Chlorotrioctylstannane

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:

11 February 2003 to 28 March 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see below

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

yes

Remarks:

see below

Qualifier:

according to guideline

Guideline:

other: ISO 10634

Deviations:

no

Principles of method if other than guideline:

Additional NaNO3 was added to the mineral medium to prevent nitrogen limitation occurring. Additionally, the temperature was temporarily too low; the temperature was in the range 13.3 to 18 °C between 13 to 23 days.
These deviations are not considered to affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Physical appearance: Colourless liquid
- ThOD(NH3): 2.38 mg O2/mg
- Solubility in water: <0.1 mg/L
- Melting point: <-15 °C
- Relative density (at 25 °C): 1.04 g/cm³
- Storage conditions: <-18 °C and protected from light
- Expiry date: 31 July 2004

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): A sample of activated sludge was taken from an oxidation ditch situated in the municipality of Hazerswoude, the Netherlands, on February 11, 2003. The oxidation ditch is used to treat domestic waste water.
- Storage conditions: The activated sludge was transported in a plastic flask and aerated until use.
- Storage length: The sludge was used on the 17 February 2003.
- Concentration of sludge: Before the start of the test, the dry weight of the sludge was determined to be 3.6 g/L. In order to get a concentration of solids of approximately 30 mg/L, 2.5 mL sludge was added to 300 mL of mineral medium.

Duration of test (contact time):

39 d

Initial conc.:

21.6 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium was prepared from concentrated nutrient stock solutions in Milli-Q water: One mL of each of the nutrient stock solutions was combined with Milli-Q water to a final volume of one litre. The mineral medium was aerated vigorously before use.
Nutrient stock solution a: 8.5 g KH2PO4, 21.8 g K2HPO4, 33.4 g N2HPO4.7H2O and 0.5 g NH4Cl were dissolved in and made up to 1000 mL with Milli-Q water. The pH of this solution was set to 7.4 ± 0.1.
5.0 g NaNO3 (added to Milli-Q water from a separate stock solution of 5 g of NaNO3 in 500 mL of Milli-Q water) was dissolved in and made up to 500 mL with Milli-Q water.
Nutrient stock solution b: Nutrient stock solution B per 1000 mL Milli-Q water: 22.5 g MgSO4.7H2O dissolved in and made up to 1000 mL with Milli-Q water.
Nutrient stock solution c: Nutrient stock solution C per 1000 mL Milli-Q water: 36.4 g CaCl2.2H2O dissolved in and made up to 1000 mL with Milli-Q water.
Nutrient stock solution d: Nutrient stock solution D per 1000 mL Milli-Q water: 0.25 g FeCl3.6H2O dissolved in and made up to 1000 mL with Milli-Q water. Solution D was prepared immediately before use.
To prevent nitrogen limitation, additional NaNO3 was added to the mineral medium as specified in the Guideline OECD 301. The nitrate and nitrite contents in the mineral medium were not confirmed by analysis.
- Solubilising agent (type and concentration if used): The test material was only slightly soluble in water and was introduced into the test system on an inert carrier according to the international standard ISO 10634. The test material was thoroughly mixed, after which an amount was dissolved in ethanol. The final test concentration was prepared by dissolving 0.2594 g of the test material in 10 mL ethanol (Stock I). An aliquot of 250 µL of Stock I was added onto a glass fibre filter (Whatman GF/C Φ 47 mm) by pipette. The ethanol was allowed to evaporate. Once the test flasks were filled with mineral medium, the filters were subsequently added.
- Test temperature: 20 ± 2
- pH: 7.4 ± 0.2
- pH adjusted: Yes. After addition of the test material and mineral medium, the pH was measured in each treatment (before inoculation). If necessary, the pH was adjusted to 7.4 ± 0.2 with 0.1 M HCl or 0.4 M NaOH solution.
- Aeration of dilution water: Yes. The test media were saturated with oxygen.
- Concentration of solids per test flask: 30 mg (d.w.)/L
- Continuous darkness: Yes
- ThOD(NH3) of test material: 51.4 mg O2/L, or 15 mg O2/flask/L for the 21.6 mg/L concentration.
- Duration: After 28 days of incubation it was decided to prolong the test by at least one week because the oxygen consumption seemed not to have reached the plateau phase. The actual termination was after 39 days (928 h). After termination of the test, the pH was measured in each flask.

TEST SYSTEM
- Culturing apparatus: Glass flasks approximately 500 mL. The test flasks were filled with 300 mL mineral medium and the filters were subsequently added. 2.5 mL of sludge was used for inoculation per flask.
- Number of culture flasks/concentration: 3 for blank with filter and test material, 2 for inoculum activity control and two for toxicity control.
- Measuring equipment: Columbus Instruments Micro Oxymax respirometer.

SAMPLING
- Sampling frequency: Oxygen concentration was determined every five hours.
- Sampling method: The respirometer attached to the test flasks was used to measure the oxygen concentration.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Containing mineral medium only and a filter that was treated with ethanol.
- Reference control: Containing 100 mg/L sodium acetate in Milli-Q water. To each replicate flask a blank glass fibre filter (treated with ethanol only) was also added.
- Toxicity control: Containing 21.6 mg/L test material and 100 mg/L sodium acetate.

Reference substance:

acetic acid, sodium salt

Remarks:

nominal test concentration of 100 mg/L

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

39 d

Details on results:

TEST CONDITIONS
The pH of the medium in the test flasks decreased to between 6.71 and 6.84 after 39 days of incubation. In the reference and the toxicity controls the pH at the end of the test was in the range of 7.45 to 7.59. The higher pH in these flasks is due to the higher CO2 production. The pH value was within the limits of the validity criteria.

The average temperature in the flasks was 21.2 ± 0.7 °C during most of the incubation time (ranged from 18.7 to 22.9 °C), with a negligible amount of measurements above 22 °C. However, between 13 and 23 days of incubation the temperature was in the range of 13.1 to 18 °C, due to technical malfunction of the incubator. In general a lower temperature will lead to lower degradation rates. Inspection of the oxygen consumption rate in that time period did not show significant changes. After 13 days of incubation the reference test, as well as the toxicity control are already in the plateau phase, so not much influence is expected there. The cumulative consumption rate in the test material flask was never above the blank value, indicating that even at higher temperature, no biodegradation will have occurred due to inhibiting effects of the test material. It was therefore decided that although the temperature was temporarily too low, it did not influence the results of the study.

BLANK AND TOXICITY CONTROL TESTS
The oxygen consumption in the blanks was 5.67 to 6.91 mg/flask after 28 days of incubation (blank 1 not included), which is equal to 18.9 to 23.0 mg/L. This is within the validity limits given in the guideline.
The cumulative oxygen consumption in the toxicity control (sodium acetate and test material) was 21.73 mg O2/flask after 14 days, which was slightly lower than that of the inoculum activity control with sodium acetate only (24.12 mg O2/flask). This indicated that the test material did slightly inhibit the degradation of sodium acetate at the concentration tested. Based on the combined ThOD of both substances, a biodegradation >25 % was reached, which, according to the guidelines, means that the test material is considered not toxic to the inoculum.

BIODEGRADATION TEST
The percentage of degradation of the test material after 28 days of incubation did not exceed 0 % in a manometric respiration test at a concentration of 21.6 mg/L, calculated from the ThOD(NH3) (2.38 mg O2/mg) of the test material. The biodegradation did not exceed the pass level of 60 % ThOD(NH3) within 28 days and, therefore, is classified as not readily biodegradable.

DATA CORRECTIONS
At some points the data were corrected. The correction comprised replacing the outlier with the average of the measurements before and after the sampling point in question. If this resulted in more than 20 % outliers, no corrections were carried out at all. One of the blanks (Blank/1) was overall considered outlier and not taken into account in calculations of mean values

Results with reference substance:

The reference material was sufficiently degraded within 14 days of incubation.
After 28 days the biodegradation ThOD % was 101 (mean of two samples).

Table 1: Biodegradation of the test material (expressed as the BOD (mg O₂/L) and as percentage of its ThOD(NH₃))

Time	BOD	% Biodegradation
(days)	mg O₂/mg	ThOD(NH₃)
14	-0.07	-3
28	-0.10	-4
39	-0.17	-7

Table 2: Results of the blank, reference and toxicity control tests (mean values of the cumulative oxygen consumption (mg O₂/flask))

Time (days)	Inoculum blank*	Inoculum Activity Control*		Toxicity Control*
	mg O₂/flask	mg O₂/flask**	Biodegradation ThOD %	mg O₂/flask	Biodegradation ThOD %
14	4.04	24.12	98	21.73	49
28	6.29	26.92	101	24.32	50
39	8.34	29.49	103	27.40	53

*Mean value of two samples

**Not corrected for blank

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The percentage degradation of the test material did not exceed 0 % in a manometric respiration test.

Executive summary:

The biodegradability of the test material was determined in accordance with the standardised guidelines OECD 301 F and EU Method C.4-D under GLP conditions. A minor deviation from the guidelines took place as additional NaNO₃ was added to the mineral medium to prevent nitrogen limitation occurring.

The test material (a colourless liquid) was only slightly soluble in water. Therefore, glass fibre filter carriers were used to introduce the test material into the inoculated medium according to the International Standard ISO 10634. One concentration of 21.6 mg/L (based on the test material as received) was tested, corresponding to a ThOD(NH₃) of 51.4 mg O₂/L.

An inoculum was prepared from activated sludge taken from an oxidation ditch used to treat domestic sewage (30 mg (d.w.)/L).   The test was incubated at the target temperature of 20 ± 2 °C and the test duration was prolonged to 39 days as it was judged that the oxygen consumption had not reached the plateau phase after 28 days. Blank, reference (sodium acetate) and toxicity (sodium acetate and test material) controls were carried out in parallel.

The test material was not degraded after 28 or 39 days.

The inoculum activity appeared to be sufficient; the reference material, sodium acetate, reached the 60 % pass level of degradation within 14 days. In the toxicity control test with 21.6 mg/L of test material and 100 mg/L sodium acetate, a slight inhibition of the degradation of sodium acetate was found. The results of the biodegradability test indicated that any biodegradability of the test material could not be demonstrated in this test due to its inhibiting effect to the inoculum; however, the test material is not toxic to the inoculum as defined by the guidelines. The test met the conditions of validity given by the guidelines.

In this study, the 60 % degradation criterion after 28 days was not met and therefore the test material was considered not readily biodegradable.

Description of key information

Not readily biodegradable; OECD 301 F, EU Test Method C.4-D, Hanstveit (2003)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The biodegradability of the test material was determined in accordance with the standardised guidelines OECD 301 F and EU Method C.4-D under GLP conditions. A minor deviation from the guidelines took place as additional NaNO₃ was added to the mineral medium to prevent nitrogen limitation occurring. The study was assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997).

The test material (a colourless liquid) was only slightly soluble in water. Therefore, glass fibre filter carriers were used to introduce the test material into the inoculated medium according to the International Standard ISO 10634. One concentration of 21.6 mg/L (based on the test material as received) was tested, corresponding to a ThOD(NH₃) of 51.4 mg O₂/L.

An inoculum was prepared from activated sludge taken from an oxidation ditch used to treat domestic sewage (30 mg (d.w.)/L).   The test was incubated at the target temperature of 20 ± 2 °C and the test duration was prolonged to 39 days as it was judged that the oxygen consumption had not reached the plateau phase after 28 days. Blank, reference (sodium acetate) and toxicity (sodium acetate and test material) controls were carried out in parallel.

The test material was not degraded after 28 or 39 days.

The inoculum activity appeared to be sufficient; the reference material, sodium acetate, reached the 60 % pass level of degradation within 14 days. In the toxicity control test with 21.6 mg/L of test material and 100 mg/L sodium acetate, a slight inhibition of the degradation of sodium acetate was found. The results of the biodegradability test indicated that any biodegradability of the test material could not be demonstrated in this test due to its inhibiting effect to the inoculum; however, the test material is not toxic to the inoculum as defined by the guidelines. The test met the conditions of validity given by the guidelines.

In this study, the 60 % degradation criterion after 28 days was not met and therefore the test material was considered not readily biodegradable.