (R1) and within 5 days in the functional control with silicone oil (RS1). The validity criterion that the degradation should be ≥ 60% after 14 d was fulfilled.

Table 2. Biodegradation [%] of the Test Item in Comparison to the Functional Control and Toxicity Control

	Biodegradation [%]
[d]	Functional Control	Test Item		Toxicity Control Test Item + Reference Item
	R	P1	P2	T
1	10	0	0	5
2	55	0	0	19
3	71	5	1	27
4	81	7	2	33
5	87	7	3	37
6	89	6	3	41
7	94	8	4	46
8	96	9	6	52
9	98	8	12	56
10	100	10	14	60
11	100	11	17	64
12	100	12	21	68
13	100	17	26	71
14	100	28	35	74
15	100	39	44	77
16	100	48	50	78
17	100	54	56	79
18	100	57	60	80
19	100	59	63	81
20	100	60	66	82
21	100	62	69	83
22	100	62	71	84
23	100	64	73	86
24	100	64	75	87
25	100	65	76	87
26	100	66	77	88
27	100	68	79	88
28	100	69	80	88

 

Table 3. Biodegradation [%] of the Test Item with silicone oil in Comparison to the Functional Control with silicone oil and Toxicity Control with silicone oil

	Biodegradation [%]
[d]	Functional Control with silicone oil	Test Item with silicone oil		Toxicity Control Test Item with silicone oil + Reference Item
	RS	PS1	PS2	TS
1	9	0	0	6
2	39	0	0	25
3	53	0	4	32
4	58	0	6	35
5	62	0	5	39
6	69	0	6	43
7	74	0	7	45
8	78	1	8	47
9	80	4	10	49
10	81	6	10	50
11	82	10	13	52
12	83	19	17	55
13	84	30	25	61
14	85	42	39	67
15	87	52	50	73
16	89	59	58	77
17	91	63	64	79
18	94	67	69	81
19	95	70	72	82
20	98	74	75	85
21	99	75	77	86
22	99	77	78	88
23	100	81	82	90
24	100	84	85	92
25	100	85	86	93
26	100	88	90	95
27	100	89	92	97
28	100	91	94	98

 

Table 4. Nitrite and Nitrate Concentrations at Test End

	Nitrite-N	Nitrate-N	corresponding Oxygen consumption	Biodegradation calculated without nitrification after 28 days
	[mg/L]	[mg/L]	[mg/L]	[%]
Inoculum Control C1	0.0	10.1	46.2	-
Inoculum Control C2	0.0	10.0	45.7	-
Inoculum Control CS1	0.06	8.89	40.8	-
Inoculum Control CS2	0.13	9.61	44.4	-
Test item repl. P1	0.0	6.93	31.7	69
Test item repl. P2	0.0	7.39	33.8	80
Test item repl. PS1	0.12	7.85	36.3	91
Test item repl. PS2	0.05	7.13	32.8	94

At test end, the concentration of nitrite and nitrate was determined by photometric measurement from aliquots of the test item replicates and inoculum control. The corresponding oxygen consumption was calculated with the factors 3.43 for nitrite and 4.57 for nitrate, as given in OECD TG 301F. Since the measured nitrite and nitrate concentration in the test item replicates are equal or lower than the background from the mineral medium, it was considered that no formation of oxidized nitrogen in the test item replicates occurred. Accordingly, the biodegradation was calculated with Theoretical Oxygen Demand (ThOD).

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The mean test item biodegradation in duplicate was 75% at day 28 without silicone oil and 93% at day 28 with silicone oil. The 10-day window criteria was met. All validity criteria were considered to be met.

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test item was tested in accordance with OECD TG 301F Annex III concerning test procedures for use of silicone oil emulsifying agent for poorly soluble compounds. The test utilised a non-adapted activated sludge from a municipal sewage treatment plant for a period of 28 days. The test item concentration selected was 17 mg/L, corresponding to a ThOD of 51.2 mg O2/L per test vessel. The study was conducted including both neat test item and the test item solved in silicone oil. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day. Corresponding inoculum control, functional control (with sodium benzoate at 30 mg/L) and/or toxicity controls were completed with and without silicone oil, as applicable. The mean oxygen depletion in the inoculum control was 19.6 mg O2/L on day 28. The mean oxygen depletion in the inoculum control with silicone oil was 15.8 mg O2/L on day 28. To check the activity of the test system sodium benzoate was used as functional control and functional control with silicone oil. The pass level for ready biodegradation (≥ 60% degradation) was reached within 3 days in the functional control and within 5 days in the functional control with silicone oil. In the toxicity control containing both test item and reference item, the biodegradation achieved 74% after 14 days, with 88% after 28 days. In the toxicity control containing both test item (in silicone oil) and reference item, the biodegradation achieved 67% after 14 days, with 98% after 28 days. pH was measured at the start of the test and at the end of the test was within the required range of 7.51 to 7.67 without silicon oil and/or 7.52 to 7.66 with silicone oil in all vessels. All validity criteria of the guideline were fulfilled. The biodegradation of the test item was determined in comparison to the readily biodegradable functional control and the toxicity control. Both test item replicates reached the 10 % level (beginning of biodegradation) within 9 days (mean of replicates). The 60% threshold was reached after 19 days. The mean biodegradation on day 28 was 75% in the test item replicates without silicone oil and meeting the 10-day window. The biodegradation of the test item with silicone oil was determined in comparison to the readily biodegradable functional control with silicone oil and the toxicity control with silicone oil. Both test item replicates containing silicone oil reached the 10% level within 11 days. The 60% threshold was reached after 17 days. The mean biodegradation on day 28 was 93% in the test item replicates with silicone oil and meeting the 10-day window. Under the conditions of the study, test item is considered as readily biodegradable.

Description of key information

Biodegradation: mean biodegradation 75% without silicone oil and/or 93% with silicone oil emulsifying agent, (28-days; 10-day window met), OECD TG 301F, 2020

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Key study : OECD TG 301F, 2020 : The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test item was tested in accordance with OECD TG 301F Annex III concerning test procedures for use of silicone oil emulsifying agent for poorly soluble compounds. The test utilised a non-adapted activated sludge from a municipal sewage treatment plant for a period of 28 days. The test item concentration selected was 17 mg/L, corresponding to a ThOD of 51.2 mg O2/L per test vessel. The study was conducted including both neat test item and the test item solved in silicone oil. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day. Corresponding inoculum control, functional control (with sodium benzoate at 30 mg/L) and/or toxicity controls were completed with and without silicone oil, as applicable. The mean oxygen depletion in the inoculum control was 19.6 mg O2/L on day 28. The mean oxygen depletion in the inoculum control with silicone oil was 15.8 mg O2/L on day 28. To check the activity of the test system sodium benzoate was used as functional control and functional control with silicone oil. The pass level for ready biodegradation (≥ 60% degradation) was reached within 3 days in the functional control and within 5 days in the functional control with silicone oil. In the toxicity control containing both test item and reference item, the biodegradation achieved 74% after 14 days, with 88% after 28 days. In the toxicity control containing both test item (in silicone oil) and reference item, the biodegradation achieved 67% after 14 days, with 98% after 28 days. pH was measured at the start of the test and at the end of the test was within the required range of 7.51 to 7.67 without silicon oil and/or 7.52 to 7.66 with silicone oil in all vessels. All validity criteria of the guideline were fulfilled. The biodegradation of the test item was determined in comparison to the readily biodegradable functional control and the toxicity control. Both test item replicates reached the 10 % level (beginning of biodegradation) within 9 days (mean of replicates). The 60% threshold was reached after 19 days. The mean biodegradation on day 28 was 75% in the test item replicates without silicone oil and meeting the 10-day window. The biodegradation of the test item with silicone oil was determined in comparison to the readily biodegradable functional control with silicone oil and the toxicity control with silicone oil. Both test item replicates containing silicone oil reached the 10% level within 11 days. The 60% threshold was reached after 17 days. The mean biodegradation on day 28 was 93% in the test item replicates with silicone oil and meeting the 10-day window. Under the conditions of the study, test item is considered as readily biodegradable.