3-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]- 2-hydroxypropyl methacrylate and 1-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]-3-hydroxypropan-2-yl methacrylate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 19 June 2008 and 24 June 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/l (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.

For the purpose of the definitive test, the test material was dispersed directly in water.
An amount of test material (500 mg) was dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 15 minutes. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentration of 1000 mg/l.
This method of preparation was performed for the three separate replicate test vessels.

- Controls:
The control group was maintained under identical conditions but not exposed to the test material.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.9 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC 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 4.1 g/l prior to use.

Synthetic Sewage:
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in 1 litre of water with the aid of ultrasonication.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Hardness:

Test water hardness: 140 mg/l as CaCO3

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.

pH:

The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.

pH values of test material preparations: 7.9

Dissolved oxygen:

Observations were made on the test preparations throughout the test period, and the pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.

Salinity:

Not applicable

Nominal and measured concentrations:

In the range-finding test activated sewage sludge micro-organisms were exposed to nominal test concentrations of 100 and 1000 mg/l.
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/l (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 500 ml conical flask
- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 litre per minute.

- No. of vessels per concentration (replicates):
3

- No. of vessels per control (replicates):
2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3.
After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

- Total organic carbon:
1.429 mg/l

- Pesticides:
0.019 µg/l

- Chlorine:
0.274 mg/l

- Conductivity:
403.577 µS/cm at 20°C

- Culture medium different from test medium:
No

EFFECT PARAMETERS MEASURED:
As each vessel reached 30 minutes contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/l and 2.5 mg O2/l). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. After measurement the contents of the BOD bottle were returned to the test vessel.

This procedure was repeated after 3 hours contact time. The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.



TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline:
Not applicable

- Range finding study
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test activated sewage sludge micro-organisms were exposed to nominal test concentrations of 100 and 1000 mg/l. The test material was dispersed directly in water.

Using this approach the effect of an excess amount of undissolved test material can be assessed in order to determine whether the test material has any effect on the activated sewage sludge micro-organisms exoenzymes, or whether the uptake of undissolved test material by processes such as phagocytosis has any adverse effect on the activated sewage sludge.

Amounts of test material (50 and 500 mg) were each separately dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 15 minutes. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentrations of 100 and 1000 mg/l.

The control group was maintained under identical conditions but not exposed to the test material.

A reference material, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 15402DH-367), was included in the range-finding test at concentrations of 3.2 and 32 mg/l in order to confirm the suitability of the inoculum.

An amount of reference material (160 mg) was dissolved in water with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 1 litre to give a 160 mg/l stock solution. Aliquots (10 and 100 ml) of the 160 mg/l stock solution were removed and dispersed with activated sewage sludge and water to give the final concentrations of 3.2 and 32 mg/l. The volumetric flask containing the reference material was inverted several times to ensure homogeneity of the solution.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Range-finding Test:
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials in the range-finding test are given in Table 1.

A study to determine the General Physico-Chemical Properties of the test material gave a water solubility level of less than 1.1 x 10-3 g/l at 20.0 ± 0.5°C, using a procedure based on the flask method. Although the test material was not in solution in the range-finding test, no significant effect on respiration was observed at either test concentration employed and given the extremely low water solubility of the test material it was considered that no inhibition would occur at this concentration. It was therefore considered justifiable to conduct the definitive test at a single nominal test concentration of 1000 mg/l which was in excess of the water solubility of the test material.

Using this approach the effect of an excess amount of undissolved test material can be assessed in order to determine whether the test material has any effect on the activated sewage sludge micro-organisms exoenzymes, or whether the uptake of undissolved test material by processes such as phagocytosis has any adverse effect on the activated sewage sludge. Additionally, the effect of the test material as a whole on a waste water treatment facility and any possible contact toxicity can be determined.

Definitive Study:
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials in the definitive test are given in Tables 2 and 3. The pH values of the test preparations at the start and end of the exposure period are given in Table 4, and observations made on the test preparations throughout the study are given in Table 5.

The following results were derived:

ECx (30 Minutes)(mg/l) ECx (3 Hours) (mg/l)
EC20 >1000 >1000
EC50 >1000 >1000
EC80 >1000 >1000
NOEC 1000 1000

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/l and 2.5 mg O2/l respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Observations made throughout the study period (see Table 5), showed that at the test concentration of 1000 mg/l, oily globules of undissolved test material were visible on the surface of the test media at 0 hours, 30 minutes and 3 hours contact time. This was considered to be due to the insoluble nature of the test material in the test media. However the test material was dispersed with the aid of ultrasonication in the test diluent for approximately 15 minutes prior to the addition of the synthetic sewage, activated sewage sludge and water, and each vessel was aerated with compressed air at a rate of approximately 0.5-1 litre per minute to ensure that there

Results with reference substance (positive control):

3,5-dichlorophenol:
ECx (30 Minutes)(mg/l) 95% CL (mg/l) ECx (3 Hours) (mg/l) 95% CL (mg/L)
EC20 4.4 - 3.1 -
EC50 11 8.9-14 9.0 7.1-11
EC80 28 - 26 -



Variation in respiration rates of controls 1 and 2 after 30 minutes was ± 2%, and ± 0% after 3 hours contact time.
The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.

Reported statistics and error estimates:

None

Table1               Oxygen Consumption Rates and Percentage Inhibition Values in the Range-findingTest

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	3.8	3	2.4	0.47	-
	R2	5.5	7	2.2	0.47	-
Test Material	100	3.6	3	2.2	0.47	0
	1000	3.6	3	2.2	0.47	0
3,5-dichlorophenol	3.2	5.9	10	2.2	0.37	21
	32	8.0	10	7.2	0.08	83

R₁– R₂= Replicates 1 and 2

Table2               Oxygen Consumption Rates and Percentage Inhibition Values in the
Definitive Test after 30 Minutes Contact Time

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	4.0	3	2.4	0.53	-
	R2	3.2	2	2.1	0.55	-
Test Material	1000 R1	3.4	2	2.4	0.50	7
	1000 R2	3.3	2	2.2	0.55	[2]
	1000 R3	3.1	2	2.0	0.55	[2]
3,5-dichlorophenol	3.2	5.9	7	2.4	0.50	7
	10	7.0	10	4.4	0.26	52
	32	7.8	10	6.8	0.10	81

[Increase in respiration rate as compared to controls]

R₁– R₃= Replicates 1 to 3

Table3               Oxygen Consumption Rates and Percentage Inhibition Values in the
Definitive Test after 3 Hours Contact Time

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	3.8	3	2.4	0.47	-
	R2	3.6	3	2.2	0.47	-
Test Material	1000 R1	3.7	3	2.2	0.50	[6]
	1000 R2	3.2	2	2.2	0.50	[6]
	1000 R3	2.8	2	1.8	0.50	[6]
3,5-dichlorophenol	3.2	5.8	9	2.3	0.39	17
	10	6.9	10	5.0	0.19	60
	32	7.9	10	7.1	0.08	83

[Increase in respiration rate as compared to controls]

R₁– R₃= Replicates 1 to 3

Table 4               pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal Concentration (mg/l)		pH
		0 Hours	3 Hours
Control	R1	7.7	8.1
	R2	7.8	8.0
Test Material	1000 R1	7.9	7.9
	1000 R2	7.9	7.9
	1000 R3	7.9	7.9
3,5-dichlorophenol	3.2	7.7	8.2
	10	7.8	8.4
	32	7.8	8.4

R₁– R₃= Replicates 1 to 3

Table5               Observations on the Test Preparations Throughout the Test Period

Nominal Concentration (mg/l)		Observations on Test Preparations
		0 Hours	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Material	1000 R1	Dark brown dispersion with oily globules of test material on surface	Dark brown dispersion with oily globules of test material on surface	Dark brown dispersion with oily globules of test material on surface
	1000 R2	Dark brown dispersion with oily globules of test material on surface	Dark brown dispersion with oily globules of test material on surface	Dark brown dispersion with oily globules of test material on surface
	1000 R3	Dark brown dispersion with oily globules of test material on surface	Dark brown dispersion with oily globules of test material on surface	Dark brown dispersion with oily globules of test material on surface
3,5-dichlorophenol	3.2	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	10	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	32	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible

R₁– R₃= Replicates 1 to 3

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/l (three replicate flasks) for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.

The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀ of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000mg/l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

The reference material gave a 3-Hour EC₅₀ value of 9.0 mg/l, 95% confidence limits 7.1 - 11 mg/l.

Description of key information

To assess toxicity to microorganisms of the substance (SiMAA2), an experimental result has been read-across from a structural analgoue substance (EC 700-043-1).

Introduction.

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test material at a concentration of 1000 mg/l (three replicate flasks) for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.

The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀ of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

The reference material gave a 3-Hour EC₅₀ value of 9.0 mg/l, 95% confidence limits 7.1 - 11 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

In addition to the ASRI study on structural analogue, additional evidence on the toxicity to microorganisms comes from the ready biodegradation study on SiMAA2.

In this study the toxicity control attained levels of degradation confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.