Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-)

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:

05 Dec 2018 - 16 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was conducted in accordance with International Guidelines (OECD 301b) and in accordance with OECD Principles of Good Laboratory Practice as incorporated into the United Kingdom Statutory Instrument for GLP. All validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Treatment of test material prior to testing:
A nominal amount of test item (1426 mg) was dissolved in 10 mL of acetone to give a 142.6 mg/mL solvent stock solution. An aliquot (444 μL) of this solvent stock solution was dispensed onto a filter paper 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, 5 minutes) prior to addition to inoculated mineral medium. The volume was then adjusted to 3 liters to give a final concentration of 21.1 mg/L, equivalent to 10 mg carbon/L.
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: None
- Final preparation of a solid: As described

FORM AS APPLIED IN THE TEST (if different from that of starting material): As described

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): Severn Trent Water plc sewage treatment plant, Loughborough, Leistershire, UK.
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: continous aeration, used on day of collection
- Storage length: used on day of collection
- Preparation of inoculum for exposure: washed twice in mineral medium, then maintained on continuous aeration
- Pretreatment: N/A
- Concentration of sludge: 2.3 g/L
- Initial cell/biomass concentration: not determined
- Water filtered: yes; deionised reverse osmosis water
- Type and size of filter used, if any: Whatman GF/A filter paper

Duration of test (contact time):

>= 28 d

Initial conc.:

ca. 10 mg/L

Based on:

other: of carbon (calculation of carbon content based upon molecular formula)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Solution a: KH2PO4 8.5 g/L, K2HPO4 21.75 g/L, Na2HPO4.2H2O 33.4 g/L, NH4Cl 0.5 g/L, Solution b: CaCl2 27.5 g/L, Solution c: MgSO4.7H2O 22.5 g/L, Solution d: FeCl3.6H2O. To 1L of purified water, the following volumes were added: 10 mL solution a, 1mL solution b, 1 mL solution c, 1mL solution d.
- Additional substrate: N/A
- Solubilising agent (type and concentration if used): 1426 mg test item in 10 mL acetone. 444 μL aliquot of this stock solution added to filter paper and left to dry. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to inoculated mineral medium. This method of test item addition was recommended by International Standards Organisation (ISO 10634, 1995) and published literature (Handley et al, 2002) due to low solubility in the test medium. The volume was then adjusted to 3 liters to give a final concentration of 21.1 mg/L, equivalent to 10 mg carbon/L.
- Test temperature: 21-23 °C.
- pH: pH values were adjusted to 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all of the vessels being adjusted
- pH adjusted: yes
- CEC (meq/100 g): not reported
- Aeration of dilution water: yes; CO2-free air
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes
- Other: total volume per test flask: 3L. Test flasks stirred continuously by magnetic stirrer during 28 day test period.

TEST SYSTEM
- Culturing apparatus: N/A
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Test vessels aerated continuously with CO2-free air at a rate of 30-100 mL/min.
- Method used to create anaerobic conditions: N/A
- Measuring equipment: pH of all vessels measured using a Hach HQ40d Flexi handheld meter
- Test performed in closed vessels due to significant volatility of test substance: N/A
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.
- Other: The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.

SAMPLING
- Sampling frequency: Observations for appearance of the test preperations was conducted on days 0, 7, 14, 21 and 28. Samples from the first CO2 absorber were taken on days 0, 2, 5, 7, 9, 14, 21, 28 and 29.
- Sampling method: Inorganic Carbon analysis using a Shimadzu TOC-L CSH TOC Analyser
- Sterility check if applicable: Not reported/applicable
- Sample storage before analysis: Day 0 samples frozen, all other samples analysed immediately
- Other: Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the IC content in the test media. The samples were filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL used to pre-condition the filter was discarded) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL used to pre-condition the filter was discarded) prior to DOC analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: An inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper
- Abiotic sterile control: N/A
- Toxicity control: The test item on a filter paper plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
- Other: The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a filter paper to give a final concentration of
10 mg carbon/L.

STATISTICAL METHODS: Statistical analysis of the Day 29 IC values for the inoculum control and test item vessels was carried out using a Student’s t-test to determine any statistically significant differences (P <0.05) between the test and control groups. All statistical analyses were performed using the SAS computer software package.

Reference substance:

benzoic acid, sodium salt

Remarks:

Batch SLBT3039, >99.5% purity

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 0

Sampling time:

28 d

Results with reference substance:

Sodium benzoate attained 74% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. After 28 days 83% biodegradation was attained. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Table 1 Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.75	1.40	1.40	1.75	1.40	1.40	1.40	1.52	1.40	1.75	1.40	1.52	1.40	1.40
2	6.73	-	6.73	-	15.77	-	19.25	-	6.38	-	6.96	-	21.69	-
5	13.26	-	12.22	-	26.30	-	32.18	-	11.76	-	12.11	-	35.06	-
7	13.30	-	13.53	-	26.49	-	34.17	-	11.24	-	11.12	-	34.86	-
9	17.33	-	14.36	-	36.82	-	36.71	-	12.20	-	13.79	-	38.53	-
14	19.15	-	17.23	-	39.89	-	41.03	-	14.51	-	16.21	-	38.42	-
21	22.08	-	19.83	-	46.08	-	43.71	-	17.24	-	18.70	-	43.94	-
28	21.95	-	21.73	-	47.38	-	46.37	-	19.49	-	23.07	-	48.72	-
29	24.38	2.44	24.61	2.78	48.76	2.44	50.21	2.44	20.48	2.44	22.60	2.44	49.06	2.67

R = Replicate
Abs = CO2absorber vessel

Table 2 Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	36	0	25
5	55	0	37
7	56	0	36
9	70	0	38
14	74	0	34
21	80	0	38
28	83	0	45
29*	83	0	41

* Day 29 values corrected to include any carry-over of CO2detected in Absorber 2

Table 3 Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon Corrected for Control Values∗ (mg/L)	Inorganic Carbon Corrected for Control Values (mg/L)	IC Content (% of TC)
Test Item 10 mg C/L R1	10.23	0.31	3
Test Item 10 mg C/L R2	10.82/9.70**	0.91/0.31**	8/3**

R = Replicate

* TC value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item.
** Results from re-analysis of frozen sample due to high IC content as a percentage of TC content

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the conditions of OECD Guideline No. 301B.

Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.

The toxicity control attained 34% biodegradation after 14 days and 41% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 74% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. After 28 days 83% biodegradation was attained. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Description of key information

Not readily biodegradable; OECD 301B; Best N (2019)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to readily biodegradable under the conditions of the OECD Guideline No. 301B.

Statistical analysis of the Day 29 IC values for the control and test item vessels showed therewere no statistically significant differences (P≥0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.

The toxicity control attained 34% biodegradation after 14 days and 41% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 74% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. After 28 days 83% biodegradation was attained. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.