Dioctadecyl disulphide

Biodegradation in water and sediment: simulation tests

Administrative data

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Remarks:

It was not possible to conduct an OECD 309. Alternatively an enhanced OECD 310 was conducted. Detailed information are to be found under section 5.2.1. For some further information also see attached justification.

Adequacy of study:

other information

Justification for type of information:

According to a Compliance Check of ECHA, the identification of degradation products was required. The OECD TG 309 was recommended as the preferred test, if technically feasible.

Based on experiments demonstrating very low water solubility and likewise low solubility in other solvents, it was concluded that the test item is very poorly soluble and therefore OECD TG 309 is not feasible.
Therefore, an enhanced OECD TG 310 test in combination with LC-HRMS (high resolution mass spectrometry) analysis for detection and identification of metabolites was performed. Detailed information are to be found under section 5.2.1.

Some further information can be found in the attached justification.

Cross-reference

Reference

Endpoint:

biodegradation in water: screening test, other

Remarks:

enhanced biodegradability including Identification of degradation products

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted from 2021-11-23
to 2022-01-27, following OECD 310 at the test facility.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other:

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Version / remarks:

2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item Hostanox SE 10
CAS No. 2500-88-1
EC No. 219-702-5
Purity (certified) 97.5 m/m% (sum of DOS1, DOS2, DOS3, DOS4)
dioctadecyl monosulfide (DOS1) 1.5 (m/m%)
dioctadecyl disulfide (DOS2) 96.0 (m/m%)
dioctadecyl trisulfide (DOS3) n.n (m/m%)
dioctadecyl tetrasulfide (DOS4) n.n (m/m%)
Water solubility < 0.5 mg/L (17 – 22 °C)
TOC* 0.74 mg C/mg test item
Appearance White powder
Expiry date 2022-03-10
Recommended storage Store cool and dry, in a well-ventilated place
* The TOC was calculated at the test facility.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Municipal sewage treatment plant, D-31137 Hildesheim, Germany
- Receipt: 2021-11-17
- Pretreatment/Concentration of sludge:
The activated sludge was washed twice with chlorine free 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 for six days until test start. 4.40 mL/L of this mixture were used to initiate inoculation (25.0 mg/L dry weight). pH-adjustment was not necessary.

Duration of test (contact time):

62 d

Initial conc.:

27.1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Application Once at test start
- Headspace to liquid ratio 1:2
- Test vessels / volume Gastight glass bottles / 250 mL
- Test medium / volume Mineral salts medium acc. to OECD 310 and inoculum / 170 mL
- Dispersion treatment Continuous stirring
- Temperature 20 - 24 (constant at ± 1 °C) actually measured 20.7 -22.8 °C
- Photoperiod Low light conditions

STATISTICAL METHODS:
The amount of TIC produced was calculated by correcting the results of the test item and functional control for endogenous TIC production of the control groups and the blind value of the sodium
hydroxide solution.
The biodegradation was calculated from the ratio theoretical TIC concentration at test start (ThIC = TOC) to net TIC production.
The 95 % confidence interval for the mean percentage biodegradation at end of exposure was calculated for the test item and the functional control.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

E-Fate Study Requirements based on compliance check by the ECHA
Identification of degradation products (Annex IX, 9.2.3.) using an appropriate test method with the substance (as specified under Appendix A, Section 2). A simulation test according to OECD TG 309 is recommended to fill the data gap.

Water Solubility
The water solubility of the test item is lower than 0.5 mg/L at 20°C [2]. No exact value for the water solubility could be derived for the test item in an OECD 309 study due to a limited sensitivity of the used analytical method.
In general, a simulation study according to OECD TG 309 should be forced down to a water solubility of 1 µg/L rather than the conduction of other simulation studies as OECD TG 307 or 308 [3]. It is anticipated that a study is technically not feasible below this threshold concentration. QSAR calculation predicts a water solubility << 1µg/L. For this reason, efforts were first made to obtain a more accurate value for the water solubility of the test item to verify if a study according to OECD TG 309 is technically feasible.

Preliminary Test Water Solubility – Column Elution Method
A preliminary study for the determination of the water solubility according to OECD TG 105 with the column elution method was performed. A GC-MS method was implemented as analytical method to monitor the water solubility of the test item. This preliminary test provided as result a water solubility of approximately 0.6 mg/L for dioctadecyl disulfide (DOS2). This value is higher than the value described in section 2 (< 0.5 mg/L) and much higher than the QSAR prediction. Nevertheless, this result was considered as trigger to go forward with the implementation of a study according to OECD TG 309 and to look if this study is technically feasible.

Test performance:

Test Item Hostanox SE 10
TOC 0.74 mg C / mg test item
Test concentration 27.1 mg test item/L (corresponding to 4.6 mg test item / 170 mL test solution)
Carbon content 20.0 mg C/L in the vessel
Pre-treatment None

Functional Control
Reference item Sodium benzoate (SIGMA ALDRICH)
Test facility ID 70144
Batch number BCBX0430
CAS No. 532-32-1
Purity 100 %
Expiry date 2026-09-03
Test concentration 30 mg/L
TOC 0.58 mg C/mg
Carbon content 17.5 mg C/L in the vessel

Inoculum Control
Mineral salts medium with inoculum without test / reference item

Day Test Inoculum Functional
Item Control Control
(P) (C) (R)
TIC LC-HRMS TIC LC-HRMS TIC
0 2 2 2 2 2
16 2 2 2 2 2
28 2 2 5 2 5
45* 2 2 2 2 -
62 5 2 5 2 -
Total number of replicates measured 23 26 9

Key result

Parameter:

% degradation (CO2 evolution)

Value:

48

Sampling time:

62 d

Details on results:

Carbon Content of the Test Item
The test item was tested at a concentration of 27.1 mg/L, corresponding to a carbon content of 20.0 mg C/L.

TIC Production in the Inoculum Controls
The mean amount of TIC present in the inoculum controls (C1 - C5) on day 28 is 4.96 mg C/L.

CO2-Production and Biodegradation
The total amount of CO2 produced in 62 days was analysed by TIC measurements at 5 sampling times.
The adaptation phase of the functional control changed within 2 days to the degradation phase (degradation > 10 %). The course of the degradation phase was rapid and the pass level of 60 % was reached within 10 days. The biodegradation came to a maximum of 92 % after 16 days. The validity criterion degradation > 60% after 14 d was fulfilled. The 95% confidence interval on day 28 was 84 – 96 %.
The test item replicates reached the 10 % level (beginning of biodegradation) within 10 days. The biodegradation on day 28 was 20 % and reached 48 % until test end (day 62). The 95 % confidence interval on day 62 was 30 – 66 %.

The test item is classified as not readily biodegradable within the 62-day period of the study.


Results Metabolite Identification
No metabolites could be detected during the sample analysis. In contrast, the performance marker could be detected on each day of analysis with a sufficient data quality as shown in attachments demonstrating that the used analytical method including data analysis was suitable for the purpose of the investigations.

Preliminary Investigations and Evaluation of Study Design (non-GLP) - Part II

Simulation Study in Surface Water (OECD TG 309)
Procedures for the application of the test item to surface water were investigated. A solubility of 1.4 g/L in 1-octanol at 23°C is described. This solvent cannot be used for the application of the test item to surface water because this solvent is not volatile enough. The residual concentration of this solvent, even after careful removal from the test vessels after application, could exceed the test concentration of the test item. 1-Octanol is readily biodegradable and could be preferably degraded and underestimate the biodegradation potential of the test item. Therefore, more suitable organic solvents were tested to verify if an accurate and reproducible application of the test item to surface water is possible.
The amount of solvent added to the test solution must be as small as possible (<< 1%) to avoid any adverse effects on the microbial activity. Therefore, a stock solution of 1 g/L (better 10 g/L) is necessary for application. Acetone, methanol, ethanol and acetonitrile were considered as suitable solvents. Solubility data for methanol (provided by the sponsor) indicated that the solubility is for too low to prepare a suitable stock solution.
The solubility in acetone, acetonitrile and ethanol was investigated at a concentration of 5 g/L. The test item was not soluble in any of these solvents. Even after up to 7 days agitation on a shaker, the test item was present as fine powder. It was concluded that the test item is highly insoluble and therefore OECD TG 309 is not feasible to conduct.

Alternative Testing
Other Simulation Studies
Studies according to OECD TG 308 or OECD TG 307 are described in the decision as alternative simulation studies when a study according to OECD TG 309 is not possible. However, these simulation studies are also not technically feasible, as it is not possible to prepare an adequate application solution.

Enhanced Test OECD TG 310
Therefore, an enhanced OECD TG 310 test in combination with LC-HRMS (high resolution mass spectrometry) analysis for detection and identification of metabolites was performed. This test simplifies the detection and identification of metabolites due to the higher test concentration. Previous studies (301F over 28 and 60 days) showed an incomplete mineralization of the test item. These results let assume that metabolites of the primary biodegradation pathway should be still present in the test solutions and that this approach should be suitable to get knowledge about the chemical structures of the metabolites.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

After a test period of 62 days the test item was not readily biodegradable. No metabolites were detected within the 62-day period of the study.

Executive summary:

According to a Compliance Check of ECHA, the identification of degradation products was required. The OECD TG 309 was recommended as the preferred test, if technically feasible.

Based on experiments demonstrating very low water solubility and likewise low solubility in other solvents, it was concluded that the test item is very poorly soluble and therefore OECD TG 309 is not feasible.
Therefore, an enhanced OECD TG 310 test in combination with LC-HRMS (high resolution mass spectrometry) analysis for detection and identification of metabolites was performed.

Metabolites were investigated and mineralisation of the test item was determined in an enhanced screening test with a non-adapted activated sludge over a test period of 62 days in the Headspace Test. The study was conducted from 2021-11-23 to 2022-01-27, following OECD 310 at the test facility. The test item was tested at a concentration of 27.1 mg/L in duplicates (test end: 5 replicates), corresponding to a carbon content of 20 mg C/L.
Investigations about metabolites were done by LC-HRMS analysis. The mineralisation of the test item was followed by TIC analyses of the quantity of CO2 produced by the respiration of bacteria. The amount of TIC produced was calculated by correcting the results of the test and reference item for endogenous TIC production of the control groups and the blind value of the sodium hydroxide solution. The biodegradation was calculated for each sampling time.
To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 10 days and came to a maximum of 92 % on day 16. The 95 % confidence interval on day 28 was 84 – 96 %.
The test item replicates reached the 10% level (beginning of biodegradation) within 10 days. The biodegradation on day 28 was 20 % and reached 48 % until test end (day 62). The 95 % confidence interval on day 62 was 30 – 66 %.
A plateau phase did not appear to have been reached after 62 days, and one replicate even reached 70% degradation after 62 days. This reinforces the assumption that the substance would be degraded even further.

The validity criteria according to the guideline are fulfilled.
No metabolites were detected. Putatively, the alkyl chains of the test item can be completely mineralised. But, higher mineralisation of the test item will be very slow due to the low water solubility of the test item linked to a limited bioavailability.

	Biodegradation [%]					Confidence
	at test end*					Interval at test end*
Replicates	1	2	3	4	5	P = 95 %
Functional Control	95	85	93	86	93	84 - 96
Test Item	51	48	33	37	70	30 - 66