Dimethylbis[(1-oxoneodecyl)oxy]stannane

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:

10 October 2017 to 08 November 2017

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)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK (which treats predominantly domestic sewage).
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper (which was rinsed 3 times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- The suspended solids concentration was equal to 3.4 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

18.6 mg/L

Based on:

test mat.

Initial conc.:

10 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD Guidelines. To 1 litre (final volume) of purified water (Reverse osmosis purified and deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)) was added the following volumes of solutions a – d: 10 mL of Solution a, 1 mL of Solution b, 1 mL of Solution c and 1 mL of Solution d. Solution a: KH2PO4 8.50 g/L, K2HPO4 21.75 g/L, Na2HPO4.2H2O 33.40 g/L and NH4Cl 0.50 g/L, pH = 7.4. Solution b: CaCl2 27.50 g/L. Solution c: MgSO4.7H2O 22.50 g/L. Solution d: FeCl3.6H2O 0.25 g/L (In order to avoid having to prepare solution (d) immediately before use, one drop of concentrated HCl per litre was added as a preservative).
- The deionised reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).
- Solubilising agent: no, the test material was dispersed directly in mineral medium. An amount of test material (55.8 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 litres to give a final concentration of 18.6 mg/L, equivalent to 10 mg carbon/L. This was the most suitable method of preparation determined by preliminary solubility work.
- Test temperature: 22 - 24 °C
- pH: 7.4 ± 0.2
- pH adjusted: yes, on Day 0 the test and reference materials were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter and the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 litres by the addition of mineral medium which had been purged overnight with CO2-free air.
- Aeration of dilution water: Approximately 24 hours prior to addition of the test and reference materials the vessels were filled with 2400 mL of mineral medium and 26.5 mL of inoculum and aerated overnight.
- Suspended solids concentration: 30 mg suspended solids (ss)/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 litre test culture vessels, each containing 3 litres of solution
- Number of culture flasks: An inoculated control, in duplicate, consisted of inoculated mineral medium. The procedure control contained the reference material (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L. The test material, in duplicate, was in inoculated mineral medium to give a final concentration of 10 mg carbon/L. The test material plus the reference material in inoculated mineral medium to give a final concentration of 20 mg carbon/L acted as a toxicity control (one vessel only).
- Method used to create aerobic conditions: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Test performed in closed vessels: yes
- 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.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
- All samples were analysed for Inorganic Carbon (IC) immediately. The remainder of all samples with the exception of the Day 0 samples were frozen for further analysis if required. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
- The samples were analysed for IC using either a Shimadzu TOC-VCSH TOC analyser or a Shimadzu TOC-LCSH TOC analyser. Samples (50 or 135 μL) were injected into the IC channel of the TOC analyser. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in at least triplicate with 3 replicates being used in the calculation/reported.
- Inorganic Carbon/Total Carbon Ratio: Samples (30 mL) were removed from the test material vessels on Day 0 prior to the addition of the test material 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 discarded in order to pre-condition the filter) 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 discarded in order to pre-condition the filter) prior to DOC analysis. Inorganic Carbon/Total Carbon analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water. The samples were analysed for IC and Total Carbon (TC) using a Shimadzu TOC-VCPH TOC Analyser. Samples (50 μL) were injected into the TC and IC channels of the TOC analyser. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature.
- Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in at least triplicate with 3 replicates being used in the calculation/reported.
- The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27. The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.

CONTROL AND BLANK SYSTEM
- Inoculum Control: An inoculated control, in duplicate, consisting of inoculated mineral medium only was used in the test.
- Test Material Preparation: The test material was dispersed directly in mineral medium. An amount of test material (55.8 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 litres to give a final concentration of 18.6 mg/L, equivalent to 10 mg carbon/L. A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.
- Reference Material Preparation: A reference material, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference material directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference material was inverted several times to ensure homogeneity of the solution. The procedure control containing the reference material, was performed in duplicate.
- Toxicity Control: A toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test. An amount of test material (55.8 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 18.6 mg test material/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L. One vessel only was used for the toxicity control.

DATA EVALUATION
- Calculation of Carbon Content: The theoretical amount of carbon present in the test material for was calculated as follows:
((No. of C atoms x mol wt of C) / mol wt of the test material ) x 100
((22 x 12.011) / 491.3) x 100 = 53.78 %
- The theoretical amount of carbon present in the reference material, sodium benzoate (C6H5COONa) was calculated as follows:
((No. of C atoms x mol wt of C) / mol wt of sodium benzoate ) x 100
((17 x 12.011) / 144.11) x 100 = 58.34 %
Thus for a 10 mg C/L test concentration the total organic carbon present for sodium benzoate was 30 mg C.

- Percentage Biodegradation: The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values into the following equation. The values of Replicates 1 and 2 are meaned for the inoculum control, test and reference materials before substitution into the following equation:
%ThCO2 = (%biodegradation) = [ (mg IC in test flask – mg IC in control flask) / mg TOC added as test material ] x 100

- Total CO2 Evolution: The total CO2 evolution in the inoculum control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:
Total CO2 evolution (mg C/L) = mg IC in control x (100 / %C of CO2) x (1/test volume)
= mg IC in control x (100/27.29) x (1/3)

VALIDATION CRITERIA
- The results of the degradation test are considered valid if in the same test the reference material yields ≥ 60% degradation (in a 10-Day window) by Day 14.
- The test material may be considered to be readily biodegradable if ≥ 60 % degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
- The toxicity control (test material and sodium benzoate) should attain ≥ 25 % degradation by Day 14 for the test material to be considered as non-inhibitory.
- The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the time the plateau is reached, at the end of the test or at the end of the 10-Day window, as appropriate, is less than 20%.
- The total CO2 evolution in the inoculum control vessels at the end of the test should not normally exceed 40 mg/L medium (= 120 mg/3 litres, corresponding to 33 mg C per flask), however values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained should be critically examined.
- The IC content of the test material suspension in the mineral medium at the beginning of the test should be < 5% of the TC.

Reference substance:

benzoic acid, sodium salt

Test performance:

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Details on results:

DEFINITIVE TEST
- Inorganic carbon values for the test material, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference materials and the toxicity control are given in Table 2.

VALIDATION CRITERIA AND BIODEGRADATION
- The total CO2 evolution in the inoculum control vessels on Day 28 was 33.80 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
- The IC content of the test material suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
- The difference between the values for CO2 production at the end of the test for the replicate vessels was < 20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
- Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test material.
- The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of procedure control Replicate 1, test vessel Replicate 2 and the toxicity control.
- Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
- The test material 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.
- The toxicity control attained 17% biodegradation after 14 days and 1% biodegradation after 28 days thereby confirming that the test material did exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. The decrease in biodegradation between Days 14 and 28 was considered to be due to the inhibitory nature of the test material on the sewage treatment micro-organisms used in the test.
- Sodium benzoate attained 69% biodegradation after 14 and 28 days, satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%, thereby confirming the suitability of the inoculum and test conditions.

Results with reference substance:

- Sodium benzoate attained 69% biodegradation after 14 and 28 days, satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%, thereby confirming the suitability of the inoculum and test conditions.

Table 1: Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Material				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs1	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	2.10	2.10	2.10	2.45	2.10	2.10	2.10	2.10	2.10	2.45	2.10	2.10	2.10	2.45
2	4.87	-	7.66	-	16.01	-	10.09	-	6.85	-	7.54	-	18.79	-
6	9.34	-	13.61	-	34.14	-	10.84	-	14.42	-	18.34	-	27.57	-
8	9.40	-	19.15	-	36.92	-	33.37	-	16.40	-	22.47	-	30.27	-
10	14.48	-	19.95	-	43.09	-	41.15	-	17.44	-	22.46	-	31.46	-
14	20.29	-	20.74	-	42.95	-	39.55	-	17.57	-	19.04	-	30.71	-
21	26.93	-	26.93/24.00*	-	49.69	-	48.22	-	20.96	-	23.32	-	33.57	-
28	33.15/27.78**	-	35.84/27.55**	-	53.42	-	48.61	-	21.39	-	28.45	-	31.92	-
29	28.72	2.78	29.06	2.55	50.32	2.78	48.65	2.78	24.38	2.78	26.39	2.78	29.57	2.78

R = Replicate

Abs = CO₂ absorber vessels

*  Result from analysis of frozen sample as original result deemed to be erroneous

**  Result from re-analysis of sample as original result deemed to be erroneous

Table 2: Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Material	Toxicity Control
0	0	0	0
2	23	3	21
6	37	16	27
8	70	17	27
10	83	9	24
14	69	0	17
21	78	0	14
28	78	0	7
29*	69	0	1

* = Day 29 values corrected to include any carry-over of CO₂ detected in Absorber 2

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study the test material is not readily biodegradable. Care should be taken in the interpretation of these results as the test material exhibited inhibitory effects in the study.

Executive summary:

The ready biodegradability of the test material in an aerobic aqueous medium was investigated in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and EPA OCSPP 835.3110, under GLP conditions.

The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at measured temperatures of between 22 and 24°C for 28 days. The biodegradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material 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. The toxicity control vessel containing both the test material and sodium benzoate, attained less than 25% biodegradation after 28 days, thereby indicating that the test material could be classed as exhibiting an inhibitory effect on the activated sewage sludge micro-organisms.

Sodium benzoate attained 69% biodegradation after 14 and 28 days, satisfied the 10-day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%, thereby confirming the suitability of the inoculum and test conditions. 

Under the conditions of this study the test material is not readily biodegradable. Care should be taken in the interpretation of these results as the test material exhibited inhibitory effects in the study.

Description of key information

Under the conditions of this study the test material is not readily biodegradable. Care should be taken in the interpretation of these results as the test material exhibited inhibitory effects in the study.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test material in an aerobic aqueous medium was investigated in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and EPA OCSPP 835.3110, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at measured temperatures of between 22 and 24°C for 28 days. The biodegradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material 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. The toxicity control vessel containing both the test material and sodium benzoate, attained less than 25% biodegradation after 28 days, thereby indicating that the test material could be classed as exhibiting an inhibitory effect on the activated sewage sludge micro-organisms.

Sodium benzoate attained 69% biodegradation after 14 and 28 days, satisfied the 10-day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%, thereby confirming the suitability of the inoculum and test conditions. 

Under the conditions of this study the test material is not readily biodegradable. Care should be taken in the interpretation of these results as the test material exhibited inhibitory effects in the study.

[Type of water: freshwater]